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Introduction ============ Brain metastases from hepatocellular carcinoma (HCC) were very rare \[[@B1],[@B2]\]. However, with the development of neuroimaging studies and improved survival due to the recent advanced therapy to the liver, the incidence has been increasing \[[@B3],[@B4]\]. Brain metastases from HCC are well-known to be accompanied by intracranial hemorrhage at the time of diagnosis. Its incidence has been reported to be 42.8%-74.7% \[[@B2],[@B5],[@B6],[@B7]\]. Whereas optimal management of intracranial hemorrhage from brain metastases has not been established, surgical resection, radiosurgery and whole brain radiotherapy (WBRT) were reported to have own role in the control of the hemorrhagic brain metastases \[[@B8],[@B9],[@B10]\]. Regarding the brain metastases from HCC, Han et al. \[[@B11]\] reported that 30% of the patients with brain metastases from HCC experienced post-treatment hemorrhage after treatment, so recurrent bleeding was the most problematic event during the management of the patients with brain metastases. However, few studies were reported addressing the control of intracranial hemorrhage of the brain metastases from HCC. Recently, Yamakawa et al. \[[@B10]\] reported that WBRT could prevent intracranial hemorrhage. However, this study enrolled only 15 patients and the patients undergoing WBRT were compared with those receiving conservative management only. In this study, authors evaluated the incidence and risk factor of post-treatment intracranial hemorrhage (defined as hemorrhagic brain metastases developed after initial treatment to brain metastases), and the impact of WBRT in the control of hemorrhage of the brain metastases from HCC. Materials and Methods ===================== After Institutional Review Board approval, medical records of 105 patients who have been diagnosed with brain metastases from HCC between January 2000 and December 2013 were retrospectively reviewed. The patients who received conservative treatment only and who did not complete WBRT were excluded. A total of 81 patients were enrolled in this study. All patients were diagnosed as brain metastases radiologically using computerized tomography or magnetic resonance imaging. Clinical data including presenting symptoms, performance status, liver function, whether primary lesion controlled or not, presence of extracranial metastases, number of brain lesion, treatment modality and clinical courses were assessed. Because there was no prospective protocol, the patients were under the variable follow-up schedule depending on the physicians. In general, the patients underwent brain images when there was a change in neurologic symptoms. The presence of post-treatment hemorrhage was identified by reviewing all follow-up brain images of the patients. Post-treatment hemorrhage was defined as progression of hemorrhage in the initial lesion, or newly developed hemorrhagic brain metastases. Neurologic death was defined following the study by Patchell et al. \[[@B12]\]. Time to intracranial hemorrhage (TTH) after treatment was calculated from the first day of any active treatment for brain metastases until the radiologic evidence of intracranial hemorrhage. Log-rank test for each prognostic factor was applied to compare the Kaplan-Meier curves for TTH. Cox proportional hazard model analysis was used for multivariate analysis. Log minus log survival plots were used for identifying proportional hazards assumptions. A chi-square test was used to examine the association between categorical variables. A p-value less than 0.05 was considered to indicate a statistically significant difference. Analyses were performed using PASW Statistics ver. 18.0 for Windows (SPSS Inc., Chicago, IL, USA) and the R Project for Statistical Computing ver. 3.1.2. Results ======= 1. Patient characteristics -------------------------- A total of 81 patients were treated with surgery, radiosurgery or WBRT for brain metastases from HCC. Median age was 56.3 years. Motor weakness (48.1%) was the most frequent symptom followed by headache (32.1%) at the time of diagnosis of brain metastases. Intracranial hemorrhage was detected in 64 patients (79.0%). Brain metastasis presented as a single lesion in 37 patients. Median value of alpha-fetoprotein (AFP) level was 1,700 ng/mL and primary tumor was controlled in 33 patients. Seventy-six patients had extracranial metastases, and the lung was the most frequent involved organ (n = 74). Transcatheter arterial chemoembolization (TACE), surgical resection, percutaneous ethanol injection therapy (PEIT), radiofrequency ablation (RFA), transplantation and/or radiation therapy were performed for the primary therapy to the liver. Chemotherapy was given to 55.6% of the patients. The Eastern Cooperative Oncology Group performance status (ECOG PS) for 20 patients was greater than 2. Child-Pugh classification was evaluated as A for 65 patients, B or C for 16 patients ([Table 1](#T1){ref-type="table"}). 2. Treatment ------------ Fifty-seven patients underwent WBRT. While 47 patients underwent WBRT alone, 7 patients had surgery followed by adjuvant WBRT and 3 patients had radiosurgery plus WBRT. Of 24 patients who had not undergone WBRT, 18 patients had radiosurgery alone, 3 patients had surgery alone, and 3 patients had surgery plus radiosurgery. Details of treatment are summarized in [Table 2](#T2){ref-type="table"}. 3. Outcome and risk factor analysis ----------------------------------- Median length of follow-up was 3.9 months for all patients and 9.3 months for survivors at the last follow-up. During follow-up, 12 post-treatment intracranial hemorrhage were identified. Three-month post-treatment hemorrhage rate was 16.1% ([Fig. 1](#F1){ref-type="fig"}). Median TTH of the patients with post-treatment hemorrhage was 1.9 months (range, 0.2 to 9.0 months). On the univariate analysis, AFP and treatment were significant risk factors ([Table 3](#T3){ref-type="table"}). Primary tumor control, extracranial metastases and initial hemorrhage were not satisfied with proportional hazard assumption. Using backward selection, ECOG PS, AFP, number of brain lesion, and treatment were incorporated into multivariate analysis. Final analysis with these 4 variables revealed that ECOG PS \> 2, AFP ≥ 1,700 ng/mL, and no WBRT were significant risk factors for post-treatment hemorrhage (p = 0.013, p = 0.013, and p = 0.003, respectively). Post-treatment hemorrhage probability according to WBRT was shown in [Fig. 2](#F2){ref-type="fig"}. Distribution of post-treatment hemorrhage lesion according to the treatment was listed in [Table 4](#T4){ref-type="table"}. Post-treatment hemorrhage of the new lesion was identified in 4 out of 24 patients treated without WBRT, while it was identified in 3 out of 57 patients treated with WBRT. Kaplan-Meier analysis showed that 3-month post-treatment hemorrhage rate of new lesion was higher in patients treated without WBRT (18.6% vs. 4.6%; p = 0.104). When we compared the patients treated with surgery and/or radiosurgery without WBRT (n = 24) and those treated with surgery and/or radiosurgery with WBRT (n = 10), the latter group showed lower 3-month post-treatment hemorrhage rate although there was not statistical significance (30.9% vs. 0%; p = 0.107). 4. Cause of death and recurrent intracranial hemorrhage ------------------------------------------------------- Overall survival was 3.4 months (95% confidence interval \[CI\], 0.0 to 6.8) for patients with post-treatment hemorrhage and 4.0 months (95% CI, 3.2 to 4.9) for patients without it (p = 0.236). Of 73 patients who were dead at last follow-up, cause of death was identified as neurologic for 45 patients or systemic for 28 patients. Thirty-five of 61 patients (57.4%) without post-treatment hemorrhage died with neurologic cause, while 10 of 12 patients (83.3%) with post-treatment hemorrhage died with neurologic cause (p = 0.114). Discussion and Conclusion ========================= In our study, post-treatment hemorrhage of brain metastases from HCC was observed in 15% of patients with 3-month rate 16.1%. ECOG PS \> 2, AFP ≥ 1,700 ng/mL, and no WBRT were significant risk factors for post-treatment hemorrhage. Recently, Yamakawa et al. \[[@B10]\] reported that radiotherapy could prevent intracranial hemorrhage in patient with brain metastases from HCC. In this study, however, the patients undergoing WBRT were compared to those receiving conservative treatment only. In our study, the patient treated with WBRT had lower 3-month post-treatment hemorrhage rate than those treated with surgery and/or radiosurgery without WBRT. Besides, post-treatment hemorrhage was not confined to the initial hemorrhage lesion. Post-treatment hemorrhage rate in the new lesion was higher in patients treated without WBRT, although statistical significance was not reached. Moreover, surgery and radiosurgery could not guarantee to prevent intracranial hemorrhage outside of the target lesion, WBRT would be effective to prevent it in the \'whole brain\'. Generally, more frequent follow-up with brain imaging was done in patients undergoing radiosurgery than WBRT alone. One might argue that frequent follow-up could result in the early detection of the asymptomatic intracranial hemorrhage. To refute this assumption, we compared the post-treatment hemorrhage rate while excluding the patients treated WBRT alone. The patients treated with surgery and/or radiosurgery without WBRT had higher 3-month post-treatment hemorrhage rate than those treated with surgery and/or radiosurgery with WBRT although statistical significance was not reached. In this sense, we can conclude that WBRT prevent bleeding of brain metastases from HCC unbiased by follow-up strategy. In this study, overall survival was not different in relation to post-treatment hemorrhage. Prognostic factors for overall survival of the patients with brain metastases from HCC were identified as ECOG PS, Child-Pugh class, AFP level, and number of brain lesion in our previous report \[[@B7]\]. Considering the advanced stage of disease, the impact of the recurrent hemorrhage on the overall survival might be limited. However, recurrent hemorrhage could be associated with neurologic death. Although the causal relationship between post-treatment hemorrhage and death was not evident due to the retrospective nature of our study, 10 of 12 patients with post-treatment hemorrhage died with neurologic cause. As uncontrolled recurrent hemorrhage eventually results in neurologic deficit, control of intracranial hemorrhage could be important in the management of brain metastases from HCC. Major limitation of this study is the insufficient follow-up data of the patients with poor survival outcome and inconsistent follow-up strategy according to the treatment modality due to the retrospective nature of the study. Post-treatment hemorrhage rate of the patients treated with WBRT alone could be underestimated. As discussed earlier, the interpretation of the results should be done with caution. Moreover, single institutional data and heterogeneous population are other limitations of the study. However, considering low incidence of brain metastases from HCC, this is the first report evaluating the post-treatment hemorrhage of brain metastases from HCC with substantial number of population. In conclusion, post-treatment hemorrhage occurs in 15% of the patients with brain metastases from HCC even after the active treatment, such as surgery, radiosurgery and/or WBRT. Poor ECOG PS, higher AFP, and no WBRT were risk factors for post-treatment hemorrhage. WBRT should be considered to prevent post-treatment hemorrhage in the treatment of brain metastases from HCC. **Conflict of Interest:** No potential conflict of interest relevant to this article was reported. {#F1} {#F2} ###### Clinical characteristics of 81 patients  HBV, hepatitis B virus; HCV, hepatitis C virus; NBNC, non-B and non-C; ECOG PS, Eastern Cooperative Oncology Group performance status; TACE, transcatheter arterial chemoembolization; PEIT, percutaneous ethanol injection therapy; RFA, radiofrequency ablation. ^a)^Each symptom was counted regardless of other symptoms of each patient. ^b)^Patients could be treated with more than one modalities. ^c)^Each metastasis was counted regardless of metastasis of other site. ###### Treatment of brain metastases  WBRT, whole brain radiotherapy. ###### Univariate and multivariate analyses for post-treatment hemorrhage  ECOG PS, Eastern Cooperative Oncology Group performance status; AFP, alpha-fetoprotein; WBRT, whole brain radiotherapy. ^a)^Log-rank test, ^b)^Cox regression analysis, ^c)^statistically significant. ###### Distribution of post-treatment hemorrhage lesion according to the treatment  WBRT, whole brain radiotherapy.

Background ========== Nanotechnology is a prioritized research topic and triggers great interest among scientists, engineers and energy researchers around the world \[[@B1],[@B2]\]. Among them, surface nanotexturing has been extensively utilized in the recent years for enabling new functionalities and tailoring excellent physical and chemical properties. A wide range of examples explored recently include antireflective coatings \[[@B3],[@B4]\], superhydrophobic surfaces \[[@B5],[@B6]\], bio-engineered thin film \[[@B7]\], anti-stiction surfaces \[[@B8]\] and bio-mimic gecko adhesives \[[@B9]\]. Experimentally, artificially fabricated inverted surface patterns of NHA and high fidelity nanopillar arrays have been proposed for substrates with structural antireflective and enhanced light management properties and practical applications include high-efficiency solar cells and synthetic gecko adhesives. In particular, antireflective coating technology using thin-film stacks are mainstream tools maturely used in many optical systems \[[@B10],[@B11]\]; however, critical limitations in the coating materials such as adhesion, thermal mismatch, and instability are the main drawbacks \[[@B12],[@B13]\]. Therefore, nanotexturing antireflective surfaces and associated fabrication technology is booming and in great demand. The major nanotexturing methods can be divided into the following three categories: micro-replication process (MRP) for combining micro/nanostructure masters, metallic mold electroplating, and replication into plastics \[[@B14]-[@B19]\]. The first primary method of MRP process can be nanoimprinting or injection nanomolding such that the mass-produce ability to functional surfaces can be implemented rapidly and is of profound technological interest \[[@B20]\]. The second method is roll-to-roll (R2R) manufacturing for printing organic light emitting diodes (OLED), thin-film solar cells, optical brightness enhancement films, or organic thin film transistors (OFET) \[[@B18],[@B21]-[@B27]\]. The third method utilized the templates such as anodic aluminum oxide (AAO) \[[@B28],[@B29]\] for anodizing high-purity aluminum to generate a porous alumina membrane as templates such that a closed-packed hexagonal array of columnar cells can be obtained. A summary for the fabrication method for the antireflective coatings is presented in Table [1](#T1){ref-type="table"}. ###### Fabrication method for the antireflective coatings **Method** **Characteristics** **Applications (other than antireflective coatings)** **References** --------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- -------------------------- Micro-replication process (MRP) Capable of creating nano/micro features on substrates of slicon or plastics. By combining three major steps of micro/nanostructure masters, metallic mold electroplating and replication into plastics. Backlight guide plate, grating, micro-mirror arrays, photonic crystals and other micro/nano features \[[@B14]-[@B19]\] Roll-to-roll (R2R) printing Capable of creating electronic devices on flexible substrates (plastics or metal foil) Typically includes steps of coatings, printing, laminating, re-reeling, and rewinding processes Organic light emitting diodes (OLED), thin film solar cells, optical brightness enhancement films or organic thin film transistors (OFET) \[[@B18],[@B21]-[@B27]\] Anodic aluminum oxide (AAO) By anodizing high-purity aluminum to generate a porous alumina membrane as templates such that a closed-packed hexagonal array of columnar cells can be obtained. Typically, can be categorized as a self-ordering synthesis of nanopores Molecular separation, energy generation and storage, electronics, photonics, sensors (biosensors), drug delivery, and template synthesis \[[@B28],[@B29]\] In this paper, we present a facile and fast fabrication route for high-throughput, low-cost nanotexturing of surfaces with tunable NHA depths. The optical properties of the textured films were systematically characterized as a demonstration to validate the proposed technique for enabling substrates with functional performance of tunable reflectivities. In addition, this NHA can be integrated with multilayer coatings of both metallic and dielectric layers to further tailor the optical properties such as ultra-sensitive sensing applications. Methods ======= Preparation of the PC film via precision injection nanomolding -------------------------------------------------------------- Precision injection nanomolding processes were routinely used to fabricate optical disks in large quantities such as CD, DVD, and blue-ray disks (BD) with subwavelength features. Therefore, we chose precision injection nanomolding to fabricate the optical element with submicron holes. Due to high optical transparency in the visible and near-infrared wavelengths, polycarbonate (PC) pellets (TAIRILITE, MD1500, 99.5% pure) were chosen as the polymer materials. A critical issue of nanoimprint or nanostructure replication is the fabrication of nanostructured stamp. Previously, the nickel imprint stamp using electroforming process and features as small as 50-nm-sized patterns of original silicon master were faithfully transferred \[[@B30]\]. The details of the electroforming process such as composition of the chemical solution and operating parameters can be found in \[[@B31]\]. For the Ni mold used for the injection nanomolding, similar to the optical disk production and prior studies, electroforming is adopted to transfer the nanostructures with the original master silicon molds. Figure [1](#F1){ref-type="fig"} shows both scanning electron microscope (SEM) and atomic force microscope (AFM) images of the Ni mold used. The period of the Ni mold array is in the range of 650 to 700 nm and the nanopillar heights are about 400 nm. Precision injection nanomolding machine (Sumitomo SD35E) used for the experiments were shown in Figure [2](#F2){ref-type="fig"} and the feeding and injection units can be clearly seen respectively in Figure [2](#F2){ref-type="fig"}a. The mold region where the Ni mold resides is also indicated in Figure [2](#F2){ref-type="fig"}b. Furthermore, Figure [2](#F2){ref-type="fig"}c illustrated the importance of precisely replicated NHA being carefully controlled by the nanoinjected substrate thickness. The experimental results reveal that the standard deviations of 50 selected samples for substrate thickness can be reliably minimized to 0.02%, demonstrating the highly consistent capability in the nanoreplication process. {#F1} {#F2} Characterization of the replication process and operating parameters -------------------------------------------------------------------- To characterize the nanotextured surfaces, both SEM (LEO 1530 Gemini, Zeiss, Oberkochen, Germany) and AFM (Digital Instruments nanoscope, Tonawanda, NY, USA) were utilized. For the optical reflectivity measurements, spectrophotometer STEAG ETA-Optic (Heinsberg, Germany) and n&k analyzer 1280 (n&k Technology, Inc., San Jose, CA, USA) were used at the angle of 90°. Results and discussion ====================== The precision injection nanomolding process has been widely accepted as one of the rapid replication methods to transfer nanostructures and is considered a major mass production technique for a wide range of commercial products \[[@B13]\]. In particular, the major processing parameters can be classified into the following: injection and mold temperatures, packing time and pressure, injection speed, etc. The diameter of the injection nanomolded film is a disk shape which geometric dimension is 120 mm in diameter and 0.6-mm thick. For a typical injection nanomolding operation, the following parameters apply: mold temperature is intentionally controlled in the range of 115 to 130°C, respectively, while the following parameters are fixed: 0.5-s packing time and 130-MPa packing pressure, injection speed 120 cm/s while the PC viscous flow was maintained at 320°C, total clamping force is fixed at 350 KN. Total cycle time for one shot of process including automatic transfer can be as low as 4 s while maintaining a high-fidelity replication. An automatic monitoring system is included in the injection process and deviation for the molding temperature is within ±0.5°C. In previous studies, the molding and PC flow temperature play a significant role on the replicated structure, both in terms of precise fidelity of depth and pitch. Other experimental work can be briefly explained as following: a stock PC pellets is fed into the system and used as the supply material. The mold holds a temperature controlled water circulation system for the purpose of heating and cooling function that facilitates the continuous operation and to ensure uniformity of viscous flow. The NHA stamp is held in the machine firmly and symmetrically about the mold geometric center while the transfer mechanism is concurrently applied. Upon finishing the molding process, the molded part is transferred to a conveyer for later rinsing deionized (DI) water bath. The system allows the user to control all the above parameter settings, and in particular, both the material and the molding temperatures are the most crucial ones. Figure [3](#F3){ref-type="fig"} shows AFM image of a typical replication of submicron holes with a scan area of 6 × 6 μm^2^. Submicron holes can be reliably and swiftly replicated for the scanned areas, and typically, we select five to seven measurements for the uniformity consideration. The fidelity of replication is experimentally validated to be extremely good and deviations are routinely maintained with 10% of the fabricated NHA depths. Previous experiences from CD/DVD/BD manufacture assist us in choosing the molding temperature as the dominating factor in the nanoreplication process. In order to investigate the impact of different molding temperatures, temperatures in the range of 110°C to 130°C are selected for the PC film replication process. From Figure [3](#F3){ref-type="fig"}, a uniformity of NHA can be demonstrated in cross-sectional view of a 700-nm pitch sample, showing consistently conformal NWA diameters of approximately 400 nm and 300-nm depth can be fabricated. {#F3} It is noticeable and worth pointing out that the NHAs fabricated here have geometrically hemispherical bottom which can be potentially served as the backside reflector one-end open cavities for photon trapping. Next, a wide range of nanohole depths in the range of approximately 200 to 420 nm can be quickly and reliably replicated simply by changing the mold temperature as shown in AFM measurements of Figure [4](#F4){ref-type="fig"}a,b,c,d. It experimentally scanned five to seven areas for each sample from the center to the circumference and variation in fabricated NHAs in terms of replication depth, diameter and periodicity and was found to be negligible, showing a consistent replication over an area of 100-mm-diameter PC film. The section analysis and associated top views for various depths as a function of molding temperature reveals that the depth is linearly proportional to the molding temperature. Note that the injection nanomolding is widely controlled in the compact disk industry, which is technically proven to be a fast, large area with a high-throughput manufacturing process. The density of surface features can be readily tuned simply by changing another Ni stamp of different periodicity. The manufacture of Ni stamp adopts the commercially available electroforming process which is described elsewhere \[[@B30],[@B31]\]. Generally, other anti-reflection nanotextured surfaces such as etching utilized anodization voltage to control the pitch over the surface feature density, while uniformity can be a serious issue over a large area. Notably, the depths of NHAs can be independently tuned by molding temperature in the present study. {#F4} Based on the above reliable replication of injection nanomolded textured PC film, we subsequently focus on the utility and potential practical applications as anti-reflection layers. Given the controlled geometry of the surface features with prescribed diameters, depth, and periodicity, textured PC film can be utilized as ideal nanoscale replication tools for template-assisted replication of nanostructured materials using nanoimprinting process. Furthermore, another important application of surface texturing is the enhancement and/or tunability of photon management. Bio-inspired structures include "moth eye" antireflective coatings and intentionally textured back contacts are two specific examples which have been shown as promising candidates to enhance the absorption and/or carrier collection efficiency of solar cells. In particular, large-area subwavelength surface texturing with tunable capability is highly desired. Measured reflection spectra of both bare and textured PC films are shown in Figure [5](#F5){ref-type="fig"}. Bare PC films can be considered as the mirror surface and exhibit a high average reflection of 9% to 10% over the explored wavelength range of 350 to 800 nm. The light reflection can be dramatically decreased to approximately 1.3% for the approximately 410-nm depth holes at the optical frequency of 420 nm. For other nanotextured surfaces with the same periodicity, the light reflection for different depths can be clearly discernible and approximately proportional to light reflection. The low reflectivity of nanotextured surfaces is vividly attributed to the bio-inspired NHA, without resorting to other methods such as tunability of refractive index typically utilized as light trapping in the deep trenches of the pores. The tendency for the reflection decrease due to the increase of NHA depth over the solar spectrum of 350 to 800 nm may be attributed to the smaller refractive index gradient with respect to structure depth \[[@B32]\]. Theoretically, the refractive index gradient plays a critical role in the significant suppression of broadband reflection through destructive interference such that the continuous change in refractive index causes the incident light to be reflected at different depths from the interface of air and anti-reflection coatings. Figure [6](#F6){ref-type="fig"} shows the AFM measured depth of the replicated nanohole arrays on PC film as a function of the injection nanomolding temperature. It can be experimentally determined that molding temperature is an effective parameter to reliably control the depths of NHA over a large area. {ref-type="fig"} and [6](#F6){ref-type="fig"}, respectively.](1556-276X-8-407-5){#F5} {#F6} In the experimental implementation of the metallic and dielectric layers deposited on the PC substrate, the method of high-vacuum plasma-assisted deposition was used and both the metallic layer Al and dielectric layer ZnS-SiO~2~ films were deposited sequentially under the conditions of Class 100 cleanroom. The thickness of Al film is approximately 100 ± 20 nm and was measured by atomic force microscope with use of the kapton tape technique. Figure [7](#F7){ref-type="fig"}a shows reflection spectrum of the mirror surface, as well as the reflection spectrum of NHA with metallic and dielectric layers calculated with the use of the finite difference time domain (FDTD) approach. The increased reflection was measured due to extra coating layers of Al (100 nm) and ZnS-SiO~2~ (100 nm), resulting in the highest reflection at 520 nm and reflection value of almost 0.73 for the mirror surface. It is observed that a similar trend can be obtained from the FDTD analysis. Again, the reflection from the similar coating layers on the NHA is significantly lower as compared to the mirror surface, which corresponds to the highest reflection at 589 nm and reflection value of almost 0.33 as shown in Figure [7](#F7){ref-type="fig"}b. Despite the similar coating layers on the same PC substrate and the same refractive index, NHA configuration does exhibit one important feature of shifted peak of reflection and can potentially function as an ultrasensitive sensing device. {#F7} Conclusions =========== In summary, a versatile and rapid process is presented based on the well-established injection nanomolding of PC polymer for the controlled nanotexturing of NHA surfaces over large areas with tunable depth topography. In addition, with the change of master Ni stamp, feature size diameter and density/periodicity can also be adjusted accordingly. The NHA-engineered surfaces exhibit a functional optical property that can be optimized for anti-reflection coatings. The proposed technology of rapidly replicated NHA surfaces may be used for efficient and cost-effective solar cells, highly light extracted light-emitting diodes (LED) and self-cleaning surfaces. The scalability of the process can be sufficiently addressed due to the reduced cycle time of 4 s and is fully compatible with the well-established mass production of DVD/BD industries. This work presents an important advance in the rapidly growing field of nanomanufacturing. Furthermore, we have also experimentally demonstrated an approach to quantitatively control transmission of light through NHA and multilayer coating of both dielectric and metallic layers with the potential use of sensing applications. The future work can be extended to the transmission of light through current NHA/multilayer structures and geometry-dependent selectivity in terms of both frequency and resonant width. Competing interests =================== The authors declare that they have no competing interests. Authors' contributions ====================== YKF designed the experiments, analyzed the data, and wrote the paper. CCP performed the experiments and measurements. CTH performed the simulations, helped with the revisions of the manuscript and preparation of response letters. All authors discussed the results, commented on, and approved the final manuscript. Acknowledgement =============== This work was supported by the Taiwan National Science Council under contract no. NSC 101-2221-E-008-014 and NSC 102-2221-E-008 -067.

INTRODUCTION ============ Since the first commercial lithium-ion battery (LIB) appeared in the early 1990s, LIBs have been widely used in portable electronic devices and have recently been successfully applied in electric vehicles, unmanned aircraft, and large-scale deployment ([@R1]). However, the graphite anode of LIBs only has a theoretical specific capacity of 372 mAh g^−1^. This inherent limitation makes it unlikely to meet the growing demand for high-energy storage ([@R2], [@R3]). Li metal is thought to be one of the most promising alternative anodes because of its step-change improvement in capacity (3860 mAh g^−1^) and its low redox potential (−3.04 V versus the standard hydrogen potential electrode) ([@R2], [@R4]). In addition, lithium-metal anode is an essential component for lithium-sulfur and lithium-air batteries, which are currently under intensive research for next-generation high-energy lithium batteries ([@R5]--[@R7]). Unfortunately, many challenges need to be overcome before Li metal batteries become a reliable technology, the most serious of which are the poor cycling stability and safety issues ([@R8]). During a typical charge step of cell cycling in lithium-metal batteries, lithium cations gain electrons on the anode surface and become metallic lithium. The reductive electrodeposition is usually nonuniform and hard to control because of the complicated surface chemistry and morphology of the anode. This heterogeneous nature of the electrodeposition or so-called Li dendrite becomes more marked during the subsequent cycles because of the uneven charge distribution along the rough surface. The growth of lithium dendrites is known as the source of battery failure. On the other hand, the electrolyte and the lithium metal could react irreversibly and form a solid electrolyte interface (SEI) on the metal surface to passivate the highly active Li metal and reduce further consumption of active materials. The fragile SEI could be ruptured during cycling because of the huge volume expansion of the anode. The instability of an SEI is another reason for the propensity of lithium to deposit in dendritic structures ([@R9], [@R10]). Once the fresh Li encounters the electrolyte, chemical reactions happen again and form a new SEI, leading to irreversible consumption of Li and electrolyte. As a consequence, the coulombic efficiency of Li metal batteries decreases, and the capacity fades quickly. The sharp dendrites on the surface of Li metal anode could pierce the separator and cause short circuit, generating heat or even igniting the organic solvents ([@R11]). In addition, the huge volume change of Li metal anode leads to internal stress and interface fluctuation ([@R12]). These problems have hindered the practical applications of rechargeable Li-metal--based batteries over the past 40 years. Many experimental studies have emerged to regulate the Li metal anodes and stabilize the electrochemical behavior of lithium-metal batteries ([@R13]--[@R20]). In addition, the formation of lithium dendrites has been monitored at various length scales. Researchers have reported and theoretically predicted the conditions of dendrite nucleation and propagation. The dendrite growth is understood to be driven by the diffusion and electromigration of both Li ion and the counter ion under electric driving force. During charging, lithium cations and their counter ions transport in opposite directions and cause ion concentration gradient. At high current densities, the situation for the anions is more marked because they cannot form electrodes. Eventually, anions pile up near the anode and deplete near the cathode. This phenomenon is called "space charge," which determines the onset time of lithium dendrites ([@R21]--[@R25]). To fundamentally understand the mechanisms of electrolyte transport properties and suppress or even eliminate dendrite growth from the nucleation step, we report on the relaxation of ion concentration gradient via pulse current charging instead of common constant current charging and the stabilization of lithium-metal batteries via simply charging cells with square-wave pulse current ([Fig. 1A](#F1){ref-type="fig"}). Pulse current charging was first used for lead acid battery to remove lead sulfate compound and extend battery lifetime ([@R26]). It was later used as an advanced charging technique for LIBs for the homogeneity of ion distribution between the two electrodes ([@R27], [@R28]). These reports showed that the pulse current charging could lower the impedance and temperature increment during the charging process and reduce side reactions compared to constant current charging. Only a few studies for the influence of pulse current charging in rechargeable lithium-metal batteries have been reported. To the best of our knowledge, the only experimental work for pulse current charging contains limited cycling information without appropriate simulation support ([@R29]). The underlying mechanism at the molecular level, which is essential for the rational design of pulse current charging, is also limited so far. Only two recent studies by Aryanfar *et al*. ([@R30]) and Mayers *et al*. ([@R31]) performed coarse-grained molecular simulations for lithium dendrite growth under pulse current charging. They both revealed a competition between the time scales for Li^+^ diffusion and reduction at the anode-SEI interface to understand the dendrite formation, and theoretically suggested that lithium dendrites could be inhibited by pulse charging with appropriate charging frequency. {#F1} To understand dendrite growth mechanism both theoretically and experimentally, we used rectangular cathodic pulses with various frequencies and rest time, and monitored their growth propensity by all-atom molecular dynamics (MD) simulations and voltage-time profiles. MD simulations were performed to examine the diffusivity of Li ions in an electrolyte under pulsed electric fields with different frequencies and strength. The solvation structures of Li ions were also considered to further understand the effectiveness of transport properties on dendrite growth. The model predicts that loose association between cations and anions can enhance the diffusion of Li^+^. We also evaluated the cycling behavior by galvanostatic cycling measurements and found that the cell lifetime can be more than doubled using certain pulse current waveforms. Both experimental and MD simulation results demonstrated that the effectiveness of pulse current charging can be optimized by choosing proper time- and frequency-dependent pulses. This work provides a molecular basis for understanding the mechanisms of pulse current charging for mitigating lithium dendrites and designing pulse current waveforms for stable performance in advanced lithium-metal batteries, such as high-energy lithium-sulfur and lithium-oxygen batteries. RESULTS ======= Diffusion of Li^+^ in pulsed electric fields -------------------------------------------- Pulsed electric fields may affect both the diffusion and electrodeposition of Li^+^. Although it is still challenging to examine the electrodeposition of Li^+^ using classic all-atom MD at a large scale, it is, however, feasible to examine the diffusion of Li^+^ at the molecular level, which is the focus of this work. We abstract a local area in an electrolyte and analyze the microscopic motion of Li^+^ based on all-atom MD simulations. In all-atom models, the position and velocity of each individual atom are explicitly represented in the simulations, which allow high-resolution analysis of simulation trajectories. The simulation system is constructed with 51 Li^+^, 51 TFSI^−^ \[bis(trifluoromethanesulfonyl)imide anion\], and 600 PC (propylene carbonate) molecules, which gives a total of 8616 atoms and represents LiTFSI \[lithium bis(trifluoromethanesulfonyl)imide\]--PC solution with a concentration of 1.0 M lithium salt. The system is in a cubic box with a dimension of 43.88 Å in length (*x*), width (*y*), and height (*z*) ([Fig. 1B](#F1){ref-type="fig"}). The self-diffusion coefficient *D* of Li^+^ can be computed from the mean square displacement (MSD) of MD trajectories based on the Einstein relation$$\mathit{D}^{\text{MSD}} = \underset{\mathit{t}\rightarrow\infty}{\text{lim}}\frac{\langle{|\overset{\rightarrow}{\mathit{r}}(\mathit{t}) - \overset{\rightarrow}{\mathit{r}}(0)|}^{2}\rangle}{6\mathit{t}}$$where $\overset{\rightarrow}{\mathit{r}}(\mathit{t})$ is the location of the center of mass of a specific ion at time *t*; the brackets indicate the ensemble average. [Figure 2](#F2){ref-type="fig"} shows that the diffusion coefficients of Li^+^ in all cases are sensitive to applied electric fields. Pulsed electric fields may significantly enhance the diffusion of Li^+^, which is larger than that in a static electric field or in the control case, where no electric field is applied. The pulsed electric field with a *T*~on~/*T*~off~ ratio of 1:3 (*T*~on~ means the period of constant current charging or discharging, and *T*~off~ means the rest period followed by the constant current charging or discharging) is observed to offer the highest diffusion coefficient of Li^+^ among all the cases examined in this study. Moreover, in an electric field with a higher strength of 3 × 10^6^ V m^−1^, a *T*~on~/*T*~off~ ratio of 1:3 is also found to be the optimal condition for Li^+^ diffusion (fig. S1A). This observation suggests that an innate nature of LiTFSI-PC solution may determine the optimal pulse current waveform for achieving the highest diffusivity of Li^+^, which is independent of the strength of the electric field within a certain range. For instance, in electric field with an even higher strength, such as 3 × 10^8^ V m^−1^, the diffusion coefficient was observed to be dominated by the strength of electric field (fig. S1B). This is consistent with a recent experimental study by Pei and co-workers ([@R32]), suggesting that high overpotentials are capable of effectively reducing the dendrite formation of lithium-metal batteries. Because the only intramolecular interactions of Li^+^ in the solutions are the ones with TFSI^−^ and PC, the solvation structures of Li^+^ and TFSI^−^ in PC solutions need to be examined for further insights. {#F2} It should be noted that the diffusion coefficients of Li^+^ obtained from this work are similar to those from other MD studies using the OPLS-AA (optimized potentials for liquid simulations for all-atom) force field ([@R33], [@R34]), which are typically about one order of magnitude lower than the experimental findings measured with the pulse gradient spin echo nuclear magnetic resonance ([@R35]). Despite the difference in value, the diffusion coefficients derived from simulations were understood to qualitatively agree with those revealed by experiments ([@R33]). Solvation structure of Li^+^ in PC solutions -------------------------------------------- As shown in the simulation snapshot ([Fig. 1](#F1){ref-type="fig"}, B and C), Li^+^ may associate with TFSI^−^ and PC, leading to certain solvation structures. The structures can be described with the radial distribution function (RDF), which shows how density of atom *j* varies as a function of distance from a reference atom *i* (*i* in this case is Li^+^, and *j* could be either a PC molecule or TFSI^−^)$$\mathit{g}_{\mathit{i}\mathit{j}}(\mathit{r}) = \frac{\langle\mathit{N}_{\mathit{i}\mathit{j}}(\mathit{r})/\mathit{V}(\mathit{r})\rangle}{\rho_{\mathit{j}}}$$where *N*~*ij*~(*r*) is the ensemble averaged number of atoms *j* in a spherical shell of volume *V*(*r*) at a distance *r* from atom *i*, and ρ~*j*~ is the bulk density of atom *j*. The coordination numbers of the PC solvent molecules surrounding the solvation shell of the ions are calculated from the following integral$$\mathit{n}(\mathit{R}) = 4\pi\rho_{\mathit{j}}\int_{0}^{\mathit{R}}\mathit{r}^{2}\mathit{g}_{\mathit{i}\mathit{j}}(\mathit{r})\mathit{d}\mathit{r}$$where *R* is the minimum after the first peak in the RDF *g*~*ij*~(*r*). The different coordination numbers may affect the dynamic properties of the Li^+^, such as their diffusivity ([@R36]). [Figure 3](#F3){ref-type="fig"} (A and B) shows that the first peak in *g*(*r*) for Li^+^ appears at around 2.1 Å for both carbonyl oxygen atoms in PC and oxygen atoms in TFSI^−^. These results agree with previous reported studies of LiPF~6~ in PC ([@R37]) and LiTFSI-acetamide systems ([@R36]). It also indicates that Li^+^ has strong solvation structures with PC molecules. This association is more obvious between Li^+^ and TFSI^−^ ion pairs because the peak intensity in [Fig. 3B](#F3){ref-type="fig"} is stronger than that in [Fig. 3A](#F3){ref-type="fig"}. ![The solvation structures of Li^+^ in PC solutions.\ RDFs of (**A**) cation-solvent \[Li^+^-O(carbonyl in PC)\] and (**B**) cation-anion \[Li^+^-O(TFSI^−^)\] and the coordination numbers of (**C**) cation-solvent \[Li^+^-O(carbonyl in PC)\] and (**D**) cation-anion \[Li^+^-O(TFSI^−^)\] under conditions of no electric field, static electric field, and pulsed electric fields with a strength of 3 × 10^5^ V m^−1^. The *T*~on~/*T*~off~ ratios of pulsed electric fields are 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, and 1:10. For convenience, the static electric field is shown as the one with a *T*~on~/*T*~off~ ratio of 1:0. Pulse current charging can reduce the coordination number of TFSI^−^ around Li^+^ ions.](1701246-F3){#F3} As shown in [Fig. 3](#F3){ref-type="fig"} (C and D), the coordination number of oxygen atoms around Li^+^ is approximately six, among which four are from PC and one or two are from TFSI^−^. Similar results have been previously reported with Raman intensity experiments ([@R38]) and classical MD simulations ([@R39]--[@R41]) that, when a static electric field is applied, the coordination number of TFSI^−^ around Li^+^ gets smaller. In addition, [Fig. 3](#F3){ref-type="fig"} (C and D) demonstrates that both coordination numbers of cation-solvent \[Li^+^-O(carbonyl in PC)\] and cation-anion \[Li^+^-O(TFSI^−^)\] can be affected by applying pulsed electric fields. When a pulsed electric field with a *T*~on~/*T*~off~ ratio of 1:3 is used, Li^+^ has a relatively larger coordination number of PC and a smaller coordination number of TFSI^−^ compared with the control case where no electric field is applied, leading to a larger diffusivity of Li^+^ ([Fig. 2](#F2){ref-type="fig"}). It is demonstrated that the pulse current charging affects the structural properties of the electrolyte, which decreases the coordination number of TFSI^−^ around Li^+^ ions. That is, the number of TFSI^−^ anions around Li^+^ is reduced, and the repulsion effect between Li^+^ and Li^+^ is promoted. As a result, the Li^+^ can transport easily, and the diffusion coefficient of Li^+^ is increased. This enhanced diffusion would reduce the cluster behavior of Li^+^ in the bulk electrolyte, promoting uniform distribution of ions and reducing ion concentration gradient. Cycling behavior ---------------- Galvanostatic cycling measurements were used to mimic the real condition of battery cycling. Cells were cycled at 3 mA cm^−2^ for 20 min in each half-cycle. Same symmetrical cells were cycled with pulse current at various *T*~on~ durations and *T*~on~/*T*~off~ ratios (*T*~on~/*T*~off~ = 1:0.5, 1:1, 1:2, 1:3, 1:5, and 1:10; *T*~on~ = 1, 5, and 10 s). All pulse waveforms are galvanostatic square-wave pulses. The current density and total capacity passed through in each half-cycle were kept the same for cells with and without pulse current. To obtain a quantitative measure of the relative performance of the two cells, we arbitrarily defined the cell lifetime as the time before peak voltage amplitude increases to 1 V. The voltage becomes unstable, and postmortem scanning electron microscopy (SEM) analysis of the lithium electrode from cells interrupted at 1 V also indicates that the voltage change coincides with the appearance of lithium dendrites on the electrode surface. [Figure 4A](#F4){ref-type="fig"} reports the voltage-time profiles for cells without pulse current at 3 mA cm^−2^, and it shows a gradual increase in hysteresis (overpotential between Li plating and stripping) as the number of cycles increases. It is interesting to notice that a sudden increase in voltage appears at the 20th cycle, and the voltage profile becomes unstable in the following cycles. The sudden increase is known as an indication for cell instability that the internal impedance changes as the lithium dendrite shoots out from the substrate. [Figure 4B](#F4){ref-type="fig"} shows the voltage profile for cells with pulse current (*T*~on~/*T*~off~ = 1:5; *T*~on~ = 1 s) at the same current density. The cell can be cycled for at least 54 cycles without sudden increase or drop in voltage. The cutoff lifetime for cells with pulse current is 48 cycles (192 hours), which is 2.4 times higher than that for cells with constant current cycling. Their detailed comparison in voltage at the 11th cycle can be found in [Fig. 4](#F4){ref-type="fig"} (C and D), which shows enlarged views of the rectangular areas corresponding to [Fig. 4](#F4){ref-type="fig"} (A and B, respectively). Cells under pulse current cycling exhibit a stable voltage response with small increase in hysteresis (about 0.8 V at the 11th cycle) compared to cells cycled without pulse current (about 1 V at the 11th cycle). [Figure 4](#F4){ref-type="fig"} (E and F) shows the details about the voltage response, current density, and *T*~on~/*T*~off~ settings in [Fig. 4](#F4){ref-type="fig"} (C and D). Generally, when cycled with pulse current, the cell shows lower voltage hysteresis and great improvement in cycling stability and cell lifetime. {#F4} To demonstrate the growth of lithium dendrites after galvanostatic cycling, we used SEM to analyze the surface and cross-section morphologies of Li-metal electrodes before cycling and after being cycled with constant current or pulse current. Symmetric Li cells were cycled at a fixed current density of 3 mA cm^−2^ with a string/plating capacity of 1 mAh cm^−2^ for each half-cycle. Then, the cells were disassembled in an Ar-protected glove box, and the Li-metal electrodes were washed by PC and dried rigorously before SEM test. As shown in fig. S2A, the surface of a pristine Li-metal electrode is uniform in general except for some small manufactural defects. After being cycled eight times, lithium foil harvested from cells without using pulse current shows rough, fibrous, and irregular jaggy-shaped structures, as displayed in fig. S2B. These nonuniform and sharp structures of lithium could eventually become Li dendrites, penetrate the separator, and cause cell short circuit. Figure S2C displays the surface morphology of the Li electrode obtained from pulse current cycling and shows uniform microstructure compared with that of the Li-metal anode with constant current cycling. We also tracked the thickness of the Li electrode from cross-section SEM analysis to study the electrode volume change upon cycling. The thickness of the pristine Li electrode is around 420 μm. After eight cycles, the electrode thickness increases by 30 and 84 μm for cells with and without pulse current cycling, respectively. The smaller increment in electrode thickness indicates that the electrode structure is more compact using pulse current cycling and the electrode volume expansion is significantly reduced. The results are consistent with the findings in galvanostatic cycling measurements and suggest that fewer lithium dendritic structures form in cells with pulse current cycling. Cycling with pulsed current can increase the lithium coulombic efficiency and ultimately improve the cycling stability and cell lifetime. To find the optimal conditions for dendrite suppression under pulse current cycling, we provide additional details about the effectiveness of pulse current cycling and statistically calculated the cell lifetime using galvanostatic measurements in Li/Li cells. The cell lifetime (*T*~c~) is defined as the time when the amplitude of voltage exceeds 1 V. A high current density of 3 mA cm^−2^ was implemented for practical rate capability. [Figure 5](#F5){ref-type="fig"} summarizes *T*~c~ as a function of *T*~on~ and frequency. The voltage of cells with constant current cycling exceeds 1 V at about the 20th cycle, whereas all cells with pulse current cycling show improvement in cell lifetime. It can also be seen in general that the cell lifetime increases with the increment of *T*~off~ at fixed *T*~on~ and increases when reducing the period of *T*~on~ at a fixed ratio of *T*~on~/*T*~off~. The optimal enhancement in cell lifetime appears when *T*~on~/*T*~off~ = 1:5, indicating that more rest time between the two periods of charging is not necessary for longer cell lifetime. The results also show that the cell lifetime depends on the pulse frequency and high-frequency pulse cycling is more efficient in promoting uniform Li electrodeposition and extending the cell lifetime. {#F5} DISCUSSION ========== Pulse current charging was demonstrated to be a promising method for stabilizing lithium-metal batteries compared with common constant current charging. We have used MD simulations to study the diffusion of Li^+^ at the molecular level. The results suggest the existence of an optimal ratio, *T*~on~/*T*~off~, for achieving the highest diffusion coefficient of Li^+^. We also analyzed the solvation of Li ions and revealed that a lower coordination number of TFSI^−^ near Li^+^ promotes the diffusivity of Li^+^. In pulsed electric fields, Li^+^ has an increased coordination number of PC and a reduced coordination number of TFSI^−^ compared to the control case where no electric field is applied. The simulation results indicate that the transport of Li^+^ in the bulk electrolytes and the underlying ion concentration gradient play a critical role in reducing dendrite formation and promoting stable cell cycling. A weaker association between Li cation and its counter ion is preferred. Electrochemical cycling performance was also evaluated for further evidence of the effectiveness of pulse current charging on cell stability. Consistent with the findings in MD simulations, all cells cycled with pulse current show enhanced cycling stability and lower hysteresis. Results from SEM analysis provide support of the cycling measurements and conclude that Li dendrites could be effectively hindered with pulse current and the volume expansion of the Li electrode can also be reduced. We found from both simulation and experimental results that the ratio of *T*~on~/*T*~off~ is critical for stabilizing the cycling behavior, and pulse charging with *T*~on~/*T*~off~ = 1:5 or *T*~on~/*T*~off~ = 1:3 is an optimal condition examined in this study. Considering the difference in time scales and other factors, such as ion migration and electrodeposition of Li ions on electrode surface, it is reasonable that some results are unable to have a one-to-one correspondence between simulations and experiments. To further enhance the benefits brought by the pulse current charging, future studies could include finding an anion with a lower coordination number around Li^+^ to promote the Li diffusivity and eventually improve cycling stability and extend the lifetime of high-energy Li-metal batteries. MATERIALS AND METHODS ===================== Simulation details ------------------ The simulation system was constructed with 51 Li^+^, 51 TFSI^−^, and 600 PC molecules, which give a total of 8616 atoms and represent the LiTFSI solution with a concentration of 1.0 M lithium salt. The system is in a cubic box with a dimension of 43.88 Å in length (*x*), width (*y*), and height (*z*). The whole system, including Li^+^, TFSI^−^, and PC, were initially energy-minimized for 10,000 steps using the conjugate gradient algorithm to remove abnormally close contacts between molecules. Following the minimization, the system was heated from 50 to 300 K using a short MD run of 1 ns or 1.0 fs time steps and 50 K increments for every 100 ps until the system reached 300 K. After heating, an additional 1 ns or 1.0 fs MD steps were run to allow further isothermal equilibration of Li^+^ and TFSI^−^ in PC. The velocity Verlet method was used for the integration of the Newtonian equations in NPT (a simulation system that consists of a constant number of atoms at a constant pressure, in which the temperature of the system is controlled by using a thermostat) ensemble for equilibration. A pressure of 1 atm was used in all simulation cases. A cutoff of 1.2 and 1.0 nm was used for the Lennard-Jones and electrostatic terms, respectively. A neighbor searching was made up to this same distance from the central ion and was updated every five simulation steps. The electrostatic interactions beyond 1.0 nm were accounted for by the computationally efficient K-space method based on particle-particle particle-mesh (PPPM). It is important to use the PPPM method in the case of ionic systems, because electrostatic energy beyond the cutoff usually contributes a lot to the total electrostatic energy ([@R42]). The final configuration of the system has a density of \~1.329 g cm^−3^, which agrees with the experimentally determined density of PC solution of LiTFSI (1.3205 g cm^−3^, 1.11 M) ([@R43]). For production runs, a series of 12-ns simulations at 300 K were carried out in the NVT (a simulation system that consists of a constant number of atoms at a constant volume, in which the temperature of the system is controlled by using a thermostat) ensemble using a Nosé-Hoover thermostat with a coupling constant of 0.1 ps. A pulsed or static electric field was applied to the system in the *z* direction. The strength of the static electric field was set at 3 × 10^5^ V m^−1^. The pulsed electric field was implemented as a square wave, with the strength of the electric field during its "on" and "off" periods equal to 3 × 10^5^ and 0 V m^−1^, respectively. A pulsed electric field with the strength of the electric field during its on and off periods equal to 3 × 10^6^ and 0 V m^−1^, respectively, was also examined. The off duration of the pulsed electric field was set to be 0.01 ns. The on duration was set in a way so that the on/off value was 0.5, 1, 2, 3, 4, 5, and 10, respectively. Moreover, a simulation case with no applied electric fields was studied as a control. The simulation results were obtained by analyzing the last 4 ns trajectories of production runs. The OPLS-AA force field ([@R44], [@R45]) was used. This force field has been widely used for biomolecules ([@R46]), hydrocarbon molecules ([@R47], [@R48]), and ionic liquids ([@R49], [@R50]). The basic functional form of potential energy in the OPLS-AA force field is defined as follows$$\mathit{E} = \sum\limits_{\mathit{i}}\mathit{K}_{\mathit{b},\mathit{i}}{\lbrack\mathit{r}_{\mathit{i}} - \mathit{r}_{0,\mathit{i}}\rbrack}^{2} + \sum\limits_{\mathit{i}}\mathit{K}_{\mathit{b},\mathit{i}}{\lbrack\theta_{\mathit{i}} - \theta_{0,\mathit{i}}\rbrack}^{2} + \sum\limits_{\mathit{i}}\left\lbrack \frac{1}{2}\mathit{V}_{1,\mathit{i}}(1 + \text{cos}(\phi_{\mathit{i}})) + \frac{1}{2}\mathit{V}_{2,\mathit{i}}(1 - ~\text{cos}(2\phi_{\mathit{i}})) + \frac{1}{2}\mathit{V}_{3,\mathit{i}}(1 + ~\text{cos}(3\phi_{\mathit{i}})) + \frac{1}{2}\mathit{V}_{4,\mathit{i}}(1 - ~\text{cos}(4\phi_{\mathit{i}})) \right\rbrack + \sum\limits_{\mathit{i}}\sum\limits_{\mathit{j} > \mathit{i}}\left\{ \frac{\mathit{q}_{\mathit{i}}\mathit{q}_{\mathit{j}}\mathit{e}^{2}}{\mathit{r}_{\mathit{i}\mathit{j}}} + 4\varepsilon_{\mathit{i}\mathit{j}}\left\lbrack \left( \frac{\sigma_{\mathit{i}\mathit{j}}}{\mathit{r}_{\mathit{i}\mathit{j}}} \right)^{12} - \left( \frac{\sigma_{\mathit{i}\mathit{j}}}{\mathit{r}_{\mathit{i}\mathit{j}}} \right)^{6} \right\rbrack \right\}$$ The total energy *E* of the system is evaluated as the sum of individual energies for the harmonic bond stretching and angle bending terms, a Fourier series for torsional energetics, and Coulomb and 12-6 Lennard-Jones terms for the nonbonded interactions. *K*~*b,i*~ is the force constant, and *r*~0~ and θ~0~ represent the reference values. The Fourier coefficients *V* and the partial atomic charges *q* were fixed on each atom center. *i* and *j* represent all pairs of atoms (*i* \< *j*). *r*~*ij*~ is the distance between atoms *i* and *j.* σ~*ij*~ and ε~*ij*~ represent the Lennard-Jones radii and potential well depths, respectively, which are obtained from parameters for each type of atom by using geometric combination rules ε~*ij*~ = (ε~*ii*~ε~*jj*~)^1/2^ and σ~*ij*~ = (σ~*ii*~σ~*jj*~)^1/2^ ([@R48]). The Lennard-Jones parameters and partial charges for Li^+^ and PC were taken from the OPLS-AA parameterization ([@R37], [@R49], [@R51]). The bonded stretching, angle bending, dihedral torsion, and nonbonded parameters of TFSI^−^ anions were obtained from Canongia Lopes and Pádua ([@R49]). The partial charge of atoms in TFSI^−^ anions was calculated with the following protocol. First, geometry optimizations were carried out at the Hartree-Fock (HF) level by using a moderately sized polarized basis set at the 6-31G\* level. The partial charges were computed by fitting the molecular electrostatic potential at the atomic centers. The effects of electron correlation and basis set extension of HF structures were considered by performing single-point calculations with second-order Møller-Plesset perturbation method MP2/cc-Pvdz and the correlation consistent polarized valence cc-pVTZ(-f) basis set. This MP2/cc-pVTZ(-f) //HF/6-31G(d) method is a common practice for OPLS-AA parameterization ([@R50], [@R52], [@R53]). The calculated restrained electrostatic potential charges for TFSI^−^ agree with other studies ([@R49]). All MD simulations were performed with the LAMMPS (Large-scale Atomic/Molecular Massively Parallel Simulator) package on a 20-node Linux cluster ([@R54]). All quantum chemistry calculations were carried out with the Gaussian 09 package ([@R55]). Electrochemical measurements ---------------------------- CR2032 coin cells were assembled in an argon-filled glove box. Bare Li metal was used as the reference and counter electrodes, and 1 M LiTFSI in PC was used as the electrolyte. Symmetrical lithium cells were galvanostatically cycled at 3 mA cm^−2^ for 20 min in each half-cycle. The same symmetrical cells were cycled with pulse current charging and discharging at various *T*~on~ and *T*~on~/*T*~off~ ratios (*T*~on~/*T*~off~ = 1:0.5, 1:1, 1:2, 1:3, 1:5, and 1:10; *T*~on~ = 1, 5, and 10 s). *T*~on~ means the period of constant current charging or discharging, and *T*~off~ means the rest period followed by the constant current charging or discharging. The current density and total capacity in each half-cycle were kept the same for cells with and without using pulse current. Neware electrochemical testing system (CT4008) and Solartron Analytical Electrochemical Workstation were used for the electrochemical measurements. SEM analysis ------------ After galvanostatic cycling, the cells were carefully disassembled in a dry glove box filled with argon to obtain Li-metal electrodes. Then, Li-metal electrodes were gently washed with PC three times to remove residual lithium salts and fully dried under vacuum. To access the cross-section images of the Li-metal electrodes, the obtained lithium foils were cut with a sharp surgical knife blade and placed on a vertical sample holder. SEM analysis was conducted on a field-emission SEM (Hitachi SU8000 FE-SEM) at 5 kV. Supplementary Material ====================== ###### http://advances.sciencemag.org/cgi/content/full/3/7/e1701246/DC1 We thank F. Chen (Department of Chemistry, Zhejiang University) for SEM analysis. **Funding:** This work was supported by the Natural Science Foundation of China (grants 21676242, 21476191, 91434110, and 21676245) and Ministry of Science and Technology of the People's Republic of China (grant 2016YFA0202900). **Author contributions:** Y.L. and Y.H. designed the experiment, conceived the project, and wrote the manuscript. Q.L. performed the experimental work and analyzed the results with the help of Y.L. and S.T. S.T. conducted the simulation work and analyzed the results with the help of Y.H. and L.L. **Competing interests:** The authors declare that they have no competing interests. **Data and materials availability:** All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. Supplementary material for this article is available at <http://advances.sciencemag.org/cgi/content/full/3/7/e1701246/DC1> fig. S1. The impact of pulsed electric fields on the diffusivity of Li^+^. fig. S2. Surface and cross-section SEM images of Li-metal electrodes cycled with or without pulse current. fig. S3. Cell lifetime at various *T*~on~ with fixed frequencies. fig. S4. Voltage-time profiles of cells cycled with pulse current at fixed *T*~on~ = 1 s. fig. S5. Voltage-time profiles of cells cycled with pulse current at fixed *T*~on~/*T*~off~ = 1:5. [^1]: These authors contributed equally to this work.

T. Nguyen, M. F. Montemor, Adv. Sci. 2019, 6, 1801797 10.1002/advs.201801797 1. Introduction {#advs1009-sec-0010} =============== Energy consumption has been continuously increasing and presently energy supply poses important challenges. The International Energy Agency report (2017) revealed that the worldwide energy consumption in 2015 was 9384 Mtoe (million tonnes of oil equivalent; 1 toe = 11.63 MWh), which was about 108% and 203% higher than the values in 2010 and 1973, respectively.[1](#advs1009-bib-0001){ref-type="ref"} Energy demand is expected to escalate by more than two times in 2050 and more than three times by the end of the 21st century.[2](#advs1009-bib-0002){ref-type="ref"} Fossil fuels are the main energy production source and have supplied 81.6% of global energy in 2015, but the associated greenhouse gas emission, in particular CO~2~, has raised serious environmental concerns.[3](#advs1009-bib-0003){ref-type="ref"} CO~2~ emissions increased from 15 548 Mt in 1973 to 30 326 Mt in 2010 and to 32 294 Mt in 2015, causing global warming, a major environmental issue.[1](#advs1009-bib-0001){ref-type="ref"} As a consequence, sustainable and renewable energy sources are acknowledged as central theme in the 2030 Agenda for Sustainable Development of United Nations, which underpins the economic and social roadmaps and the strategic plan of Department of Energy.[2](#advs1009-bib-0002){ref-type="ref"}, [4](#advs1009-bib-0004){ref-type="ref"} Presently, the world is assisting very fast growth of renewable energy sources; for example, hydropower increased from 1296 TWh in 1973 to 3978 TWh in 2015, wind electricity production increased from 104 TWh in 2005 to 838 TWh in 2015, and photovoltaic energy increased from 4 TWh in 2005 to 247 TWh in 2015. Decentralized renewable energy production and its intermittency cause mismatch between energy supply and energy demand, a fact that results in energy waste and poor efficiency. Thus, energy storage is essential in the energy chain, and must be integrated with energy production to balance and stabilize energy mismatch by storing and supplying energy on demand. Moreover, voltage fluctuations in renewable energy supply require energy storage systems with fast response time, particularly for grid frequency and peak shaving. Besides grid applications, high energy and high power density energy storage devices are also essential to supply off‐grid applications such as electric vehicles, microelectronics, medical devices, and portable electronic devices. A wide array of energy storage systems tailored to the application needs are being developed, such as magnetic, electrochemical, chemical, cryogenic, mechanical, and thermal systems.[5](#advs1009-bib-0005){ref-type="ref"} Among those, supercapacitors, superconducting magnetic energy storage (SMES), and flywheels display quick response time, ranging from few milliseconds to few seconds,[6](#advs1009-bib-0006){ref-type="ref"} being the prime choice for high‐power requirements. However, SMES impose high operational costs and flywheels are bulky in size and, as a consequence, supercapacitors are becoming the most promising energy storage solutions for applications requiring high power. For supercapacitors based on carbon electrodes, fast double‐layer charging provides high specific power (above 10 kW kg^−1^) and long operational life.[7](#advs1009-bib-0007){ref-type="ref"} However, the charge storage mechanism, essentially based on adsorption, is limited by the electrode material surface area, resulting in rather low specific energy, typically below 10 Wh kg^−1^ (3--30 times lower compared to batteries), and high self‐discharge rate.[8](#advs1009-bib-0008){ref-type="ref"} Improvement of the energy density of supercapacitors, while maintaining their high power response, is nowadays a major challenge. Charge storage in pseudocapacitive metal oxides such as cobalt oxides, RuO*~x~*, MnO~2~, and V~2~O~5~ is governed by Faradaic charge storage processes that involve sub‐valence state changes and enabled the development of high rate and energy density supercapacitors. For example, RuO~2~ and MnO~2~ display theoretical specific capacitance values above 1000 F g^−1^ in working potential windows of 1 V. More recently, hybrid supercapacitors employing redox‐active materials, which store charge via reversible redox reactions and display hybrid charge storage mechanisms, different from double‐layer charging and pseudocapacitance, are able to deliver high capacity and are considered efficient solutions to enhance energy density. Hybrid supercapacitor designs include cell assemblies in asymmetric configurations and make use of redox‐active electrodes for hybrid charge storage. Presently, redox‐active transition metal compounds, in particular oxides and hydroxides of different metals such as Ni, Co, Fe, Ti, V, Mo, and Nb, are emerging as promising materials for electrodes used in hybrid supercapacitors.[9](#advs1009-bib-0009){ref-type="ref"} In these materials, metal sites act as redox‐active centers and sustain processes involving different valence states, a property that enables increased charge storage capacity. Moreover, since the valence changes of those compounds occur at different potentials, there is a wide choice of metal oxide/hydroxide materials that can be combined to assemble hybrid supercapacitors displaying optimal performance. For instance, in aqueous electrolytes, valence changes of Ni^2+^/Ni^3+^ and Co^2+^/Co^3+^ occur in a potential range of ≈0--0.6 V, while those of Mn^3+^/Mn^4+^ occur in the range of ≈0--1 V and those of Fe^2+^/Fe^3+^ occur in the range of −1 to 0 V.[10](#advs1009-bib-0010){ref-type="ref"} Metal oxides and hydroxides present different crystalline structures, either with open tunnels or with open interlayers, which enable ion insertion and extraction during the redox reactions and provide charge storage both in bulk sites and in surface sites. Some hydroxides contain intercalated anions for charge balancing in their layered structure; these anions are exchangeable, providing extra routes for tailoring the charge storage properties of the electrode material. Aqueous electrolytes including acid, neutral, or alkaline electrolytes and nonaqueous electrolytes, such as organic or ionic liquids, are currently being utilized for supercapacitors.[11](#advs1009-bib-0011){ref-type="ref"} The electrolyte nature largely determines the working voltage of supercapacitors that in nonaqueous organic electrolytes can reach up to 3 V and in ionic liquids electrolytes can reach even higher values.[12](#advs1009-bib-0012){ref-type="ref"} The charge capacity increases with voltage; thus, wider working voltage obtained in nonaqueous electrolytes can support the development of supercapacitors with higher capacity. However, nonaqueous electrolytes suffer from low ionic conductivity (10--100 mS cm^−1^ for organics and \<10 mS cm^−1^ for ionic liquids), leading to decreased power response. Moreover, the large molecular sizes of organic electrolytes decrease the capacitance and generally nonaqueous electrolytes bring higher costs (including high‐cost purification of water content to 3--5 ppm in organic electrolytes) and require controlled atmosphere for operation.[13](#advs1009-bib-0013){ref-type="ref"} Organic electrolytes are also toxic and flammable, raising serious safety issues during operation and disposal. Despite these serious drawbacks, current commercial supercapacitors use nonaqueous electrolytes, taking advantage of wider working voltages. Presently, supercapacitors based on aqueous electrolytes are part of many electrochemical energy storage agendas because of several advantages compared to nonaqueous electrolytes. Aqueous electrolytes present high ionic conductivity, nearly 1 S cm^−1^, which is two orders of magnitude above organic and ionic liquid electrolytes, and the hydrated ion sizes are smaller than organic, achieving higher capacity.[14](#advs1009-bib-0014){ref-type="ref"} Moreover, aqueous electrolytes are low cost, safe, and easy to handle. Although the working voltage in aqueous electrolytes (aqueous supercapacitors) is limited within the water splitting window, theoretically 1.23 V, by functionalization and engineering of the electrode materials, it is possible to widen the redox potential ranges and to expand the overpotential for water splitting. Thus, aqueous supercapacitors with working voltages up to 2.6 V have been reported. The storage capability of supercapacitors is governed by the electrode material and the charge storage mechanisms that determine the charge transfer or charge accumulation processes at electrode--electrolyte interface. Charge storage performance, response rate, and operation lifetime can be tailored by different engineering and functionalization routes that lead to optimal material physicochemical properties such as active charge storage sites, conductivity, and redox activity. The ultimate research frontier is to develop high energy density supercapacitors to fill the energy gap between supercapacitors and batteries, while keeping high power and long cycling lifetime. Functional electrodes assembled in devices tailored to the real need specificities are presently at the research forefront. The three research streams leading to the latest advances on supercapacitors are overviewed in **Figure** [**1**](#advs1009-fig-0001){ref-type="fig"}. {#advs1009-fig-0001} In this review, authors aim at addressing the most recent research advances in metal oxide and hydroxide--based aqueous supercapacitors, spanning from fundamental charge storage studies and electrode materials engineering to need‐tailored device assemblies. Note that this review will not cover supercapacitor basics that readers may find in many dedicated reviews, perspectives, or books; instead, it highlights the latest materials functionalization and engineering trends, the respective charge storage mechanisms, and need‐tailored assemblies. 2. Charge Storage Mechanisms {#advs1009-sec-0020} ============================ The charge storage mechanisms generally accepted to explain the electrochemical behavior of metal oxide and hydroxide electrodes in aqueous electrolytes are based on valence changes of the metallic species through Faradaic proton/alkali metal intercalation/deintercalation, surface adsorption/desorption,[15](#advs1009-bib-0015){ref-type="ref"} or surface redox reactions with anions.[16](#advs1009-bib-0016){ref-type="ref"} Some typical examples are highlighted below$$\left. {RuO}_{2} + xH^{+} + xe^{-}\leftrightarrow{RuO}_{2 - x}\left( {OH} \right)_{x} \right.$$ $$\left. {MnO}_{2} + C^{+} + e^{-}\leftrightarrow{MnOOC} \right.$$ $$\left. {NiO} + {OH}^{-}\leftrightarrow{NiOOH} + e^{-} \right.$$ $$\left. {Co}_{3}O_{4} + H_{2}O + {OH}^{-}\leftrightarrow 3{CoOOH} + e^{-} \right.$$ $$\left. {Co}\left( {OH} \right)_{2} + {OH}^{-}\leftrightarrow{CoOOH} + H_{2}O + e^{-} \right.$$ $$\left. {CoOOH} + {OH}^{-}\leftrightarrow{CoO}_{2} + H_{2}O + e^{-} \right.$$ $$\left. {Ni}\left( {OH} \right)_{2} + {OH}^{-}\leftrightarrow{NiOOH} + H_{2}O + e^{-} \right.$$where C^+^ is an alkaline ion or a proton in the electrolyte. Although these mechanisms are widely accepted, they are rather general and lack detail on how the physicochemical properties of the electrode material change during the charge storage and charge release processes. Relevant materials\' properties include morphology, structure, and valence states (Figure [1](#advs1009-fig-0001){ref-type="fig"}, storage mechanism axis); their changes during the electrochemical processes that store and release charge govern electrode performance such as charge capacity, response rate, and operational lifetime. Thus, understanding such changes is essential to design electrodes tailored to meet the specificities of the final application. Preliminary understanding of valence and local structure changes of MnO~2~ electrodes has been obtained using in situ X‐ray absorption near‐edge structure (XANES) and extended X‐ray absorption fine structure (EXAFS).[17](#advs1009-bib-0017){ref-type="ref"} The shift of Mn K‐edge peak in XANES revealed that MnO~2~ valence gradually increased from +3.23 to +3.95 when charging, and gradually decreased to 3.27 after discharging in aqueous 2 [m]{.smallcaps} KCl electrolyte.\[\[qv: 17a\]\] The irreversibility of Mn valence states during charge storage is responsible for capacity decay after cycling; however, reversibility of the valence states and the local structure of MnO~2~ depend upon the working potential window.\[\[qv: 17b\]\] The formation of low‐valence states after discharge resulted in Mn dissolution, leading to capacity decay. Recently, several in situ*/*operando observations addressing morphological, valence, and structural changes of metal oxides/hydroxides have been reported and have provided new insights into the charge storage mechanisms of metal oxide/hydroxide electrodes in aqueous electrolytes. Thus, in the following subsection the most recent achievements on important metal oxides and metal hydroxides are overviewed. 2.1. Metal Oxides {#advs1009-sec-0030} ----------------- ### 2.1.1. Mn Oxides {#advs1009-sec-0040} Morphological and mechanical changes of γ‐MnO~2~ during the charge/discharge process in 1 [m]{.smallcaps} KCl were studied by in situ atomic force microscopy (AFM) and nanoindentation built in within AFM.[18](#advs1009-bib-0018){ref-type="ref"} This study evidenced the MnO~2~ particle contraction/expansion during the charge/discharge process (**Figure** [**2**](#advs1009-fig-0002){ref-type="fig"}a,b). The expansion was inhomogeneous (Figure [2](#advs1009-fig-0002){ref-type="fig"}b), and localized contractions of grains were also observed (Figure [2](#advs1009-fig-0002){ref-type="fig"}c). Pores among the particles were shrunken during particle expansion and localized grain contractions and interparticle pore shrinkage compensated such expansion. This behavior was explained by proton intercalation, reaction [(2)](#advs1009-disp-0002){ref-type="disp-formula"}, which induced lattice expansion and changes in Mn ionic radius upon valence changes that increased from 0.530 Å for Mn^4+^ to 0.645 Å for Mn^3+^, in the discharge process. In situ nanoindentation revealed softening of MnO~2~ by proton intercalation, due to reduction of the elastic modulus and hardness of MnO~2~ after discharging, in agreement with particle expansion. This contraction/expansion during the charge/discharge process rendered for morphological changes, which are often observed in metal oxide--based electrodes after cycling. Thus, it is worth stating that MnO*~x~*‐based electrodes with porous structures are the key to overcome contraction/expansion‐related drawbacks and to design active materials displaying increased cycling stability. {ref-type="ref"} Copyright 2013, John Wiley and Sons. d) Reproduced with permission.[22](#advs1009-bib-0022){ref-type="ref"} Copyright 2014, Elsevier Ltd. e) Reproduced with permission.[23](#advs1009-bib-0023){ref-type="ref"} Copyright 2015, American Chemical Society.](ADVS-6-1801797-g002){#advs1009-fig-0002} Mn valence changes in different MnO~2~ polymorphs have been recently reported and, for example, Mn valence state changes in δ‐MnO~2~ nanosheets were investigated by in situ X‐ray absorption spectroscopy (XAS) in the near‐edge region.[19](#advs1009-bib-0019){ref-type="ref"} The as‐prepared δ‐MnO~2~ displayed Mn valence state of +3.16, which is lower than the theoretical valence state of +4 due to the presence of preintercalated cations, which attracted electron from oxygen sites in good agreement with results published elsewhere.\[\[qv: 17a\]\] After immersion in 0.5 [m]{.smallcaps} Na~2~SO~4~, the Mn valence state decreased to +3.09, effect explained by intercalation of Na^+^. The Mn valence state progressively increased to +3.23 when charging up to 0.6 V versus saturated calomel electrode (SCE) and continuously decreased to +3.05 upon discharging to 0 V versus SCE. The changes in the valence states were due to deintercalation and intercalation of cations on charging and discharging, respectively. Similarly, Mn valence state of K--birnessite MnO~2~ nanosheets increased from +3.01 to +3.12 upon charging from 0 to 0.8 V versus SCE and decreased to +3.01 upon discharging to 0 V versus SCE.[20](#advs1009-bib-0020){ref-type="ref"} Overcharging in the oxygen evolution region resulted in irreversible valence changes.[21](#advs1009-bib-0021){ref-type="ref"} These results were confirmed by different reports addressing Mn valence changes in MnO~2~ electrodes during the charge storage and release processes. It has been suggested that the valence changes are not influenced by MnO~2~ polymorphs, but it has been evidenced that valences of +3.23 and +3.12 at full charge state are still far from full oxidation (+4). Summarizing, the reported work demonstrates that there is still insufficient exploration of the full Mn valences for charge storage purposes, a research line that is likely to be pursued in the near future, thanks to the attractiveness of Mn oxides as charge storage materials. Structural changes of α‐Mn~0.98~O~2~ during the charge/discharge process in Na~2~SO~4~ were monitored by operando Raman spectroscopy.[22](#advs1009-bib-0022){ref-type="ref"} The results revealed structural transformations due to insertion of hydrated Na^+^ in the tunnel of MnO~2~, in agreement with the valence changes proposed elsewhere.[19](#advs1009-bib-0019){ref-type="ref"} The transformation from MnO~2~ to Mn~3~O~4~ phase was also noticed, resulting in the coexistence of Na--MnO~2~ and Mn~3~O~4~ after immersion in the electrolyte. The hydrated proton intercalation into the tunnel and hydrated Na^+^ deintercalation occurred when the electrode was slowly charged to 0.06 V, resulting in the formation of MnOOH. The proton and Na^+^ were fully deintercalated when charging to 0.6 V (Figure [2](#advs1009-fig-0002){ref-type="fig"}d, top). At higher charging voltage, from 0.6 to 1 V, Mn~3~O~4~ was transformed into MnO~2~ phase, phenomenon associated with deintercalation of Mn^2+^ at octahedral sites in Mn~3~O~4~ (Figure [2](#advs1009-fig-0002){ref-type="fig"}d, bottom). During the discharge process, from 1 to 0 V, reversible reactions occurred, associated with Mn^2+^ intercalation in the voltage range from 1 to 0.6 V and proton and Na^+^ intercalation/deintercalation in 1 × 1 tunnel under the voltage range from 0.6 to 0 V. The effect of different cation sizes (Li^+^, Na^+^, and K^+^), in aqueous electrolytes, on the structural changes and charge storage performance of MnO~2~ electrodes was also studied by operando electrochemical Raman spectroscopy.[23](#advs1009-bib-0023){ref-type="ref"} In the high charge state of MnO~2~, water molecules intercalated in the tunnels/interlayer, resulting in structural similarity in aqueous electrolytes with different cations. The charge storage mechanism was similar when different cations were used, and during the discharge process, cations were intercalated into the tunnels, replacing water intercalation. The smaller ionic radius of cations, K^+^ (1.37 Å), Na^+^ (0.99 Å), and Li^+^ (0.59 Å), compared to water (2.8 Å) resulted in decreased interlayer spacing during discharge. The different sizes of cations intercalated into MnO~2~ tunnels are responsible for structural changes during the charge/discharge process and increased cation size augments the interlayer distance (Figure [2](#advs1009-fig-0002){ref-type="fig"}e). Structural polarizability (vibration mode polarizability) changes were noticed when the size of the intercalated cation increased. There was a polarization in the Mn---O vibration along the chains of the MnO~2~ framework compared to the symmetric stretching vibration of Mn---O bond in the MnO~6~ octahedra. Increased cation size also decreased the Jahn--Teller distortion when the electrode was fully discharged. Higher specific capacitances and rate performance were obtained with smaller cation size due to the favored cation intercalation process, which reduced charge transfer resistance, diffusion resistance, and diffusion time constant. However, electrolytes with smaller cation size (e.g., Li^+^) resulted in poorer charge--discharge cycling stability. This result was due to the high structural changes (Jahn--Teller distortion) that induced faster degradation of the electrode when it was cycled in electrolytes with smaller size cations. Summarizing, it is clear that electrolyte cations play a pivotal role in defining the valences changes and electrode capacity and stability. The optimal combination in terms of Mn oxides and electrolyte composition still represents an important gap in the state of the art and further studies are very necessary to better clarify this issue. Valence state and structural changes of Mn~3~O~4~ during charge--discharge processes in 1 [m]{.smallcaps} Na~2~SO~4~ were examined by in situ XANES and X‐ray diffraction (XRD).[24](#advs1009-bib-0024){ref-type="ref"} When charging the Mn~3~O~4~ electrode from 0 to 1.2 V versus Ag/AgCl, the Mn valence state was stable in the potential range of 0--0.75 V, but increased in the potential range of 0.75--1.2 V. This discontinuous valence change was different from the continuous change in MnO~2~ discussed above, and can be related to redox reactions occurring at high potentials. In situ XRD during the charge--discharge revealed the spinel lattice contraction--expansion, which correlated well with the oxidation--reduction processes; no phase changes were observed. The charge storage mechanisms of the Mn~3~O~4~ electrodes were also detailed by operando Raman spectroscopy[25](#advs1009-bib-0025){ref-type="ref"} and, contrarily to the previous report, an irreversible transformation from Mn~3~O~4~ to MnO~2~ phase and Na^+^ and Mn^2+^ (extracted from tetrahedral sites) intercalation into the defects or tunnel sites of the MnO~2~ in the first charge were noticed (Equation [(8)](#advs1009-disp-0008){ref-type="disp-formula"})$$\left. {Mn}_{3}O_{4}\rightarrow{Na}_{x}{MnO}_{2} \cdot nH_{2}O \right.$$ Discharge associated with the intercalation of Na^+^ cations in the tunnel induced an electrochemical transformation of the MnO~2~ phase. Studies on the transformed MnO~2~ revealed the softening of the MnO~2~ lattice due the reversible deintercalation of Na^+^ and intercalation of H~2~O due to the electrostatic repulsion--induced expansion of interlayers when charging. The partial reversible oxidation of Mn^2+^ in the interlayers to Mn^3+^ was also proposed, resulting in partial formation of Mn~3~O~4~ after complete deintercalation of Na^+^ $$\left. \left( {{MnO}_{2}\,^{2 -}{Mn}^{2 +}} \right)_{interlaminar} - e^{-}\leftrightarrow{Mn}^{3 +}{MnO}_{2}{}^{2 -} \right.$$ ### 2.1.2. V Oxides {#advs1009-sec-0050} V~2~O~5~ has commonly been thought to store charge via cation intercalation/deintercalation reactions and displayed good pseudocapacitive response, but its poor cycle life is still hindering its use. Thus, understanding its charge storage mechanism is essential to develop new V~2~O~5~ electrodes with improved performance. In situ XRD tracking of structural changes in K*~x~*V~2~O~5~ electrodes during charge--discharge in KCl electrolyte revealed that the (001) plane lattice expanded on charging and contracted on discharging.[26](#advs1009-bib-0026){ref-type="ref"} The charging was related to K^+^ deintercalation from (001) interlayers, which weakened the interaction of K^+^ with negatively charged \[VO~6~\] octahedra, leading to expansion of (001) interlayers. On discharging, the reverse process led to the lattice contraction. Structural and mass changes of α‐V~2~O~5~ nanowire electrodes during the charge storage and release processes in aqueous 1 [m]{.smallcaps} Na~2~SO~4~ were studied by operando Raman spectroscopy.[27](#advs1009-bib-0027){ref-type="ref"} Structural changes of α‐V~2~O~5~ during charge--discharge cycling revealed that no phase changes occurred and that V---O---V and V---O bond lengths were changed. These changes could be associated with the expansion/compression of (001) planes during the charge/discharge process as a result of Na^+^ (or their hydrated form) deintercalation/intercalation into V~2~O~5~ lattice, as revealed by previous work.[26](#advs1009-bib-0026){ref-type="ref"} In situ electrochemical quartz crystal microbalance showed the mass loss and the mass increase during the charge/discharge processes, which could be due to the reversible mass change from Na^+^ (or their hydrated form) deintercalation/intercalation reactions. The mass of V~2~O~5~ was reduced after one charge/discharge cycle when compared to the initial mass, suggesting an irreversible mass loss of the electrode due to dissolution into the electrolyte, which resulted in poor cycle life. The existing studies highlight the potential of this material and anticipate an important gap related to the need of developing solutions to prevent dissolution of the V~2~O~5~ active material during the charge/discharge cycling, to increase its electrochemical performance. ### 2.1.3. Co Oxides {#advs1009-sec-0060} Co oxides have attracted enormous attention as active materials for supercapacitors, providing charge storage, thanks to Faradaic and non‐Faradaic processes, and have been studied since many years. Different cobalt oxides with different morphologies are easy to synthesize and provide very high theoretical capacitances. In alkaline electrolytes, Co~3~O~4~ is converted into CoOOH, which in turn can be converted into CoO~2~. Despite its high theoretical capacitance, in practice values achieved are much lower and the main reason has been attributed to the decreased conductivity and formation of parasitic species over cycling. It is worth noting that sometimes reported capacitances are miscalculated because of the battery‐type signature of this material. Moreover, it has been noticed that the lifetime of cobalt oxide electrodes has been limited by expansion/contraction phenomena. The design of different high surface area morphologies has been the main route to overcome these drawbacks and the literature offers a variety of cobalt oxides produced by different routes such as Co~3~O~4~ nanowires, nanosheets, microspheres, nanospheres, hollow spheres, thin films, and many others. It has been shown that the electrochemical performance depends on the synthesis route, surface area pore size, and nature of the electrolyte. Despite all the advances and popularity of this material, the active potential window in alkaline electrolytes is typically limited to ≈0.5 V, an issue that limits its specific capacitance and its use. The strategy to overcome these drawbacks is nowadays focused on doping or combining cobalt oxides with other transition metal oxides or modification with heteroatoms, a research line that may enable the design of supercapacitor electrodes with increased stability in wider potential windows. Despite the research interest, scarcity of cobalt and its actual high price are limiting practical applications of Co‐based electrodes for charge storage in both the battery and supercapacitor field. 2.2. Metal Hydroxides {#advs1009-sec-0070} --------------------- ### 2.2.1. Ni Hydroxides {#advs1009-sec-0080} Structural changes of NiOOH electrodes in alkaline electrolytes have been studied by operando Raman spectroscopy and the results revealed that no relevant phase changes occurred during the charge/discharge processes.[28](#advs1009-bib-0028){ref-type="ref"} Structural evolution mainly arose in the potential ranges where redox reactions occurred (0.35--0.5 V during the charge process and 0.5--0.3 V during the discharge process) (**Figure** [**3**](#advs1009-fig-0003){ref-type="fig"}a). The charge process shortened the Ni---O bonds, which resulted from stiffening of Ni---O stretching vibration and Ni---O bending vibration modes; an increase of vibrational polarizability to Ni---O stretching modes also occurred after charging, as revealed by blueshifts of E~g~ and A~1g~ modes and an increase in A~1g~/E~g~ intensity ratio in Figure [3](#advs1009-fig-0003){ref-type="fig"}a. This process was reversible, leading to the lengthening of Ni---O bond, softening of Ni---O stretching and bending vibrations, and reduction of vibration polarizability. Changes in oxidation states were also noticed at high charge/discharge conditions. This was assigned to the reversible transformation of Ni^3+^ to Ni^4+^ as widely discussed in the literature. The structural evolution and oxidation state changes have been attributed to consecutive breaking and formation of O---H bonds during the reversible redox reactions (Figure [3](#advs1009-fig-0003){ref-type="fig"}a). Electrolyte cations (Li^+^, Na^+^, or K^+^) do not seem to influence the structural changes during charge storage. Layered NiOOH can host a number of cations in the interlayer. However, only minor structural disorders have been noticed after cation intercalation/deintercalation, and it can be considered that their contribution to the charge storage capacity was not relevant. The charge storage capacity varied with Ni---O bond length, softening/stiffening of Ni---O vibration, and their polarizability. {ref-type="ref"} Copyright 2016, John Wiley and Sons. b) Reproduced with permission.[30](#advs1009-bib-0030){ref-type="ref"} Copyright 2017, Springer Nature. c) Reproduced with permission.[31](#advs1009-bib-0031){ref-type="ref"} Copyright 2017, Elsevier Inc. d) Reproduced with permission.[32](#advs1009-bib-0032){ref-type="ref"} Copyright 2014, John Wiley and Sons.](ADVS-6-1801797-g003){#advs1009-fig-0003} Merrill et al. suggested that oxygen redox chemistry could also contribute to the charge storage besides the commonly accepted mechanisms based on valence changes of metallic ions.[29](#advs1009-bib-0029){ref-type="ref"} Using operando Raman spectroscopy and thermodynamic analysis, they proposed a charge storage mechanism for Ni(OH)~2~ and hydrated NiO, involving the oxidation/reduction of Ni^2+^/Ni^3+^ and the oxygen redox chemistry instead of Ni^4+^ involvement. This effect is still far from being well understood and further studies are still crucial to clarify how the oxygen redox chemistry contributes to the high charge storage capacity of Ni(OH)~2~ electrodes. A major drawback on the application of Ni hydroxides is the lack of conductivity over cycling, an issue that requires construction of nanoporous and nanosized architectures to enhance both electron transfer and ion diffusion. ### 2.2.2. Co Hydroxides {#advs1009-sec-0090} Co(OH)~2~ is a promising charge storage electrode material working in alkaline electrolyte, which displays high specific capacity, high response rate, and long cycle life. However, the intrinsic properties and mechanisms underlying their good charge storage capability are still very less understood. Recently, in situ electrochemical XAS and density functional theory (DFT) calculations have been carried out during the charge storage and release process on Co(OH)~2~ platelet electrode.[30](#advs1009-bib-0030){ref-type="ref"} The shifts of Co K‐edges in XANES spectra revealed that Co valence state increased on charging from −0.3 to 0.4 V and decreased upon discharging. The Co valence states reversibly changed as evidenced by the same absorption Co K‐edge positions after charge/discharge processes. The XAS oscillation pattern of the electrode after discharging showed no changes, suggesting negligible structural changes. Only some small adjustments of atoms occurred, involving the decrease of Co---O and Co---Co bond lengths and structural disorder on charging and vice versa on discharging, as revealed by the negative shifts and increased intensity of Co---O and Co---Co shells in EXAFS results during charging and reverse changes during discharging (Figure [3](#advs1009-fig-0003){ref-type="fig"}b). The bond length variations are 0.06 and 0.22 Å for Co---O and Co---Co, respectively. DFT calculations showed that the energy difference of the deprotonated Co(OH)~2~ and ground‐state CoOOH is as low as 0.59 eV, and the activation energies for the phase transformation of Co(OH)~2~ to CoOOH and CoOOH to Co(OH)~2~ are 0.76 and 0.34 eV, respectively. This low activation energy, plus the minor structural changes for the phase transformation, explained the high power response and long cycle life of Co(OH)~2~. H^+^ deintercalation/intercalation during the phase transformation processes (Figure [3](#advs1009-fig-0003){ref-type="fig"}b), which is similar to charge storage in batteries, resulted in increased specific capacity. This study evidenced the concept of developing electrode materials, which can display high power response similar to supercapacitor materials, while achieving energy densities similar to battery materials. For that, structural similarity of the redox reactants is essential for high specific power and high cycle life and the deintercalation/intercalation redox reaction is essential for high specific energy. Goodenough and coworkers explained further the results obtained for Co(OH)~2~ and extended them to Ni(OH)~2~ electrodes with structure and redox processes similar to Co(OH)~2~.[31](#advs1009-bib-0031){ref-type="ref"} While Co(OH)~2~ showed good response rate and cycling life, these properties are generally poor for its isostructural Ni(OH)~2~. The good response rate and cycle life of Co(OH)~2~ platelets were attributed to its CoO~2~ planes vertical to the large area of the platelet, providing short pathways for H^+^ diffusion for the bulk redox reactions (Figure [3](#advs1009-fig-0003){ref-type="fig"}c). Furthermore, the highly reversible transformation of Co(OH)~2~/CoOOH could prevent morphological breaking due to volume changes during H^+^ deintercalation/intercalation processes. This orientation was possibly due to the 3d orbital ordering on the octahedral Co^2+^ ions, which is absent in octahedral Ni^2+^ in Ni(OH)~2~. It could result in different orientation of NiO~2~ planes, rather than vertical to the outer surface of the nanostructures, which led to slow bulk redox processes and hence poor response rate and cycle life. ### 2.2.3. Fe Hydroxide {#advs1009-sec-0100} Iron oxy hydroxide, FeOOH, displays pseudocapacitive response mainly in the negative region of the working potential window in aqueous electrolytes, being presently studied as negative electrode in asymmetric supercapacitors. However, as for other hydroxides, the charge storage mechanism of FeOOH still remains vague and many details are lacking. Preliminary work on the charge storage mechanism of nanoplatelet lepidocrocite γ‐FeOOH in 1 [m]{.smallcaps} Li~2~SO~4~ made use of in situ electrochemical XAS during the charge/discharge process.[32](#advs1009-bib-0032){ref-type="ref"} The results showed only the shift of Fe K‐edge during the charging and discharging of FeOOH electrode, which suggests structural similarity and oxidation state changes during the redox reactions. The Fe---O bond length in the octahedral FeO~6~ decreased when the working potential increased from −0.8 to −0.1 V versus Ag/AgCl, as shown in EXAFS results in Figure [3](#advs1009-fig-0003){ref-type="fig"}d, which corresponded to a change in the Fe oxidation state from +2.78 to +2.93 (Figure [3](#advs1009-fig-0003){ref-type="fig"}d). Fe---Fe bond length for the Fe atoms between neighboring octahedral FeO~6~ also decreased, suggesting the contraction of layered FeO~6~, which is probably related to deintercalation of Li^+^. During the Li^+^ deintercalation reaction, structural order increased as revealed by the increase of Fourier‐transformed magnitude of Fe K‐edge EXAFS (Figure [3](#advs1009-fig-0003){ref-type="fig"}d), corresponding to restoration of symmetric oxygen distribution in the octahedral FeO~6~. The charge storage mechanism of FeOOH was explained considering valence changes in Fe atoms, from +2 to +3, during the Li^+^ deintercalation/intercalation reaction as$$\left. {Fe}\left( {III} \right){OOH} + {Li} + \leftrightarrow{LiFe}\left( {II} \right){OOH} \right.$$ The above discussion provides important insights into the charge storage mechanisms of metal oxide/hydroxide--based electrodes. More details about valence state changes, structural changes, and morphological changes during the operative condition of the electrodes have been reported. Important work has contributed to better understand the physicochemical changes occurring during charge storage and discharge; however, some contradictory results have also been observed and it is worth noting that during operando Raman measurements, structural changes induced by laser beam during measurements could result in the misleading results and interpretation. While many oxides and hydroxides display charge storage ability in aqueous electrolytes, only a limited number has been deeply studied and the respective charge storage mechanisms explained. Indeed, for the same oxides/hydroxides, there are different phases/polymorphs, which strongly impact the charge storage processes and mechanisms, an issue that still requires deeper studies. Moreover, the role of different electrolytes in the charge storage processes of many oxides and hydroxides is far from being well understood. Further work on these important subjects is still necessary to unveil the charge storage mechanisms in different electrolytes and to develop more reliable electrodes overcoming the drawbacks that limit the practical use of metal oxides and hydroxides as electrodes for supercapacitors. 3. Electrode Materials Engineering and Functionalization {#advs1009-sec-0110} ======================================================== Presently, most of the studies on metal oxides/hydroxides for supercapacitors focus on the design and development of electrodes that aim at achieving higher energy and power density and long‐term charge storage/release stability. On the one hand, intrinsic properties of metal oxides/hydroxides such as metal atoms, crystal structure/orientation, defect chemistries, and morphologies govern the charge storage performance. On the other hand, extrinsic materials engineering and functionalization, via atomistic doping and formation of composites with other materials, are also expected to improve electrochemical performance much further. Figure [1](#advs1009-fig-0001){ref-type="fig"} (electrode engineering axis---EEA) depicts different intrinsic (right side of EEA, Figure [1](#advs1009-fig-0001){ref-type="fig"}) and extrinsic (left side of EEA, Figure [1](#advs1009-fig-0001){ref-type="fig"}) materials engineering routes to modify supercapacitor electrode properties. These properties are briefly addressed in the following parts of this section. Concerning metal elements, cations with high number of electrons transferred during the redox reactions in aqueous electrolytes are expected to deliver high charge storage capacity. Their redox‐active potential range and overpotential for water splitting determine the working potential window. Low electronegative cations can facilitate electron transfer, improving high power density. High electronegative cations can increase the electrochemical potential of the redox reactions, resulting in high charge storage capacity. Electronegativity of metal cations can also vary the metal--oxygen bond length and ion adsorption capability, leading to different energy and power densities as suggested by the results of operando electrochemical Raman spectroscopy discussed in the previous section. Metal cations, with low molar mass, incorporated in low molar units are beneficial for high gravimetric capacity electrodes. For example, NiO (74.69 g mol^−1^) has molar mass lower than MnO~2~ (86.93 g mol^−1^) and Co~3~O~4~ (240.80 g mol^−1^), resulting in higher theoretical gravimetric capacity values. Many metal oxides and hydroxides currently under investigation for charge storage electrodes in aqueous supercapacitors display different polymorphs. Their crystal structure affects the electronic structure, namely, bandgaps and local electron density, which in turn influence electron conductivity and the redox activity of the electrodes. Crystal structures with 2D interlayer or 1D tunnel favor bulk diffusion of electrolyte ions, while close‐packed crystal structures can support only surface redox reactions, unless structural changes occur during the charge storage/release process. Thus, structural instability during the redox charging and discharging can alter the charge storage mechanisms. Incorporating metal cations with different ionic radii into mixed metal oxides and hydroxides can induce stress/strain and change the metal--oxygen bonding in the crystals, which affect directly the redox activity. Amorphous phases can facilitate charge transfer reactions due to their weak chemical bonding and presence of more surface defects.[33](#advs1009-bib-0033){ref-type="ref"} Defect chemistries, such as cation or oxygen vacancies, affect electronic structure and can change redox activity.[34](#advs1009-bib-0034){ref-type="ref"} The theoretical specific capacitance (F g^−1^), assuming that the charge storage is constant over the working potential, can be calculated based on the number of electrons that are stored/released during the redox reactions according to the following equation$$C = \left( \frac{nF}{m} \right)\frac{X}{E}$$where *n* is the number of electrons stored/released in the redox reactions, *F* is the Faraday constant (96 485 C mol^−1^), *m* is the molar mass of metal oxides, and *E* is the working potential window. *X* is the surface fraction (or electrode/electrolyte interface) of oxide material. This equation demonstrates that high experimental specific capacitance values can only be achieved when the oxide electrodes possess high specific surface area to maximize the number of active sites for the redox reactions. Therefore, it highlights how relevant is the specific surface area to improve the specific capacitance of the electrode. Electrode morphology can define surface area and porosity, affecting the active sites for the redox reactions.[35](#advs1009-bib-0035){ref-type="ref"} Electrolyte diffusion pathways, which influence the response rate or power, also depend on the electrode morphologies. These morphologies can be controlled by different preparation processes inducing different crystal growths and crystal orientations, leading to different exposed crystal facets. Surface energy of the different facets is varied, resulting in different redox activities and thus altering the charge storage performance. The morphologies should possess micro/nanoporosity to compensate the expansion/contraction of grain/particle, softening/stiffening of chemical bonds during the charge--discharge cycling, and increase the ionic diffusion of electrolyte ions into the electrodes. Nanosizing and nanostructuring are thus crucial routes to prepare electrode materials with enhanced energy storage capacity.[36](#advs1009-bib-0036){ref-type="ref"} The control of intrinsic properties in metal oxide and hydroxide electrodes is the key route to optimize electrochemical performance. Intrinsic properties such as low electron conductivity of both oxides and hydroxides and structural instability of hydroxides are not easy to improve via intrinsic materials engineering. Thus, the development of extrinsic materials engineering and functionalization approaches, via cation or anion doping, or compositing with other materials, enables new routes to enhance the charge storage performance of the electrodes further. For example, hybrid electrodes composed of electron conducting channels coated with high redox‐active metal oxides and hydroxides display boosted storage performance via extrinsically increased electron conductivity and surface area. Composites of metal oxides and hydroxides with conducting metals or carbon‐based nanomaterials are currently the main research stream to improve electrodes\' performance. Intrinsic and extrinsic materials engineering have been mainly controlled by the routes used to prepare the electrode materials as well as by the postprocessing methods. Next, in this section, recent advances in electrode materials engineering and functionalization via intrinsic and extrinsic approaches towards enhanced electrochemical performance will be highlighted. Note that for metal oxides or hydroxides displaying battery‐like behavior, the specific charge storage capacity should be presented as C g^−1^ or mAh g^−1^ (or per cm, cm^2^, or cm^3^) because the specific charge is not constant over the working potential window. Since many reports have presented specific charge capacity as F g^−1^, in this discussion, the values will be used as reported in the original literature. The main research trends have been focused on strategies to obtain high gravimetric energy density electrodes; however, for practical application of supercapacitors, length, areal, and volumetric capacities are becoming important metrics as reported in several studies. These will also be discussed in the context of the intrinsic and extrinsic materials engineering approaches. 3.1. Intrinsic Materials Engineering {#advs1009-sec-0120} ------------------------------------ ### 3.1.1. Crystal Engineering {#advs1009-sec-0130} *Phase Engineering*: Manganese dioxide, MnO~2~, crystallizes in different crystal polymorphs, such as α, β, γ, δ, and λ forms, and various of these polymorphs have been studied as charge storage electrodes for supercapacitors. These different crystal polymorphs are based on different arrangement of MnO~6~ octahedral units, forming 1D tunnel structures (α phase, 2 × 2 open tunnels; β phase, 1 × 1 open tunnels; and γ phase, 1 × 2 open tunnels), 2D interlayer structure (δ phase), and 3D spinel structure (λ phase), which display different electrochemical activity. Thus, optimization of crystal polymorphs of MnO~2~ is being considered an important route to achieve higher‐performance electrodes. Earlier work on crystal engineering of MnO~2~ has been reported in the last decade[37](#advs1009-bib-0037){ref-type="ref"} and the outcomes revealed that large tunnel size in α phase and high interlayer distance in δ phase support better intercalation/deintercalation reactions and display higher capacitance values compared to other phases. Although these results provide some initial insights into how crystal structures of MnO~2~ would affect specific capacitance, the capacities obtained are modest (200 F g^−1^). Moreover, the materials chemistry and engineering routes proposed to control MnO~2~ polymorphs and to optimize their electrochemical performance are still little understood. Recently, MnO~2~ with α, β, and δ polymorphs have been synthesized by controllable hydrothermal reactions by varying the concentration of K^+^ (*C* ~K+~) and H^+^ (*C* ~H+~) cations in the electrolyte (**Figure** [**4**](#advs1009-fig-0004){ref-type="fig"}a).[38](#advs1009-bib-0038){ref-type="ref"} The α phase formed when the *C* ~K+~ is higher than *C* ~H+~, the β phase formed when *C* ~H+~ is higher than *C* ~K+~ and the δ phase formed in excess of K^+^, as shown in a schematic diagram and XRD patterns in Figure [4](#advs1009-fig-0004){ref-type="fig"}b. K^+^ stabilized the formation of 2 × 2 tunnels during MnO~2~ dissolution--recrystallization processes and excess of K^+^ induced the formation of 2D interlayer by destructing the 2 × 2 tunnels. α and δ phases displayed, respectively, specific capacitance values of 535 and 464 F g^−1^, which are higher than the 155 F g^−1^ of the β phase due to increased intercalation reactions in 2 × 2 tunnel and interlayer structure compared to 1 × 1 tunnel structure, when the materials are tested in 1 [m]{.smallcaps} KOH electrolyte (Figure [4](#advs1009-fig-0004){ref-type="fig"}b, right). {ref-type="ref"} Copyright 2015, Royal Society of Chemistry. c) Reproduced with permission.[40](#advs1009-bib-0040){ref-type="ref"} Copyright 2016, John Wiley and Sons. d) Reproduced with permission.[41](#advs1009-bib-0041){ref-type="ref"} Copyright 2017, Royal Society of Chemistry.](ADVS-6-1801797-g004){#advs1009-fig-0004} Charge storage performance of TiO~2~ nanotube arrays with different polymorphs has been studied by thermal transformation of the anatase phase to the rutile phase.[39](#advs1009-bib-0039){ref-type="ref"} Rutile TiO~2~ nanotubes displayed capacitance values of 2.6 mF cm^−2^ at 1 mV s^−1^, which are higher than those of anatase phase due to higher conductivity and presence of Ti^+3^ after forming rutile phase. Although many metal oxides such as MoO~2~, MoO~3~, V~2~O~5~, and Fe~2~O~3~ exist in different polymorphs, only scarce work discussing controllable crystal phase engineering has been reported up to date. Ni(OH)~2~ and Co(OH)~2~ display isostructures composed of two different polymorphs: α and β (Figure [4](#advs1009-fig-0004){ref-type="fig"}c).[40](#advs1009-bib-0040){ref-type="ref"} α phases crystallize in the hydrotalcite structure, with positively charged Ni(OH)~2~ or Co(OH)~2~ layers, balanced by intercalated anions and display a high interlayer spacing of ≈8 Å. β phases crystallize in brucite‐type hydroxide, with hexagonal close‐packed structure, and show an interlayer spacing of ≈4.6 Å. The higher interlayer spacing in the α phase supports intercalation redox reactions, making this phase theoretically very interesting for supercapacitor electrodes. Nevertheless, the α phase is a metastable one, being quickly converted into the β phase in alkaline media. Thus, controlling the formation of different polymorphs and comparing their electrochemical response is very challenging. Recently, the formation of α‐Co(OH)~2~ and β‐Co(OH)~2~ has been selectively controlled by magnetic field--driven hydrothermal synthesis.[41](#advs1009-bib-0041){ref-type="ref"} The α‐Co(OH)~2~ was formed under an applied magnetic field of 2 T at 180 °C, the mixed α and β phases were formed under 0.2 T, whereas β‐Co(OH)~2~ was formed in the absence of the magnetic field, revealing the magnetic field--induced phase transformation, as shown in XRD patterns (Figure [4](#advs1009-fig-0004){ref-type="fig"}d, left). α‐Co(OH)~2~ phase showed specific capacity values of 885 F g^−1^ at 1 A g^−1^, which increased compared to the value of 388 F g^−1^ at 1 A g^−1^ of β‐Co(OH)~2~ (Figure [4](#advs1009-fig-0004){ref-type="fig"}d, right). α‐Ni(OH)~2~ and β‐Ni(OH)~2~ have also been selectively grown by varying the reaction temperature during the hydrothermal process.[42](#advs1009-bib-0042){ref-type="ref"} Some studies have focused on the preparation of the α phase due to its higher theoretical redox activity compared to the β one. Novel approaches have been proposed to produce α‐Ni(OH)~2~ such as bioinspired synthesis via two ion chamber reactors of Ni^2+^ cations and OH^−^ anions separated by a Nafion membrane.[43](#advs1009-bib-0043){ref-type="ref"} This route produced α‐Ni(OH)~2~, which delivered specific capacity of 2090 F g^−1^ at a specific current of 2 A g^−1^. The solvothermal process produced layered (4--5 nm in thickness) α‐Ni(OH)~2~ nanosheets that delivered specific capacity of 2064 F g^−1^ at 2 A g^−1^.[44](#advs1009-bib-0044){ref-type="ref"} It has been demonstrated that the β phase could also deliver high charge storage capacity. For example, microwave‐assisted hydrothermal synthesized β‐Ni(OH)~2~ presented specific capacity of 1566 F g^−1^ at 1 A g^−1^,[45](#advs1009-bib-0045){ref-type="ref"} and oriented‐attachment synthesized single‐layer β‐Co(OH)~2~ presented specific capacity of 2028 F g^−1^ at 5 A g^−1^.[46](#advs1009-bib-0046){ref-type="ref"} Despite these advances, the cycle life of Ni(OH)~2~ polymorphs is still very problematic compared to Co(OH)~2~ polymorphs, a consequence of the structural instability discussed in the previous section. Amorphous metal oxides and hydroxides have also been investigated for aqueous supercapacitor electrodes. For example, amorphous Ni--Co--Fe hydroxide and Ni hydroxide have been prepared by different routes and display good charge storage performance.[47](#advs1009-bib-0047){ref-type="ref"} However, for these materials no relevant structural--electrochemical relationship has been reported up to date, to the best of our knowledge, and further research is required to understand the charge storage behavior. *Crystal Facet Engineering*: The contribution of surface activity of the electrode materials to the redox reactions is closely related to their charge storage performance. From the crystal point of view, surface activity is associated with crystal facets, where different atomic arrangements on the outer surface alter surface energy and redox activity. Consequently, understanding the activity of different facets and controlling the formation of highly active facets are important goals to enhance the electrode performance. Whereas highly active facets play a main role in surface activity of materials with compact crystal structures such as NiO, Co~3~O~4~, and NiCo~2~O~4~, metal oxides and hydroxides with large open tunnels or interlayers, which support the bulk (intercalation) redox reactions, can also be influenced by the orientation of the open tunnels or interlayers to the outer surface. Surface energy of four low‐index crystal facets of β‐MnO~2~, including {110}, {101}, {100}, and {001}, and their proton adsorption have been studied recently by DFT calculations (**Figure** [**5**](#advs1009-fig-0005){ref-type="fig"}a).[48](#advs1009-bib-0048){ref-type="ref"} Their surface energies increased in the order of {110}, {101}, {100}, and {001} (Figure [5](#advs1009-fig-0005){ref-type="fig"}a). Facets with presence of higher number of oxygen atoms per unit area offer higher number of adsorption sites, resulting in higher adsorption pseudocapacitance. {001} facet displayed the highest calculated adsorption pseudocapacitance of 1.61 mF cm^−2^, followed by {100}, {101}, and {110} facets. The adsorption pseudocapacitance values of different facets increased proportionally according to their surface energies. Naturally, the lowest surface energy facet is the preferential grown surface; therefore, surface engineering routes to optimize the growth of high‐energy facets are essential to increase pseudocapacitance values. Moreover, energy barriers for proton diffusion into near surface and into the bulk of β‐MnO~2~ are much less in {001} facet compared to {110} facets (Figure [5](#advs1009-fig-0005){ref-type="fig"}b). The proton diffusion coefficient into near surface of {001} is 35 orders of magnitude higher than that of {110} facets, favoring the proton diffusion along {001} direction. 1D tunnel of β‐MnO~2~ vertical to {001} facets could facilitate the proton diffusion, resulting in high diffusion coefficients. Thus, optimizing tunnel orientation via facet engineering is also expected to improve the charge storage performance of the electrode materials. Experimental work addressing facet engineering has been reported. For example, Co~3~O~4~ nanocrystals with predominant high‐energy exposed facets {110}, as shown in transmission electron microscopy (TEM) results (Figure [5](#advs1009-fig-0005){ref-type="fig"}c), were prepared via hydrothermal thermal synthesis, varying precursor concentrations, followed by thermal calcinations at 250 °C.[49](#advs1009-bib-0049){ref-type="ref"} Nanorods, nanobelts, and nanosheets with high‐energy {110} exposed facets delivered higher specific capacity value compared to nanocubes or nanooctahedra with low‐energy (100) or (111) exposed facets (176.8 F g^−1^ at 1 A g^−1^ vs 20 F g^−1^ at 5 mV s^−1^). Cuboctahedral NiO mesocrystals, with increased high‐energy {001} exposed facets, were prepared by solvothermal routes and displayed a specific capacity of 1039 F g^−1^ at 1 A g^−1^, which was higher for the NiO mesocrystals with lower‐energy {111} exposed facets.[50](#advs1009-bib-0050){ref-type="ref"} α‐Fe~2~O~3~ with tunable exposed facets was prepared by a solvent‐mediated precipitation route with varied ethylene glycol concentration.[51](#advs1009-bib-0051){ref-type="ref"} NiCo~2~O~4~ microspheres surrounded by nanowires, containing different exposed reactive planes---(001) and $(01\overline{1})$, were prepared by the hydrothermal method and post‐thermal calcinations.[52](#advs1009-bib-0052){ref-type="ref"} They displayed specific capacity of 1284 and 986 F g^−1^ at 2 and 20 A g^−1^, respectively, and long‐term stability with only 2.5% capacity loss after 3000 cycles. The *c*‐axis preferentially oriented TiO~2~ nanotubes, grown by annealing amorphous TiO~2~ nanotubes in vacuum, showed increased capacity compared to randomly grown TiO~2~ nanotubes (8.21 mF cm^−2^ vs 0.32 mF cm^−2^ at 100 mV s^−1^).[53](#advs1009-bib-0053){ref-type="ref"} Note that the high number of oxygen vacancies in TiO~2~ influenced the electrochemical response; this defect chemistry will be discussed in the following section. {ref-type="ref"} Copyright 2017, American Chemical Society. c) Reproduced with permission.[49](#advs1009-bib-0049){ref-type="ref"} Copyright 2011, Springer Nature.](ADVS-6-1801797-g005){#advs1009-fig-0005} Facet engineering of metal hydroxides for supercapacitors has been scarcely reported, to the best of our knowledge; nevertheless, controlling the preferential growth of hydroxides with highly active facets would vary electrochemical activity, changing bulk ion diffusion coefficients and the corresponding electrode performance. Despite some results that have been reported for aqueous supercapacitor electrodes, facet engineering is still very little studied in the field. Further studies, looking into the charge storage ability of different facets and understanding and optimization of highly active facets, are crucial to advance knowledge and to develop metal oxide and hydroxide--based materials for aqueous supercapacitors. ### 3.1.2. Defect Engineering {#advs1009-sec-0140} Defect engineering is an effective route to modify the properties of materials as their physicochemical response changes with the formation of structural or chemical imperfections. Defects may change materials\' electronic structure, such as band structure, and form defect states in the bandgap, either as donor or as acceptor, which vary carriers\' concentration, chemical potential, and work function, affecting directly the electron conductivity and the redox activity of the materials. Surface defects change atomic configuration and electronic states at the surface, and vary the surface activity of the materials. Intrinsic defect engineering, via formation of nonstoichiometric metal oxides or hydroxides, such as oxygen or cation vacancies, without addition of other elements (extrinsic defects), can significantly alter the physiochemical properties of materials and sum up the advantages of low‐cost material modification routes. This approach has been progressively employed to enhance the supercapacitive performance of metal oxide and hydroxide materials. MnO~2~ containing controllable oxygen vacancies (MnO~2−~ *~x~*) was prepared by thermal hydrogen reduction at different temperatures.\[\[qv: 34a\]\] These defects enhanced the pseudocapacitive response in MnO~2−~ *~x~*, leading to maximized areal capacitance to 0.2 F cm^−2^ at 1 mA cm^−2^ in 0.5 [m]{.smallcaps} Na~2~SO~4~ as compared to 0.05 F cm^−2^ at 10 mV s^−1^ of the stoichiometric MnO~2~. The oxygen vacancies induced the formation of mixed valence Mn (+4, +3, and +2) for charge balancing and reduced the charge transfer resistance that is assigned to enhanced capacity. However, excess of oxygen vacancies decreased capacity due to increased charge transfer resistance, which highlights how important is to control this defect (**Figure** [**6**](#advs1009-fig-0006){ref-type="fig"}a). First principles DFT calculation on oxygen vacancy formation at β‐MnO~2~ grain boundary and single‐layered MnO~2~ revealed the existence of spin‐polarized gap states and metallic behavior at the grain boundary and half‐metallic behavior of the single layer, which could also be associated with the increased capacity of the MnO~2−~ *~x~* electrode.[54](#advs1009-bib-0054){ref-type="ref"} ![a) Left: charge--discharge curves at 1 mA cm^−2^; right: capacitance values and Mn valence states of MnO~2−~ *~x~* electrodes prepared by hydrogen reduction at different temperatures. b) Current--voltage plot of NiCo~2~O~4−~ *~x~* nanowires reduced with different hydrogen treatment times. c) Left: crystal structure of Co~3~O~4~ containing oxygen vacancy; right: electron conductivity of Co~3~O~4−~ *~x~* and Co~3~O~4~ at different temperatures (calculated by DFT method); and d) specific capacity values of Co~3~O~4−~ *~x~* and Co~3~O~4~ during charge--discharge cycling. e) XANES spectra of H*~x~*MnO~2~ and MnO~2~ reassembled at different pH values; f) top: illustration of Mn surface Frenkel; bottom: in‐plane Mn--Mn distance and Mn--Mn~surf~ revealed by pair distribution function analysis of MnO~2~ reassembled at pH 2; g) specific capacitance values at different specific currents of H*~x~*MnO~2~ and MnO~2~ reassembled at different pH values. a) Reproduced with permission.\[\[qv: 34a\]\] Copyright 2014, Elsevier Ltd. b) Reproduced with permission.\[\[qv: 34b\]\] Copyright 2016, Royal Society of Chemistry. c,d) Reproduced with permission.\[\[qv: 58a\]\] Copyright 2014, John Wiley and Sons. e--g) Reproduced with permission.[61](#advs1009-bib-0061){ref-type="ref"} Copyright 2017, Springer Nature.](ADVS-6-1801797-g006){#advs1009-fig-0006} Oxygen‐deficient α‐Fe~2~O~3~ nanorods were prepared by thermal decomposition of hydrothermally grown FeOOH in N~2~ atmosphere and evidenced improved donor density.[55](#advs1009-bib-0055){ref-type="ref"} α‐Fe~2~O~3~ displays n‐type semiconductive behavior and its donor densities increased from 5.5 × 10^18^ to 8.1 × 10^19^ cm^−3^ after oxygen vacancy formation. As a result, the equivalent series resistance and charge transfer resistance of the electrode decreased. The creation of oxygen vacancies in Fe~2~O~3~ nanorods with high mass loading of 4.3 mg cm^−2^ led to increased pseudocapacitance values of 382.7 mF cm^−2^ at 0.5 mA cm^−2^ in 3 [m]{.smallcaps} KCl electrolyte and increased stability with 95.2% capacitance retention after 10 000 cycles. Oxygen vacancies were introduced in VO~2~ by thermal reduction in H~2~ atmosphere and increased the material conductivity by almost three orders of magnitude and created essentially low V vacancies.[56](#advs1009-bib-0056){ref-type="ref"} Those intrinsically enhanced properties improved the pseudocapacitive performance of the electrode in 1 [m]{.smallcaps} Na~2~SO~4~ aqueous electrolyte, which displayed specific capacitance of 300 F g^−1^ (for a symmetric cell, the capacitance of the single electrode was not reported) compared to 76 F g^−1^ of stoichiometric VO~2~ and good cycling stability with 96% capacitance retention after 500 charge--discharge cycles (which generally quickly dropped for VO*~x~*‐based electrode working in aqueous electrolytes). Controllable oxygen‐deficient spinel NiCo~2~O~4~ electrodes were prepared by thermal reduction in H~2~ atmosphere with different treatment times.\[\[qv: 34b\]\] Electron conductivity increased for treatment up to 3 h and decreased when the H~2~ treatment time was extended for 4 h as revealed by in situ electrical scanning tunneling microscopy--TEM (Figure [6](#advs1009-fig-0006){ref-type="fig"}b). Formation of optimal oxygen vacancies and a structural disorder surface enhanced redox response of the nonstoichiometric NiCo~2~O~4~ electrode, which delivered a high capacity of 2.13 F cm^−2^ at 1 mA cm^−2^ (which is 240% increment as compared to the stoichiometric spinel oxide). Oxygen vacancies formed by H~2~ reduction on MnMoO~4~ also improved redox response.[57](#advs1009-bib-0057){ref-type="ref"} Oxygen vacancies were also produced by chemical reduction of electrodes with NaBH~4~ (a reducing agent).[58](#advs1009-bib-0058){ref-type="ref"} For example, conductivity of oxygen‐deficient Co~3~O~4~ electrodes increased as revealed by DFT calculation (Figure [6](#advs1009-fig-0006){ref-type="fig"}c), which resulted in enhanced storage capacity (978 F g^−1^) (Figure [6](#advs1009-fig-0006){ref-type="fig"}d). Monolayer NiTi‐layered double hydroxide (LDH) was grown by in situ reverse microemulsion technique.[59](#advs1009-bib-0059){ref-type="ref"} Ni^3+^ surface state in the monolayer increased compared to the bulk materials, which was probably related to the oxygen vacancies. DFT calculations revealed the transformation from semiconducting stoichiometric NiTi hydroxide to gapless half‐metallic Ni^3+^‐associated NiTi hydroxide. This property resulted in an electrode delivering high specific capacity, 2310 F g^−1^ at 1.5 A g^−1^, and 82% capacity retention after 3000 charge--discharge cycles. Oxygen vacancy engineering has shown to be an efficient route to tailor the charge storage performance of metal oxide electrodes. Oxygen vacancies on electrode materials are currently produced via two main routes: hydrogen treatment of the stoichiometric materials at elevated temperature and thermal annealing of metal hydroxides in inert atmosphere. Other engineering routes to produce oxygen vacancies, such as high‐energy particle bombardment and forming oxygen vacancies during materials\' growth processes, can also be interesting routes to tailor electrode performance. While several works addressing oxygen vacancy engineering have been reported for aqueous supercapacitors, metal vacancy engineering is much less studied. Among those, Ni^2+^ vacancies in NiO induced the formation of Ni^3+^ and enhanced the storage capacity.[60](#advs1009-bib-0060){ref-type="ref"} Recently, the effect of cation point defects on the pseudocapacitive response of δ‐MnO~2~ electrodes was studied in detail.[61](#advs1009-bib-0061){ref-type="ref"} In this work, controllable Mn point defects and Mn^3+^/Mn^4+^ ratios in δ‐MnO~2~ were implemented by exfoliation and reassembling of crystalline δ‐H*~x~*MnO~2~ nanosheets at pH of 2 or 4. Mn^3+^ content increased in the reassembled δ‐MnO~2~ compared to the crystalline δ‐H*~x~*MnO~2~; the lowest pH value increased the reduction of Mn^4+^ to Mn^3+^, which increased Mn^3+^ content. The average Mn valence states calculated from XANES were 3.59, 3.36, and 3.24 for H*~x~*MnO~2~, and δ‐MnO~2~ assembled at pH 4 and pH 2, respectively (Figure [6](#advs1009-fig-0006){ref-type="fig"}e). High‐energy X‐ray scattering and pair distribution function analysis revealed the presence and the increase in surface Frenkel Mn defects (Figure [6](#advs1009-fig-0006){ref-type="fig"}f--g) in the assembled δ‐MnO~2~ and the Frenkel defect concentration increased 30%, when lowering the pH from 4 to 2, since the in‐plane Mn was expelled preferable in higher acidic condition to form Mn vacancies. Assembled δ‐MnO~2~ nanosheets favored preferentially vacancy formation and the reduction of Mn^4+^ rather than the crystalline H*~x~*MnO~2~, due to the steric hindrance by H*~x~*MnO~2~ interlayer that consisted of protons and water. The increased Mn surface Frenkel defects in assembled δ‐MnO~2~ at low pH of 2 enhanced the pseudocapacitive response. The specific capacitance values were 306, 209, and 103 F g^−1^ at 0.2 A g^−1^ for δ‐MnO~2~ assembled at pH 2 and pH 4, and crystalline H*~x~*MnO~2~ (Figure [6](#advs1009-fig-0006){ref-type="fig"}g). Enhanced pseudocapacitance related to the formation of Frenkel defects could be associated with easier intercalation reaction, and the increased conductivity with the presence of more defect‐induced Mn^+3^ content. Although this work suggested that cation vacancy engineering would enlarge the storage capacity, the cycle life of the assembled δ‐MnO~2~ is still an important drawback that imposes further research. ### 3.1.3. Functional Architecture Engineering {#advs1009-sec-0150} Architecture engineering is currently a very dynamic research stream toward optimal charge storage performance of aqueous supercapacitors. The architecture of the materials composing the electrode affects specific surface area and electrolyte diffusion ability, which in turn control the number of surface active sites for the charge storage processes, thereby governing storage capacity as revealed by Equation [(11)](#advs1009-disp-0011){ref-type="disp-formula"}. A wide array of nanostructured metal oxides and hydroxides with different dimensionality have been designed. Low dimension lengths generally contribute to increased specific surface area and reduced diffusion length for bulk reactions. 0D nanostructures such as nanoparticles, 1D nanostructures such as nanowires, nanorods, and nanotubes, and 2D nanostructures such as layered nanosheets and nanoplatelets have been studied and in general evidence good charge storage performance. These nanostructures can be grown directly onto current collectors, forming 3D nanostructured electrode architectures containing interlinked nanostructures, which prevent agglomeration effects, accommodate volume expansion during Faradaic redox reactions, and reduce the contact resistance since there is no need of adding binders. The design of architectures based on nanostructured morphologies, accounting for optimal weight and volume distribution of the active material, has been proposed for enhancing supercapacitor performance. Architectures assembling different nanostructures are likely to provide more active sites for redox reactions. Hollow structures containing several shells with optimized empty spacing can enhance the volumetric capacity and can minimize stress induced by contraction/expansion of the material. Thus, tuning the design and production of electrode architectures based on nanostructured materials and the comprehension of their growing processes is an important route to tailor supercapacitor electrodes toward enhanced charge storage performance. Different MnO~2~ nanostructures were prepared by hydrothermal routes with varied reaction times.[62](#advs1009-bib-0062){ref-type="ref"} This route produced an active material composed of spherical microparticles containing different nanostructures, which changed morphology when increasing the reaction time, leading to the formation of nanosheets, nanofibers, and nanotubes. Urchin spheres, consisting of nanotubes and randomly distributed nanotubes, were formed when the reaction time was set to 8 and 12 h. δ‐MnO~2~ was formed under reaction times lower than 2 h, while α‐MnO~2~ was formed under longer reaction time. The MnO~2~ nanostructure growing process is schematically depicted in **Figure** [**7**](#advs1009-fig-0007){ref-type="fig"}a--f. The growth of microspheres involves nanosheets associated with the formation of layered δ‐MnO~2~ phase, which favored the growth of 2D features (Figure [7](#advs1009-fig-0007){ref-type="fig"}a,g). The δ‐MnO~2~ nanosheets were metastable states, resulting in diffusion of δ‐MnO~2~ domains into α‐MnO~2~ nanofiber nuclei (Figure [7](#advs1009-fig-0007){ref-type="fig"}b,h) and anisotropic growth of the nanofiber with increased reaction time (Figure [7](#advs1009-fig-0007){ref-type="fig"}c,i) until complete transformation of the nanosheets (Figure [7](#advs1009-fig-0007){ref-type="fig"}d,j). This process was due to the high thermodynamic stability of the 2 × 2 tunnel α‐MnO~2~ compared to the layered δ‐MnO~2~. The surface of α‐MnO~2~ nanofibers in contact with the reaction media was under Ostwald ripening process (recrystallization process), which was faster than that in the inner side, leading to the formation of hollow nanotubes upon complete growth of the nanofibers. Ostwald ripening continued with the increased reaction time, resulting in complete transformation of the nanofibers into nanotubes (Figure [7](#advs1009-fig-0007){ref-type="fig"}e,k), as revealed by 3D electron tomography. Nanotubes with open large hollows formed and dispersed in solution rather than in the urchin sphere with longer hydrothermal time due to the influence of acid etching (Figure [7](#advs1009-fig-0007){ref-type="fig"}f,l). {ref-type="ref"} Copyright 2014, John Wiley and Sons. b) Reproduced with permission.[63](#advs1009-bib-0063){ref-type="ref"} Copyright 2015, Royal Society of Chemistry.](ADVS-6-1801797-g007){#advs1009-fig-0007} Double Ni--Mn oxides were prepared by cathodic electrodeposition on stainless steel followed by thermal annealing for the supercapacitor electrodes[63](#advs1009-bib-0063){ref-type="ref"} and different morphological changes were observed when varying Ni:Mn ratios (Figure [7](#advs1009-fig-0007){ref-type="fig"}m). A novel surface morphology, with a complex nanostructure composed of the nanosheets linked with the texture particles, was formed at Ni:Mn of 1:3. The morphological changes when varying Ni:Mn ratios were assigned to the differential growth of the unitary metal oxides. Cyclic voltammograms of these oxide films evidenced redox peaks shifted to lower potentials when the Mn content increased (Figure [7](#advs1009-fig-0007){ref-type="fig"}n). The redox response increased and then decreased when the Mn content in the binary Ni--Mn oxides gradually increased, reaching the maximum response at Ni:Mn of 1:3. This response is a consequence of a synergistic effect of the redox reactions and the architecture of the Ni--Mn oxides. The specific capacity values (Figure [7](#advs1009-fig-0007){ref-type="fig"}p) calculated from the galvanostatic charge--discharge at the specific current of 1 A g^−1^ (Figure [7](#advs1009-fig-0007){ref-type="fig"}o) evidenced maximum values of 300 F g^−1^ in the Ni--Mn oxide film with Ni:Mn of 1:3 in agreement with the synergistic redox response observed in the cyclic voltammograms and with the enhanced specific surface area provided by the new electrode architecture. This electrode also displayed very good behavior under continuous charge--discharge cycling, with a capacity retention of nearly 100% after 1500 cycles. The morphology of Ni(OH)~2~ films prepared by hydrothermal routes was tailored under different reaction temperatures, which can control nucleation and growing kinetics.[64](#advs1009-bib-0064){ref-type="ref"} Different morphologies were obtained, including nanoflakes, stacked nanoflakes, nanobelts, and nanoribbons, which were formed when the reaction temperature increased, displaying surface areas of 29.04, 16.98, 15.34, and 13.29 m^2^ g^−1^, respectively. Metal oxides and hydroxides transformed from metal organic frameworks (MOFs) are currently emerging as an attractive route to design new architectures for supercapacitor electrodes. MOFs possess high surface area and high porosity due to their structural order, which consists of different metal centers and ligands. Thus, structural transformations of MOFs to oxide and hydroxide phases, while retaining high specific surface area and porosity, are expected to work as high‐performance electrode materials.[65](#advs1009-bib-0065){ref-type="ref"} For example, Ni*~x~*Co~3−~ *~x~*O~4~ nanoparticles were prepared by thermal annealing of Ni--Co--MOF‐74 nanocrystals at 400 °C, displaying a spindle‐like morphology preserved from Ni--Co--MOF‐74 with high surface area of 64--117 m^2^ g^−1^.\[\[qv: 65b\]\] The obtained electrodes presented a maximum specific capacity of 797 F g^−1^ at 1 A g^−1^ and good long‐term stability. Co, Ni, and Ni--Co hydroxide hollow nanoparticle--nanoflake architectures (NFAs) on Ni foam were prepared by immersing a ZIP‐8 nanoflake array onto Ni foams (prepared by transformation of zinc nitrate hydroxide nanoflake array in 2‐methylimidazole solution) in nickel nitrate and cobalt nitrate dissolved methanol solution (**Figure** [**8**](#advs1009-fig-0008){ref-type="fig"}a).[66](#advs1009-bib-0066){ref-type="ref"} During the transformation reaction, breaking of ZIP‐8 coordination bonds and oxidation of Co^2+^/Ni^2+^ to Co^3+^/Ni^3+^ occurred, and Zn^2+^ ions coprecipitated with Ni^2+^/Co^2+^ and Ni^3+^/Co^3+^ ions due to Kirkendall effect, resulting in the formation of the hollow morphology (Figure [8](#advs1009-fig-0008){ref-type="fig"}b--d). This novel Ni--Co hydroxide architecture delivered a capacity of 2.04 C cm^−2^ (971.4 C g^−1^) at 4 mA cm^−2^ (1.9 A g^−1^) and 0.84 C cm^−2^ (400 C g^−1^) at 48 mA cm^−2^ (22.9 A g^−1^) and a capacity loss of 5.9% after 5000 cycles (Figure [8](#advs1009-fig-0008){ref-type="fig"}e,f). {ref-type="ref"} Copyright 2016, Royal Society of Chemistry. g) Reproduced with permission.[67](#advs1009-bib-0067){ref-type="ref"} Copyright 2017, John Wiley and Sons. h--k) Reproduced with permission.[68](#advs1009-bib-0068){ref-type="ref"} Copyright 2017, John Wiley and Sons.](ADVS-6-1801797-g008){#advs1009-fig-0008} Metal oxides and hydroxides with more complex nanostructured architectures are also being developed for supercapacitor electrodes such as core--shell, yolk--shell, and multishell structures.[67](#advs1009-bib-0067){ref-type="ref"} Multishelled particles of Mn--Co oxide, Mn--Ni oxide, Zn--Mn oxide, and Mn--Co--Ni oxide were synthesized by thermal treatment of the corresponding amorphous coordination polymer precursor spheres.[68](#advs1009-bib-0068){ref-type="ref"} Amorphous coordination polymers can be incorporated with most of the transition metal cations, thereby allowing tunable composition of their derived compounds. The thermal oxidation of those compounds resulted in the formation of seven‐layered multishelled particles evidenced by TEM (Figure [8](#advs1009-fig-0008){ref-type="fig"}g--k). This process is related to the oxide spallation phenomena during oxidation, in which the particle shrinkage, due to weight loss, created residual stress in the oxide layer. When the oxide layer reached a critical thickness, the spallation occurred and a yolk--shelled structure was formed at 360 °C (Figure [8](#advs1009-fig-0008){ref-type="fig"}i). The oxide layer displayed crumpled morphologies as a result of the residual stress during phase transformation. The contraction continuously occurred with heating, forming yolk--multishelled particles at 390 °C (Figure [8](#advs1009-fig-0008){ref-type="fig"}j) and seven‐layered multishelled particles at 500 °C (Figure [8](#advs1009-fig-0008){ref-type="fig"}k). The Ni--Co oxide multishelled particles delivered a maximum specific capacity of 1908 F g^−1^ at 2 A g^−1^ and excellent cycling stability with 93.6% capacity retention after 20 000 cycles. In situ electrochemical TEM results revealed that the multishelled particles provided good volume change accommodation. Many routes, such as hydrothermal, electrodeposition, and sol--gel, or more recent ones such as MOF conversion, have been used to fabricate novel nanoarchitectures for high‐performance aqueous supercapacitors. By controlling the synthesis parameters, it has been possible to control material growth and to create novel morphologies assembled in different architectures. However, often nanoarchitectures may evolve under an unpredictable way. Advanced electrode design techniques, which can accurately control the growth of the intended nanostructures, can advance significantly the development of novel electrode architectures, not only for aqueous supercapacitors but also for many other applications. Presently, 3D printing techniques are evolving very fast and some interesting achievements have been reported for 3D printing with graphene‐based electrodes.[69](#advs1009-bib-0069){ref-type="ref"} Despite important advances, the use of this technique to design metal compound electrodes is still at early infancy, to the best of authors\' knowledge. The most important factor for current 3D printing technologies is the printable ink. Some results on hydroxide ink were recently reported and eventually these will nucleate the rising of 3D printing techniques to design metal oxide and hydroxide electrodes, for example.[70](#advs1009-bib-0070){ref-type="ref"} 3.2. Extrinsic Materials Engineering and Functionalization {#advs1009-sec-0160} ---------------------------------------------------------- ### 3.2.1. Doping {#advs1009-sec-0170} Doping is widely used to engineer materials\' properties by adding, intentionally, a small quantity of extrinsic atoms. Doping intends to control relevant properties such as electronic structure, charge carrier concentration, and phases, which greatly influence electrical conductivity, chemical potential, and surface activity. As these properties are associated with the redox activity of electrode materials and govern their charge storage performance, relevant work on either metal or nonmetal doping of metal oxide and hydroxide electrodes has been reported. Ce^3+^‐doped MnO~2~ was prepared by the hydrothermal route.[71](#advs1009-bib-0071){ref-type="ref"} Ce^3+^ doping induced phase transformation from β‐MnO~2~ to α‐MnO~2~ with increased tunnel size due to the stabilization of 2 × 2 tunnels by Ce^3+^. Optimal doping concentration (5.6% of Ce to Mn) produced finer nanorods, displaying decreased diameter and length due to inhibition of growth along and perpendicular to the \[001\] direction, with presence of Ce^2+^ in 2 × 2 tunnel that increased material electrical conductivity. Thus, Ce^3+^ doping increased charge storage capacity by one order of magnitude compared to undoped MnO~2~. Al‐doped α‐MnO~2~ materials (**Figure** [**9**](#advs1009-fig-0009){ref-type="fig"}a,b) with different dopant concentrations suffered morphological changes based on microspheres with different nanostructures such as nanoneedles, nanoparticles, and fine nanosheets.[72](#advs1009-bib-0072){ref-type="ref"} Densities of states calculated by DFT revealed an increase of the Fermi level energy and doping states near the valence and conduction bands, narrowing the bandgap and increasing conductivity (Figure [9](#advs1009-fig-0009){ref-type="fig"}c,d). Al‐doped α‐MnO~2~ microspheres (1.75 wt%) delivered specific capacitance values of 213 and 146 F cm^−3^ in 0.5 [m]{.smallcaps} Na~2~SO~4~ at 0.1 A g^−1^ and at mass loading of ≈4 mg cm^−2^, good Coulombic efficiency (nearly 100%), and good cycle life, maintaining 91% of the initial capacitance after 15 000 cycles (Figure [9](#advs1009-fig-0009){ref-type="fig"}e). Similarly, Fe acted as electron donor in Fe‐doped Co~3~O~4~ to increase the density of states near the Fermi level and the conductivity, thereby enhancing charge storage performance.[73](#advs1009-bib-0073){ref-type="ref"} Fe‐doped MnO~2~ induced significant morphological changes and a phase transformation, from mixed α‐MnO~2~ and γ‐MnO~2~ to mixed α‐MnO~2~, γ‐MnO~2~, and ε‐MnO~2~ phases.[74](#advs1009-bib-0074){ref-type="ref"} Optimal Fe dopant concentration reduced both equivalent series resistance and charge transfer resistance, as evidenced by electrochemical impedance spectroscopy, thus enhancing the pseudocapacitive performance. Fe‐doped mixed α‐, γ‐, and ε‐MnO~2~ with high mass loading of 5 mg cm^−2^ delivered specific capacitances of 267.0 and 183.1 F g^−1^ at 0.1 and 5.0 A g^−1^, respectively, with 3.2% capacitance loss after 2000 charge--discharge cycles. Mn~3~O~4~ octahedron nanocrystals were doped with different transition metal ions, including Cr, Co, Ni, and Cu.[75](#advs1009-bib-0075){ref-type="ref"} Divalent cations (Co, Ni, and Cu) occupied tetrahedral sites of Mn~3~O~4~ and a trivalent cation (Cr) occupied octahedral sites of Mn~3~O~4~. Doping with Co and Cu decreased the average rhombic length of the octahedron, whereas doping with Cr and Ni slightly increased these lengths. Cr‐doped Mn~3~O~4~ delivered enhanced specific capacitance value compared to the undoped one (272 F g^−1^ vs 202 F g^−1^ at 0.5 A g^−1^). Contrarily, Co‐doped Mn~3~O~4~ displayed similar capacity and Ni‐ and Cu‐doped Mn~3~O~4~ decreased the capacity. The role of different transition metal doping on the charge storage mechanism is still unclear and no concise mechanism has been established, but existing results highlight the importance of doping elements. Further research is thus necessary to establish the mechanism that governs the doping effects on the charge storage capacity. {ref-type="ref"} Copyright 2014, Elsevier Ltd. g) Reproduced with permission.[76](#advs1009-bib-0076){ref-type="ref"} Copyright 2016, American Chemical Society.](ADVS-6-1801797-g009){#advs1009-fig-0009} α‐Ni(OH)~2~ doped with Mg (1Mg to 13Ni) was prepared by in situ ion‐exchange reactions using Mg(OH)~2~ as sacrificial template and dopant.[76](#advs1009-bib-0076){ref-type="ref"} The spontaneous ion‐exchange reaction, due to differences in the solubility product of Mg(OH)~2~ and Ni(OH)~2~, led to simultaneous formation of Ni(OH)~2~ and doping with Mg. Mg‐doped α‐Ni(OH)~2~ displayed high Brunauer--Emmett--Teller surface area (202 m^2^ g^−1^) and pore volume of 0.56 cm^3^ g^−1^, which resulted in high specific capacity values: 1931 and 1496 F g^−1^ at 0.5 to 20 A g^−1^ in 6 [m]{.smallcaps} KOH, respectively. The capacity retained 95% of its initial value after 10 000 cycles, which is an interesting achievement considering the poor stability of Ni(OH)~2~‐based electrodes (Figure [9](#advs1009-fig-0009){ref-type="fig"}f). Morphological stableness was observed by scanning electron microscopy (SEM), thanks to the incorporation of Mg(OH)~2~ into the Ni(OH)~2~ structure that also stabilized its redox reactions. S‐doped V~6~O~13−~ *~x~* electrodes were prepared by thermal annealing of V~3~O~7~ nanowires in nitrogen atmosphere containing sulfur vapor.[77](#advs1009-bib-0077){ref-type="ref"} S doping increased electron conductivity and ion diffusion, resulting in high gravimetric capacitances, 1350 F g^−1^ (0.72 F cm^−2^) at 1.9 A g^−1^. Nevertheless, the specific capacitance decreased quickly after 200 cycles to 47.7% of the initial capacitance due to chemical dissolution of V^3+^ during charge--discharge cycling. Although a carbon coating is required to stabilize the electrode, the high capacitance obtained by S doping suggested that nonmetal doping could be employed to enhance charge storage. α‐Ni(OH)~2~ nanowires doped with S^2−^ in their interlayer gallery were produced by an anion‐exchange reaction between intercalated SO~4~ ^2−^ and S^2−^.[78](#advs1009-bib-0078){ref-type="ref"} This exchange reaction reduced the crystallinity and created nanocavities distributed over the nanowires since the radius of SO~4~ ^2−^ is larger than S^2−^, forming mesoporous nanowires. The increase of S^2−^ content resulted in decreased surface area and larger pore diameter related to the development of mesopores. The optimal S^2−^‐doped α‐Ni(OH)~2~ showed a high capacity value of 2223 F g^−1^ at 1 A g^−1^ (compared to 376 F g^−1^ of undoped ones). β‐FeOOH doped with 5.06% of F was prepared via a hydrothermal route[79](#advs1009-bib-0079){ref-type="ref"} and the material displayed low resistivity, with an area normalized electrical resistance of 2.211 Ω cm^−2^ measured by the four‐probe method. The reduction of the bandgap in β‐FeOOH from 1.05 to 0.2 eV in F‐doped β‐FeOOH, as calculated by DFT, resulted in metal‐like property significantly enhancing conduction. Thus, F‐doped β‐FeOOH enlarged the pseudocapacitive response (1.12 F cm^−2^ at 1.0 mA cm^−2^), which is higher than that of F‐free β‐FeOOH (0.63 F cm^−2^ at 1.0 mA cm^−2^) and the specific surface area was also reduced after F doping (51.896 m^2^ g^−1^ vs 63.099 m^2^ g^−1^). This material also showed excellent response with 83.0% capacitance retention under high current of 100 mA cm^−2^ and good capacitance retention after 5000 cycles. α‐(Ni/Co)(OH)~2~ doped with metaborate (BO~2~ ^−^) in their interlayers showed strong bonds with the hydroxide layers (other ions that can be intercalated such as nitrate, chloride, and sulfate showed weak bonding with the hydroxide layers), stabilized the hydroxide structure, and improved the material charge storage ability.[80](#advs1009-bib-0080){ref-type="ref"} However, a composite formed with graphene was still necessary to increase the material conductivity, an issue that will be discussed in the following section. ### 3.2.2. Compositing and Hybridizing {#advs1009-sec-0180} Metal oxides and hydroxides are wide‐bandgap materials with low electron conductivity. This property leads to the decrease of the redox‐active sites due to the inefficient electron transport to the electrode/electrolyte interface. Thus, the development of hybrid or composite materials composed of redox‐active compounds dispersed over conducting materials has been an attractive route to improve the electrode charge storage performance. Metals or metal alloys such as Ni, Ni--Cu, and Au and carbon‐based materials, such as graphene and carbon nanotubes, have been employed for fabricating composites with metal oxides and hydroxides. While composites with metals mainly aim at increasing conductivity, composites with carbon can provide additional charge storage due to the contribution of the double layer, increasing storage capacity and response rate. Agglomeration and lack of chemical stability of the redox materials could be minimized by forming composites with more stable redox activity. Moreover, composites may display different advantageous architectures that facilitate diffusion of electrolyte ions. Novel Ni--Mn oxide/Ni--Cu foam electrodes were deposited on stainless collectors by a two‐step electrodeposition route: first Ni--Cu foam and then Ni--Mn oxide. These electrodes utilized the redox response of Ni--Mn oxide for charge storage and the high electronic conductivity of the Ni--Cu foam.[81](#advs1009-bib-0081){ref-type="ref"} The Ni--Cu foams presented an open porous 3D morphology with randomly distributed micrometric pore size. The pore walls were constituted of randomly interconnected dendrites forming an open porous 3D percolation network, thus creating a good electron conducting pathway and favoring the diffusion of the electrolytes. Ni--Mn oxide uniformly covered the Ni--Cu dendrites of the foams and displayed percolating nanosheet‐like morphologies with porous structure (**Figure** [**10**](#advs1009-fig-0010){ref-type="fig"}a,b). The increase of the volume fraction of Ni--Mn oxide films obtained by electrodeposition over thicker foams increased specific capacitance of the electrode. Thanks to this architecture, the electrode delivered good redox activity and maximal specific capacity of 848 F g^−1^ at 1 A g^−1^ (Figure [10](#advs1009-fig-0010){ref-type="fig"}c,d). Interestingly, these electrodes displayed excellent response rate with capacity retention of 83% when the specific current increased from 1 to 20 A g^−1^ (Figure [10](#advs1009-fig-0010){ref-type="fig"}e). These promising results were the result of the hierarchical architecture consisting of double 3D percolation networks of Ni--Mn oxide nanosheets and Ni--Cu dendrites that enhanced the interfacial area of the active material, facilitating the accessibility of ions and charge transfer. {ref-type="ref"} Copyright 2015, Royal Society of Chemistry. f--j) Reproduced with permission.[85](#advs1009-bib-0085){ref-type="ref"} Copyright 2014, John Wiley and Sons. k--m) Reproduced with permission.[89](#advs1009-bib-0089){ref-type="ref"} Copyright 2016, Elsevier Ltd.](ADVS-6-1801797-g010){#advs1009-fig-0010} Other metal oxide/hydroxide--metal composites/hybrids have also been reported such as Fe~2~O~3~--Ni nanotubes,[82](#advs1009-bib-0082){ref-type="ref"} RuO~2~--Au foam,[83](#advs1009-bib-0083){ref-type="ref"} MnO~2~--Ni nanowires,[84](#advs1009-bib-0084){ref-type="ref"} MnO~2~--Ni nanopore arrays,[85](#advs1009-bib-0085){ref-type="ref"} Ni--Mn oxyhydroxide--nanoporous Ni--Mn alloy,[86](#advs1009-bib-0086){ref-type="ref"} and Ni--Co oxyhydroxide on Ni--Co dendrites, all displaying enhanced electrochemical response as charge storage materials.[87](#advs1009-bib-0087){ref-type="ref"} α‐Fe~2~O~3~ nanoneedles and MnO~2~ nanosheets were grown on ultrafine Ni nanotube arrays.[82](#advs1009-bib-0082){ref-type="ref"} Ni layer on a ZnO nanorod array was first formed, followed by anodic deposition of FeOOH or MnO~2~. The local pH, near the electrode, decreased during electrodeposition of FeOOH and dissolved ZnO; the post‐thermal treatment transformed FeOOH into α‐Fe~2~O~3~, leading to formation of α‐Fe~2~O~3~ nanoneedles--Ni nanotubes. HCl was used to etch the ZnO nanowires in MnO~2~--Ni--ZnO and to form MnO~2~ nanosheets--Ni nanotubes. The efficient electron transport of Ni nanotubes, together with the formation of conformably nanostructured active oxide coatings, provided good charge storage performance. α‐Fe~2~O~3~--Ni and MnO~2~--Ni nanotubes delivered capacitance values of 418.7 F g^−1^ at 10 mV s^−1^ and 440.7 F g^−1^ at 5 mV s^−1^, respectively, with 7--8% capacitance loss after 5000 charge--discharge cycles. RuO~2~--porous Au electrodes formed via the growth of porous foam‐like Au layers followed by the growth of a hydrous RuO~2~ coating.[83](#advs1009-bib-0083){ref-type="ref"} This electrode delivered areal capacitance of 3.25 F cm^−2^ at 0.1 mV s^−1^, which is comparable to the state of the art in Li microbatteries. MnO~2~ was grown by magnetic field--driven selective deposition on ultralong Ni nanowires, with length up to 1 mm, followed by anodic electrodeposition of MnO~2~.[84](#advs1009-bib-0084){ref-type="ref"} The ultralong nanowires provided high surface sites to support high mass loading of MnO~2~ on the current collector and maintained thin layers of MnO~2~ on nanowires. The electrode, with MnO~2~ mass loading of 3.51 mg cm^−2^, presented gravimetric and areal capacitances of 214 F g^−1^ and 750 mF cm^−2^ at 1 mV s^−1^, respectively, and stability under charge--discharge cycling with no capacitance loss after 20 000 cycles. Ni rectangular nanopore arrays, prepared by a replication route, from an anodic aluminum oxide membrane template, via alternative deposition and etching (Figure [10](#advs1009-fig-0010){ref-type="fig"}f,g), were used to support the growth of MnO~2~ growth.[85](#advs1009-bib-0085){ref-type="ref"} MnO~2~ coating could maintain the pores of the Ni arrays, whose size decreased with increasing MnO~2~ loading (Figure [10](#advs1009-fig-0010){ref-type="fig"}h--j), thus promoting diffusion of electrolyte ions and enhancing the response rate. As a result, the MnO~2~--Ni nanopore array electrode containing 0.08 mg MnO~2~ presented specific capacitances of 570 F g^−1^ at 2 A g^−1^ and 271 F g^−1^ at 100 A g^−1^ and maintained 47.5% of its capacitance when increasing the current for 50 times. Ni--Mn oxyhydroxide was grown on dealloyed nanoporous Ni--Mn alloy by electrochemical oxidation of a Ni--Mn alloy in alkaline media.[86](#advs1009-bib-0086){ref-type="ref"} The oxyhydroxide developed into the nanopore channels, via the paradigm of domain matching epitaxy, with a small lattice mismatch by dislocations. This electrode possessed efficient electron transport to the hydroxide layer and delivered volumetric capacitance of 505 F cm^−3^ at 0.5 A cm^−3^ and response rate with capacitance of 339 F cm^−3^ at 10 A cm^−3^. Ni--Co oxyhydroxide nanoplates were formed over vertically grown Ni--Co 3D dendrites by electrooxidation processes in alkaline media.[87](#advs1009-bib-0087){ref-type="ref"} This architecture provided a large number of active sites for redox reactions, enhanced the contact of active materials with conducting channel, favored the diffusion of electrolyte ions, and possibly accommodated volume expansion/contraction phenomena. As a result, the electrode delivered capacity of 121 mAh g^−1^ at 5 mV s^−1^ with acceptable cycle life. Electrodeposition of Co(OH)~2~ on nanoporous Au[88](#advs1009-bib-0088){ref-type="ref"} resulted in electrodes that achieved specific capacity of 1800 F g^−1^ at 20 A g^−1^. Ni(OH)~2~--nanoporous Au was fabricated by chemically dealloying of electrodeposited Ag~30~Au~70~ followed by hydrothermal growth of Ni(OH)~2~, forming corral‐like Ni(OH)~2~ films on the top of the nanoporous Au and thin Ni(OH)~2~ layer in the inner pores (Figure [10](#advs1009-fig-0010){ref-type="fig"}k,l).[89](#advs1009-bib-0089){ref-type="ref"} The maximal capacity was obtained by optimal growth of Ni(OH)~2~ on the top and in the inner pores of nanoporous Au, which lowered the interface resistance to result in high gravimetric and volumetric capacities of 3168 F g^−1^ and 2223 F cm^−3^, respectively, at 5 A g^−1^ (Figure [10](#advs1009-fig-0010){ref-type="fig"}m). Moreover, this electrode also achieved very high response rate, with 70% capacity retention at 500 A g^−1^ (Figure [10](#advs1009-fig-0010){ref-type="fig"}m), and long‐term stability with capacity loss of 10% after 30 000 charge--discharge cycles. These interesting results were obtained not only by the formation of the nanoporous Ni(OH)~2~--Au, but also by lowering the interfacial resistance between Ni(OH)~2~ (p‐type semiconductor) and Au due to low Ohmic contact as a result of their matching work function. In fact, the work function of Ni(OH)~2~ is 4.97 eV and that of Au is 5.1 eV. Thus, Ni(OH)~2~--Au formed a matched contact (Figure [10](#advs1009-fig-0010){ref-type="fig"}l). Graphene is currently among the most studied carbon materials for composite fabrication and hybridizing with metal compounds, particularly oxides and hydroxides. On the one hand, graphene confers interesting physicochemical properties that are suitable to enhance electroactivity of metal oxides and hydroxides, to increase specific surface area, to enhance electrical conductivity, and to provide high flexibility and high mechanical strength. On the other hand, graphene is the most recent discovered carbon form and it has been intensively investigated, a fact that resulted in important advancements concerning new applications. Composites or hybrid materials made of metal oxides or hydroxides with graphene in different forms have been reported for aqueous supercapacitor electrodes. Fabrication routes include dispersion or wrapping on/with graphene, exfoliation and restacking to form multilayer hybrid materials, or coating on (or with) 3D graphene films. Fe~2~O~3~--graphene hydrogel composites were prepared by a hydrothermal method, where Fe~2~O~3~ simultaneously grew together with graphene hydrogel, leading to uniform dispersion of Fe~2~O~3~ on the graphene sheets.[90](#advs1009-bib-0090){ref-type="ref"} This hydrogel composite showed higher specific surface area (173 m^2^ g^−1^) compared to individual graphene hydrogels (134 m^2^ g^−1^) and Fe~2~O~3~ particles (24 m^2^ g^−1^), thus increasing the number of sites available for the redox reactions and favoring electrolyte diffusion. Furthermore, the conducting graphene network enhanced electron transport into Fe~2~O~3~ and prevented dissolution and agglomeration of Fe~2~O~3~ nanoparticles. The Fe~2~O~3~--graphene hydrogel composite electrode displayed higher capacity and response rate compared to graphene hydrogel and Fe~2~O~3~ nanoparticles, delivering a specific capacity of 908 F g^−1^ at 2 A g^−1^ and good response rate with 69% capacity retention at 50 A g^−1^. The charge--discharge stability of the composite was enhanced compared to Fe~2~O~3~ nanoparticles. RuO~2~--graphene hybrid was prepared by disassembly/reassembly of graphene monoliths to load RuO~2~ particles inside the monolith, forming a uniform high‐density material. This high‐density electrode delivered a high volumetric capacitance (1485 F cm^−3^ at 0.1 A g^−1^).[91](#advs1009-bib-0091){ref-type="ref"} Layered NiCo~2~O~4~--reduced graphene oxide (rGO) hybrids were prepared by exfoliation and layer‐by‐layer assembly of Ni--Co hydroxide and graphene oxide (GO) followed by freeze drying and thermal treatment.[92](#advs1009-bib-0092){ref-type="ref"} This architecture (**Figure** [**11**](#advs1009-fig-0011){ref-type="fig"}a--c) contains a 3D conductive graphene network that could suppress restacking of graphene and reaggregation of NiCo~2~O~4~, resulting in increased specific surface area (167.6 m^2^ g^−1^ vs 109.7 m^2^ g^−1^ of NiCo~2~O~4~). The redox activity was enhanced (Figure [11](#advs1009-fig-0011){ref-type="fig"}d), and the electrode delivered specific capacity of 1388 F g^−1^ at 0.5 A g^−1^, good response rate (840 F g^−1^ at 30 A g^−1^), and long‐term charge--discharge stability with 90.2% capacity retention after 20 000 cycles. {ref-type="ref"} Copyright 2017, Elsevier B.V. e--g) Reproduced with permission.[97](#advs1009-bib-0097){ref-type="ref"} Copyright 2017, John Wiley and Sons. h--k) Reproduced with permission.[99](#advs1009-bib-0099){ref-type="ref"} Copyright 2014, John Wiley and Sons. l--o) Reproduced with permission.[100](#advs1009-bib-0100){ref-type="ref"} Copyright 2017, John Wiley and Sons. p,t,u,v) Reproduced with permission.[101](#advs1009-bib-0101){ref-type="ref"} Copyright 2015, American Chemical Society.](ADVS-6-1801797-g011){#advs1009-fig-0011} β‐Ni(OH)~2~/graphene hybrids were prepared by simple solvothermal reaction of a Ni precursor, GO, and water in benzyl alcohol.[93](#advs1009-bib-0093){ref-type="ref"} By controlling the amount of Ni precursor, in low quantity, single layer‐by‐layer assembly of β‐Ni(OH)~2~ and graphene was formed. Amorphous FeOOH quantum dots dispersed over functionalized graphene sheets were prepared by hydrothermal reaction and displayed good charge storage performance.[94](#advs1009-bib-0094){ref-type="ref"} Ni(OH)~2~--graphene hybrid hydrogel was prepared by a hydrothermal method.[95](#advs1009-bib-0095){ref-type="ref"} The graphene hydrogel was constructed of out‐of‐plane pores and contained in‐plane pores, which formed a hierarchical pore structure, providing efficient sites to anchor Ni(OH)~2~ and favoring access of electrolyte. The charge storage performance of Ni(OH)~2~--graphene hybrid outperformed its single components and the electrode achieved capacity of 1250.3 F g^−1^ at 5 A g^−1^. Ni--Co hydroxide--graphene monolithic composite electrodes were fabricated by a hydrothermal method, followed by vacuum filtration and contained alternate layers of NiCo--CH nanowires and graphene nanosheets.[96](#advs1009-bib-0096){ref-type="ref"} Oxygen species on graphene sheets supported the nucleation and growth of well‐dispersed Ni--Co hydroxide nanowires, which formed open porous channels between the graphene sheets, leading to high charge storage performance. Carbon nanotubes were also introduced in the composite, acting as conducting linkers between graphene sheets and Ni--Co hydroxide. Carbon nanotubes increased the mass and volume of the electrode, decreasing the specific capacity, but the enhanced electrical conductivity (16.8 S m^−1^ for the electrode with carbon nanotubes vs 4.2 S m^−1^ for the electrode without carbon nanotubes) improved the response rate. Other forms of carbon nanostructures have also been reported for composites with metal oxides and hydroxides. Ni--Mn hydroxide--porous carbon composites were prepared by the hydrothermal method.[97](#advs1009-bib-0097){ref-type="ref"} Porous carbon was carbonized from zeolitic imidazolate framework‐8 (ZIF‐8), forming a regular rhombic dodecahedral carbon shape. The deposition of Ni--Mn hydroxide uniformly covered the porous carbon, which surrounded the carbon with interlinked and disordered nanosheets (Figure [11](#advs1009-fig-0011){ref-type="fig"}e,f). This composite displayed mesoporous structure and enhanced electrical conductivity, and the agglomeration of Ni--Mn hydroxide was prevented by the presence of porous carbon, leading to an optimal capacity of 686.28 C g^−1^ at 1 A g^−1^ (Figure [11](#advs1009-fig-0011){ref-type="fig"}g). NiCo~2~O~4~--porous carbon composites were also prepared using ZIF‐8‐derived porous carbon and Ni--Co precursors followed by thermal transformation, and the electrode showed good charge storage performance.[98](#advs1009-bib-0098){ref-type="ref"} Ni--Mn hydroxide--carbon nanotube shell--core hybrids were prepared by a coprecipitation technique, forming gauze‐like NiMn‐LDH nanosheets attached along the nanotube backbone (Figure [11](#advs1009-fig-0011){ref-type="fig"}h--j).[99](#advs1009-bib-0099){ref-type="ref"} The hybrids with carbon nanotubes provided an endurance framework for grafting the hydroxide and their contact boosted electron transfer to the hydroxide layer. Moreover, this hybrid possessed a high specific surface area (198 m^2^ g^−1^) and pore volume (0.38 cm^3^ g^−1^), resulting in high‐performance electrodes (Figure [11](#advs1009-fig-0011){ref-type="fig"}k). Ni--Co hydroxide--N‐doped carbon--carbon cloth was prepared through dip coating and carbonization to form N‐doped carbon on the carbon cloth, followed by the hydrothermal growth of an interconnected Ni--Co hydroxide nanosheet layer (Figure [11](#advs1009-fig-0011){ref-type="fig"}l--n).[100](#advs1009-bib-0100){ref-type="ref"} The N‐doped carbon layer improved the hydrophilicity of the carbon cloth (contact angle decreased from 135° to 0° after coating with N‐doped carbon), favored the nucleation and growth of Ni--Co hydroxide layer, and formed a strong electronic interaction with the hydroxide layer. The composite electrode retained the superhydrophilic property, with contact angle of 0°, thus favoring the electrolyte diffusion and enhancing the electrode electrochemical response. These properties contributed to build electrode with high capacity (1817 F g^−1^ at 1 A g^−1^, above that of Ni--Co hydroxide on the carbon cloth, Figure [11](#advs1009-fig-0011){ref-type="fig"}o), high response rate, and long cycle life. A Fe~2~O~3~--carbon nanotube--graphite foam composite, composed of Fe~2~O~3~ nanoparticles grafted on a 3D graphite foam--carbon nanotube nanoforest collector, was prepared via two‐step deposition.[101](#advs1009-bib-0101){ref-type="ref"} The carbon nanotube nanoforest was grown on the graphite foam by chemical vapor deposition, to form a composite foam collector, and Fe~2~O~3~ nanoparticles were grown on this modified carbon foam by atomic layer deposition (Figure [11](#advs1009-fig-0011){ref-type="fig"}p,t,u,v). The composite carbon foam collectors possessed high surface area, high conductivity, and good flexibility, and provided not only the double‐layer charge storage, but also anchored sites for the growth of Fe~2~O~3~ and electrical contacts to increase electron transfer in the intrinsically poor conductive Fe~2~O~3~. This composite electrode achieved high areal capacity (470.5 mF cm^−2^ at 20 mA cm^−2^) and high charge--discharge stability up to 50 000 cycles (Figure [11](#advs1009-fig-0011){ref-type="fig"}v). Composites/hybrids of different metal oxides and hydroxides have also been studied, to take advantages of different redox reactions in distinct active materials in the composites or to design hierarchical structures to enhance electroactive surface and electrolyte diffusion. Such composites of MnO~2~--NiCo~2~O~4~,[102](#advs1009-bib-0102){ref-type="ref"} MnCo~2~O~4~--Ni(OH)~2~,[103](#advs1009-bib-0103){ref-type="ref"} Mn--Co hydroxide--Ni(OH)~2~,[104](#advs1009-bib-0104){ref-type="ref"} Fe--Co hydroxide--NiO,[105](#advs1009-bib-0105){ref-type="ref"} and TiO~2~--Ni(OH)~2~ have shown good charge storage performance and promising properties as electrode materials for supercapacitors.[106](#advs1009-bib-0106){ref-type="ref"} 4. Tailored Devices {#advs1009-sec-0190} =================== Materials engineering and different functionalization approaches have been proposed to fabricate electrodes for supercapacitors with increased charge storage capacity (much above the values typical of the double layers on carbon electrodes), high power response, and long‐term operation. Typically, in carbon‐based double‐layer supercapacitors, cells are assembled in the symmetric configuration, which consist of two similar capacitive electrodes (negative electrode and positive electrode) isolated by a separator immersed in the electrolyte. This assembly limits the working voltage of the devices to the working potential of the symmetric cell. The energy density of the device is proportional to the integrated area under the charge--discharge curve, which pinpoints the importance of enlarging the working voltage. Thus, asymmetric designs are attracting a lot of attention. These devices consist of two different charge storage electrodes, electrochemically active in different potential windows, that when combined result in wider working voltage. Moreover, the complementary water decomposition potential of each electrode, with different overpotentials for hydrogen and oxygen evolution, can widen the working voltage of the cell, over the theoretical water splitting potential range of 1.23 V. Therefore, asymmetric assemblies are envisioned as an important route to boost energy density of aqueous supercapacitors. It is worth noting that supercapacitors are power devices. The state of the art in developing high energy density electrodes is not only by fabricating capacitive and pseudocapacitive charge storage materials, but also by producing battery‐like and hybrid (mixed battery and capacitive responsive) responsive materials to maximize power. Thus, in the asymmetric design, to keep the maximum power response, capacitive carbon materials are used in one of the electrodes, despite the fact that energy density is modest due to limited double‐layer charging. A well‐known example is the Li supercapacitor, a device that still requires lithiation steps and organic electrodes. Metal oxides and hydroxides can store charge in either more positive or more negative regions of the potential window. Thus, it is possible to combine different metal oxides and hydroxides, with enhanced redox kinetics, in positive and negative electrodes to deliver high charge storage capacity, good power response, and long‐term stability. These asymmetric combinations can improve much further energy density of supercapacitor cells, while maintaining adequate power response, even in aqueous electrolytes. It is worth noting that asymmetric designs employing battery‐like responsive electrodes shall be referred to as hybrid supercapacitors. Charge balancing between the positive electrode and the negative electrode is required when assembling asymmetric supercapacitor cells. **Figure** [**12**](#advs1009-fig-0012){ref-type="fig"} shows the discharge profile of asymmetric cells assembled with electrodes that make use of materials with different charge storage behavior (capacitive and battery‐like). The capacity of the asymmetric cell at high currents depends on whether the charge is balanced at low or high currents due to the different response rates of negative and positive electrodes (Figure [12](#advs1009-fig-0012){ref-type="fig"}).[107](#advs1009-bib-0107){ref-type="ref"} Although charge balancing is widely applied, recently it has been shown that charge unbalancing, interestingly, could widen the working voltage of the symmetric cell, an issue that certainly will be further investigated.[108](#advs1009-bib-0108){ref-type="ref"} {ref-type="ref"} Copyright 2015, Elsevier B.V.](ADVS-6-1801797-g012){#advs1009-fig-0012} Traditionally, aqueous electrolytes, including acidic (e.g., H~2~SO~4~, HNO~3~), neutral (e.g., Na~2~SO~4~, Li~2~SO~4~, KCl), and alkaline (e.g., KOH, NaOH, LiOH), in the liquid form are used in supercapacitor cells. Operation in aqueous electrolytes is of utmost relevance, but there are still some drawbacks to overcome, such as leakage and material corrosion. To avoid that, quasi‐solid‐state gel electrolytes have emerged recently as an interesting option. In these systems, the aqueous electrolyte is trapped in polymer matrices such as poly(vinyl alcohol) (PVA), poly(acrylic acid), and poly(methyl methacrylate), supplying ions for redox reactions with active materials. Quasi‐solid‐state gel electrolytes can prevent dissolution or corrosion of active materials and the leakage of electrolyte, improving lifetime of devices. Moreover, assembling cells using quasi‐solid‐state gels offers advantages of flexibility, safety, and wider working temperate range,[50](#advs1009-bib-0050){ref-type="ref"} advancing cell package engineering for functional solid‐state supercapacitors. Supercapacitors displaying high energy density are presently a very important target and, nowadays, many applications have specific requirements, demanding specially tailored supercapacitors (Figure [1](#advs1009-fig-0001){ref-type="fig"}, device assembly axis). For example, development of miniaturized devices and flexible and wearable electronics imposes integration of reliable micro‐supercapacitors and/or flexible supercapacitors. Integration of supercapacitors with renewable electrical energy sources, such as solar cells and wind turbines, and replacement of conventional Al electrolytic capacitors require devices with short time response, leading to the development of high‐frequency micro‐supercapacitors. The replacement of multicomponents with multifunctional devices can save space, which is of utmost relevance for electronics, leading to the development of supercapacitors tailored for multiple purposes. During operation of supercapacitors, irreversible deformations or failures could occur due to intentional bending, twisting, and stretching or accidental causes. Thus, supercapacitors with restorable abilities, such as healable or shape memory to heal failures or to recover back to the original shape, are also being developed. The following subsections address the latest advances in metal oxide and hydroxide--based aqueous supercapacitors tailored to answer the application needs. For simpler description, supercapacitor cells are denoted as negative electrode material\|\|positive electrode material. These subsections are organized as follows. First, typical carbon\|\|metal oxide/hydroxide supercapacitors are described followed by new metal oxide/hydroxide\|\|metal oxide/hydroxide supercapacitors with enhanced energy density. Then, micro‐supercapacitors including high‐frequency ones and flexible supercapacitors will be discussed. Finally, some advances on multipurpose supercapacitors, and restorable and degradable supercapacitors will be discussed. 4.1. High Energy Density Supercapacitors {#advs1009-sec-0200} ---------------------------------------- ### 4.1.1. Carbon\|\|Metal Oxide/Hydroxide Supercapacitors {#advs1009-sec-0210} Many metal oxides and hydroxides, such as NiCo~2~O~4~, MnO~2~, Ni(OH)~2~, and Co(OH)~2~, display redox response in aqueous electrolytes in positive potential windows. Different materials engineering routes, as described previously, allow maximizing the redox response, making metal oxides and hydroxides suitable materials to assemble high energy density electrodes for aqueous supercapacitors. They can be assembled with capacitive carbon electrodes and various carbon‐based materials (e.g., activated carbon, graphene, carbon nanotubes, or their composites), which provide high conductivity and high specific surface area for double‐layer charging. Generally, such configurations display wider working voltages, about 1.6--1.8 V; values near 2 V were also achieved. Asymmetric cells employing pseudocapacitive materials, such as manganese oxide, can display increased energy density and high power density as a result of the pseudocapacitive response of the oxide and capacitive response of carbon, respectively. Several asymmetric cells based on this configuration and delivering high capacitive performance have been reported: MnO~2~ nanotube--activated graphene,[109](#advs1009-bib-0109){ref-type="ref"} MnO~2~--carbon nanofiber composites,[110](#advs1009-bib-0110){ref-type="ref"} and MnO~2~--graphene oxide composites.[111](#advs1009-bib-0111){ref-type="ref"} Porous MnO~2~ nanotubes consisting of MnO~2~ nanosheets were prepared by hydrothermal routes, using polycarbonate membranes as sacrificial template.[109](#advs1009-bib-0109){ref-type="ref"} When assembled into cells using activated graphene as negative electrodes, in 1 [m]{.smallcaps} Na~2~SO~4~ aqueous electrolytes, activated graphene\|\|MnO~2~ nanotubes delivered energy density of 22.5 Wh kg^−1^ and maximum power density of 146.2 kW kg^−1^ in working voltage windows of 1.8 V. MnO~2~--carbon nanofiber composites were prepared by carbonization of bacterial cellulose pellicles, followed by redox reactions of MnO~4~ ^−^ with carbon to form coatings made of MnO~2~ nanoparticles on the surface of carbon nanofibers.[110](#advs1009-bib-0110){ref-type="ref"} MnO~2~--carbon nanofiber as positive electrode was assembled with N‐doped carbon nanofiber (prepared by carbonization of bacterial cellulose pellicles in the presence of urea) as negative electrodes and the devices could operate in working voltage windows of 2 V. This N‐doped carbon nanofiber\|\|MnO~2~--carbon nanofiber cells delivered energy density of 32.91 Wh kg^−1^ and maximum power density of 284.63 kW kg^−1^, as well as good cycle life, with 95.4% capacity retention after 2000 cycles. Hierarchical porous carbon\|\|MnO~2~--graphene oxide cells were prepared combining nanoflaked MnO~2~--graphene oxide multilayers and hierarchical porous carbon prepared by carbonization of natural *Artemia* cyst shells (**Figure** [**13**](#advs1009-fig-0013){ref-type="fig"}a).[111](#advs1009-bib-0111){ref-type="ref"} These cells operated in working voltage windows of 2 V (Figure [13](#advs1009-fig-0013){ref-type="fig"}b,c), delivering energy densities of 46.7 and 18.9 Wh kg^−1^ at power densities of 100 and 2000 W kg^−1^, respectively, and evidenced good cycle life with 93% capacity retention after 4000 cycles (Figure [13](#advs1009-fig-0013){ref-type="fig"}d). {ref-type="ref"} Copyright 2014, John Wiley and Sons. e--h) Reproduced with permission.[116](#advs1009-bib-0116){ref-type="ref"} Copyright 2015, Royal Society of Chemistry. i--l) Reproduced with permission.[118](#advs1009-bib-0118){ref-type="ref"} Copyright 2017, John Wiley and Sons.](ADVS-6-1801797-g013){#advs1009-fig-0013} Asymmetric cells assembled with battery‐like responsive materials with high storage capacity in the positive electrode are currently an important route to obtain high energy density aqueous supercapacitors. For example, N‐doped graphene\|\|NiO,[50](#advs1009-bib-0050){ref-type="ref"} reduced graphene oxide\|\|Co*~x~*Ni~1−~ *~x~*O--reduced graphene oxide,[112](#advs1009-bib-0112){ref-type="ref"} activated carbon nanorod\|\|NiCo~2~O~4~,[113](#advs1009-bib-0113){ref-type="ref"} activated carbon\|\|NiMoO~4~,[114](#advs1009-bib-0114){ref-type="ref"} activated carbon\|\|Mg‐doped α‐Ni(OH)~2~,[76](#advs1009-bib-0076){ref-type="ref"} activated carbon\|\|Ni--Co hydroxide--N‐doped carbon,[100](#advs1009-bib-0100){ref-type="ref"} and carbon nanofoam paper (CNFP)\|\|Ni*~x~*Co~1−~ *~x~*(OH)~2~--CNFP cells have been proposed.[115](#advs1009-bib-0115){ref-type="ref"} It is interesting to note that despite battery‐like responsive materials, with well‐defined redox peaks, being employed as positive electrodes, the resulting asymmetric cells could display quasi‐rectangular cyclic voltammograms, suggesting good capacitive behavior.[112](#advs1009-bib-0112){ref-type="ref"}, [114](#advs1009-bib-0114){ref-type="ref"} Those supercapacitor cells generally work in alkaline electrolytes since the currently studied battery‐like responsive electrodes typically display optimal redox response in such media. The low overpotential for oxygen evolution of those positive electrodes and the low overpotential for hydrogen evolution of carbon materials in alkaline media limit the working voltage of cells to about 1.5--1.7 V. Activated carbon\|\|NiMoO~4~--NiCo~2~O~4~ cells (using NiMoO~4~ nanosheets shell--NiCo~2~O~4~ nanowire core arrays as positive electrode) operated in a voltage window of 1.75 V in 2 [m]{.smallcaps} KOH (Figure [13](#advs1009-fig-0013){ref-type="fig"}e--g) and delivered volumetric energy density of 5.64 mWh cm^−3^ at power density of 0.04 W cm^−3^ (Figure [13](#advs1009-fig-0013){ref-type="fig"}h).[116](#advs1009-bib-0116){ref-type="ref"} Activated carbon\|\|Mg‐doped α‐Ni(OH)~2~ cells operated in working voltage windows of 1.6 V in 6 [m]{.smallcaps} KOH and delivered maximum energy and power densities of 57.9 Wh kg^−1^ and 26 kW kg^−1^, respectively.[76](#advs1009-bib-0076){ref-type="ref"} Activated carbon\|\|Ni--Co hydroxide--N‐doped carbon cell operated in voltage windows of 1.6 V and displayed energy densities of 69.7 and 41.5 Wh kg^−1^ at power densities of 0.8 and 21.6 kW kg^−1^, respectively, and long life cycle up to 20 000 cycles.[100](#advs1009-bib-0100){ref-type="ref"} Quasi‐solid‐state asymmetric cells based on carbon\|\|metal oxide/hydroxide are also commonly reported, being based on activated carbon\|\|CoMoO~4~/CoNiO~2~ and carbon nanotube\|\|Co~3~O~4~--CoMoO~4~ cells working in LiOH/KOH--PVA gel electrolyte.[117](#advs1009-bib-0117){ref-type="ref"} Activated carbon\|\|CoMoO~4~/CoNiO~2~ cells using CoMoO~4~/CoNiO~2~ core/shell nanowires/nanoneedles grown on Ni foam by a two‐step hydrothermal route were used as positive electrode.\[\[qv: 117a\]\] This device displayed energy densities of 59.75 and 11.33 Wh kg^−1^ at power densities of 1464 and 14 880 W kg^−1^, respectively, in a voltage window of 1 V, with no significant capacity loss after 50 000 cycles. A carbon nanotube\|\|Co~3~O~4~--CoMoO~4~ cell was assembled with a positive electrode made of CoMoO~4~ nanosheets grown on Co~3~O~4~ nanocones/Ni foam via a two‐step hydrothermal route.\[\[qv: 117b\]\] It delivered energy densities of 45.2 and 37.0 Wh kg^−1^ at power densities of 400 and 6400 W kg^−1^, respectively, within the working voltage window of 1.6 V and the capacity retention was 96.5% after 3000 cycles. These quasi‐solid‐state asymmetric cells displayed capacitive response, evidencing quasi‐rectangular cyclic voltammograms and promising application as hybrid supercapacitors. Quasi‐solid‐state asymmetric cells working under voltage windows up to 2.4 V have also been described.[118](#advs1009-bib-0118){ref-type="ref"} This design was based on the formation of Ni~0.25~Mn~0.75~O @ C electrode with high oxygen evolution potential and displayed electrochemical response in the potential range of 0--1.4 V. Thus, an asymmetric cell made of activated carbon\|\|Ni~0.25~Mn~0.75~O \@C in LiCl--PVA electrolyte (Figure [13](#advs1009-fig-0013){ref-type="fig"}i,j) could operate in a working voltage window of 2.4 V (Figure [13](#advs1009-fig-0013){ref-type="fig"}k), delivering high energy and power densities (Figure [13](#advs1009-fig-0013){ref-type="fig"}l). ### 4.1.2. Metal Oxide/Hydroxide\|\|Metal Oxide/Hydroxide Supercapacitors {#advs1009-sec-0220} The energy density of asymmetric supercapacitor cells is limited by the lowest capacity electrode. In the case of carbon\|\|metal oxide/hydroxide cells, carbon electrodes with lower capacity reduce the energy density of the whole cell. To overcome this limitation, asymmetric cells that employ redox materials in both electrodes have been proposed for high energy density supercapacitors. Thus, it is essential to develop active materials, for both the positive and negative electrodes with well‐matched redox potentials to enable optimal energy performance. This is actually a very dynamic research line and paves the way to design new supercapacitor assemblies matching increased energy and power densities. Pseudocapacitive materials store charge via fast and reversible redox reactions, mimicking the capacitive response of carbon‐based materials. When using pseudocapacitive materials for both electrodes in asymmetric cells, their fast kinetic redox reactions can provide simultaneously high power response and high energy density. For example, FeWO~4~\|\|MnO~2~ (both electrodes are pseudocapacitive) were assembled in an asymmetric coin cell, working in 5 [m]{.smallcaps} LiNO~3~ electrolyte.[119](#advs1009-bib-0119){ref-type="ref"} This cell stored charge in a voltage range of 1.4 V and displayed specific capacitance of 8 F g^−1^, stable charge and discharge over 35 000 cycles, good Coulombic efficiency (nearly 100% over 35 000 cycles), low leakage current, and low self‐discharge rate. α‐Fe~2~O~3~ nanoneedle/Ni nanotube\|\|MnO~2~ nanosheet/Ni nanotube cells operated in Na~2~SO~4~ aqueous electrolyte or Na~2~SO~4~/PVA poly‐mer gel electrolyte[82](#advs1009-bib-0082){ref-type="ref"} and delivered better redox performance due to the higher ionic conductivity of the liquid form. These cells reached maximum energy density of 34.1 Wh kg^−1^ at power density of 3197.7 W kg^−1^, and were stable up to 5000 cycles, without significant capacity loss. Fe~3~O~4~\@Fe~2~O~3~\|\|Fe~3~O~4~\@MnO~2~ cells were assembled using core--shell structures consisting of Fe~2~O~3~ or MnO~2~ grown over Fe~3~O~4~ nanorod cores.[120](#advs1009-bib-0120){ref-type="ref"} These cell were operated in a working voltage window of 2 V and achieved energy densities of 83 mWh cm^−3^ (26.6 Wh kg^−1^) and 0.45 mWh cm^−3^ (14.5 Wh kg^−3^) at power densities of 15.6 mW cm^−3^ (500 W kg^−1^) and 500 mW cm^−3^ (16 kW kg^−1^), respectively. Using this design concept, supercapacitor cells with working voltage windows up to 2.6 V were able to reach the working voltage of organic supercapacitors, as recently reported.[121](#advs1009-bib-0121){ref-type="ref"} Based on the formation of composite materials, containing carbon cloth with increased overpotential for oxygen and hydrogen evolution in neutral electrolytes, the Na~0.5~MnO~2~/carbon cloth could operate in a working potential range of 0--1.3 V and carbon‐coated Fe~3~O~4~/carbon cloth could operate in a working potential range of −1.3 to 0 V (**Figure** [**14**](#advs1009-fig-0014){ref-type="fig"}b). Thus, the asymmetric C‐coated Fe~3~O~4~\|\|Na~0.5~MnO~2~ (Figure [14](#advs1009-fig-0014){ref-type="fig"}a) cells could store charge in a working voltage window of 2.6 V (Figure [14](#advs1009-fig-0014){ref-type="fig"}c),[121](#advs1009-bib-0121){ref-type="ref"} delivering energy density of 81 Wh kg^−1^ at power density of 647 W kg^−1^ and retaining 93% of the initial capacity over 10 000 cycles (Figure [14](#advs1009-fig-0014){ref-type="fig"}d). {ref-type="ref"} Copyright 2017, John Wiley and Sons. f--h) Reproduced with permission.\[\[qv: 122a\]\] Copyright 2016, Springer Nature. i--l) Reproduced with permission.[125](#advs1009-bib-0125){ref-type="ref"} Copyright 2016, Royal Society of Chemistry.](ADVS-6-1801797-g014){#advs1009-fig-0014} Asymmetric cells made of pseudocapacitive materials combined with battery‐like responsive materials are also being studied, following the concept of carbon\|\|battery‐like responsive material cells. For example, Fe~3~O~4~/reduced graphene oxide\|\|NiO and FeOOH\|\|NiMoO~4~ cells have been reported, showing high charge storage performance.[122](#advs1009-bib-0122){ref-type="ref"} Fe~3~O~4~/reduced graphene oxide\|\|NiO cells were assembled using triple‐shelled NiO hollow microspheres as battery‐like responsive electrode and Fe~3~O~4~ nanoparticles dispersed over reduced graphene oxide sheets as pseudocapacitive electrode in KOH electrolyte (Figure [14](#advs1009-fig-0014){ref-type="fig"}e). This cell stored charge over a working voltage of 1.6 V (Figure [14](#advs1009-fig-0014){ref-type="fig"}f,g) and delivered energy density of 51.0 Wh kg^−1^ at a power density of 800 W kg^−1^ (Figure [14](#advs1009-fig-0014){ref-type="fig"}h). FeOOH\|\|NiMoO~4~ cells were assembled using low‐crystalline FeOOH nanoparticles as pseudocapacitive electrode and NiMoO~4~ nanowires as battery‐like electrode. The FeOOH nanoparticles presented high specific capacitance of 1066 F g^−1^ at 1 A g^−1^ at mass loadings of 1.6 mg cm^−2^; even at high mass loading of 9.1 mg cm^−2^, which is comparable with the mass loading of commercialized carbon supercapacitor, they still delivered specific capacitance of 716 F g^−1^ (186 F cm^−3^) at 1 A g^−1^. Thus, these cells could deliver energy densities of 104.3 and 31 Wh kg^−1^ at power densities of 1.27 and 0.94 kW kg^−1^, respectively, in a working voltage window of 1.7 V. A device was assembled and delivered energy densities of 31.44 Wh kg^−1^ (17.24 Wh L^−1^) and 12.72 W kg^−1^ at power densities of 305 and 4.976 W kg^−1^ (2.736 W L^−1^), respectively. This cell design was stable under both charge--discharge cycling and floating tests. Interestingly, supercapacitor cells assembled with battery‐type electrodes, for both electrodes, have also been reported and displayed good performance. Probably, fast Faradaic redox reactions at each electrode, or the formation of composites with carbon‐based materials, enabled these well‐matched electrochemical responses.[123](#advs1009-bib-0123){ref-type="ref"} For example, a ZnFe~2~O~4~/stainless steel mesh\|\|Ni(OH)~2~/stainless steel mesh cell in which both electrode have well‐defined redox peaks was recently assembled[124](#advs1009-bib-0124){ref-type="ref"} in a working voltage window of 1.6 V. The cell delivered energy density of 42 Wh kg^−1^ at power density of 5 kW kg^−1^ and retained 83% of initial capacity after 8000 cycles. Fe~2~O~3~/carbon nanotube\|\|NiO--carbon nanotube cells in a 1.8 V window delivered energy densities of 137.3 and 102.2 Wh kg^−1^ at power densities of 2.1 and 39.3 kW kg^−1^, respectively (Figure [14](#advs1009-fig-0014){ref-type="fig"}i--l).[125](#advs1009-bib-0125){ref-type="ref"} Fe~2~O~3~/carbon nanotube/graphite foam\|\|CoMoO~4~/graphite foam cells were assembled in KOH electrolyte[101](#advs1009-bib-0101){ref-type="ref"} and worked under the voltage of 1.6 V, retaining 94.5% of their initial capacitance after 50 000 charge--discharge cycles. These cells delivered energy densities of 74.7 and 41.1 Wh kg^−1^ at power densities 1.4 and 11.2 kW kg^−1^, respectively, and displayed good capacitive response, evidencing quasi‐rectangular cyclic voltammograms, due to the fact that both electrodes are carbon‐based composites, favoring the redox reactions at each one. 4.2. Micro‐Supercapacitors {#advs1009-sec-0230} -------------------------- Smart electronic devices based on autonomous microsystems have evolved rapidly and presently are used in a wide range of applications. Microsystems and devices such as micro/nanoelectromechanical systems, micro/nanorobots, health sensors (including implant biosensors), and environmental and industrial sensors, with wireless sensing and communication abilities, enabled sensing and transfer of information across networks and advanced the development of smart integration systems. Such miniaturized autonomous devices require electrical energy supply to serve their purpose. Following the use of batteries for large‐scale devices, miniaturized batteries (microbatteries) with a size compatible with microsystems can be integrated in these systems. However, limited power density and lifetime have restricted their use in microsystems, when peak on/off power supply is required (e.g., sensing devices) or when long‐term use is expected (e.g., implant devices). Thus, miniaturized supercapacitors (also known as micro‐supercapacitors), which display energy storage/release characteristics similar to those of macro‐supercapacitors, have been proposed as power supply devices for microsystems. Nevertheless, the development of micro‐supercapacitors is quite behind miniaturized electronic system technologies, opening an interesting research frontier. Micro‐supercapacitor assemblies follow those of conventional electrochemical energy storage cells, including two electrodes and the electrolyte. They are assembled in different designs, frequently as sandwich structures (**Figure** [**15**](#advs1009-fig-0015){ref-type="fig"}a), in‐plane structures (Figure [15](#advs1009-fig-0015){ref-type="fig"}e), and fiber‐shaped structures. The fiber‐shaped structure, which advances the integration of micro‐supercapacitor in flexible microelectronics, is out of the scope of this section and will be discussed in the following section regarding flexible supercapacitors. Sandwich‐like micro‐supercapacitors mimic the design of bulky supercapacitors or thin‐film microbatteries, in which the electrolyte (generally in solid state) is inserted between two parallel charge storage electrodes. This simple design facilitates large‐scale fabrication, but the integration into microdevices is still very challenging. In‐plane design, micro‐supercapacitors are constructed of microelectrodes integrated on a common substrate, which are physically separated by interspacing between them. This design involves the use of microfabrication methods, taking advantage of the current state of the art in microfabrication technologies, to precisely define the architecture pattern of the supercapacitor electrode. Moreover, it makes easier integration of micro‐supercapacitors into microsystems. Note that, in micro‐supercapacitors, both mass and volume per unit area of active material are very small and occupy a nonsignificant part of the whole microsystem. The integration area of the devices in microsystems is the most significant parameter among density and geometric considerations. As a consequence, gravimetric capacity, which is widely reported, becomes an unsuitable metric and the energy storage capacity of the integrated devices per footprint area (areal capacity) is the most important capacity metric for micro‐supercapacitors. The sandwich‐like micro‐supercapacitor, in which each electrode occupies a footprint area, can deliver high areal capacity, if the electrode design enables better diffusion between the electrolyte and electrolyte/electrode contact. However, this design often displays decreased areal energy density compared to the in‐plane design, when using the same electrode materials, due to increased electrolyte diffusion into both the positive and negative microelectrodes, thanks to the interspacing between them. {ref-type="ref"} Copyright 2016, Elsevier B.V. e--h) Reproduced with permission.[127](#advs1009-bib-0127){ref-type="ref"} Copyright 2014, John Wiley and Sons. i--l) Reproduced with permission.[128](#advs1009-bib-0128){ref-type="ref"} Copyright 2017, Springer Nature. m--o) Reproduced with permission.[129](#advs1009-bib-0129){ref-type="ref"} Copyright 2017, American Chemical Society.](ADVS-6-1801797-g015){#advs1009-fig-0015} The use of redox materials based on metal oxides and hydroxides for micro‐supercapacitors can significantly enhance energy density. Nevertheless, electrode materials engineering is still required to increase surface to volume ratio and is extremely important to enlarge areal capacity. Microfabrication techniques such as printing and lithography followed (or not) by deposition or post‐processing method to form redox‐active metal oxides and hydroxides are being used to fabricate in‐plane micro‐supercapacitors. Nevertheless, microfabrication techniques to precisely control the interspacing down to nanosize and to increase the footprint areas of electrodes still require important development to enhance the electrode areal capacity. Based on micropatterning of active materials, their precursors, or current collectors (followed by depositing of active materials), the formation of active materials on microelectrodes could favor the fabrication processes. Thus, symmetric designs (which were not discussed in the previous section of this review) have been employed to construct micro‐supercapacitors. The next section highlights some advances in metal oxide and hydroxide--based micro‐supercapacitors. ### 4.2.1. Metal Oxide and Hydroxide in Micro‐Supercapacitors {#advs1009-sec-0240} To facilitate the integration of micro‐supercapacitors with electronic components on the same chip, the fabrication processes should be compatible with complementary metal--oxide--semiconductor processes that are used for construction of the integrated circuits. Thus, composites of redox materials with engineered porous Si substrate are considered the best choice to enhance charge storage performance. For example, sandwich micro‐supercapacitors were assembled in PVA--H~2~SO~4~ gel electrolyte using an atomic layer deposited coating of RuO~2~ on Si nanowire arrays prepared by metal‐assisted anodic etching of commercial wafers (Figure [15](#advs1009-fig-0015){ref-type="fig"}a--c). The device showed areal capacitance 2.9 mF cm^−2^ at 0.08 mA cm^−2^ and 2.3 mF cm^−2^ at 0.4 mA cm^−2^ (Figure [15](#advs1009-fig-0015){ref-type="fig"}d) and long‐term stability.[126](#advs1009-bib-0126){ref-type="ref"} It should be noted that despite the high cost of RuO*~x~* that hinders its wide use in bulky high energy density supercapacitors, the low quantity required in micro‐supercapacitors does not raise the costs significantly, since the main cost is mainly the consequence of the microfabrication processes. Sandwich symmetric RuO~2~·*x*H~2~O--Au foam cells assembled in silicotungstic acid--PVA gel electrolyte were integrated in a Si water.[83](#advs1009-bib-0083){ref-type="ref"} This micro‐supercapacitor delivered a remarkable areal capacitance of 1220 mF cm^−2^ at 1.5 mA cm^−2^, thanks to the formation of the porous hybrid architecture discussed in the previous section. Presently, the main approach in micro‐supercapacitors involves the design of planar configurations, with current collectors patterned on the substrate by microfabrication and deposition or post‐processing methods, to form the redox‐active materials, which enables easier integration into microsystems. For example, Ni(OH)~2~‐based planar micro‐supercapacitors have been reported (Figure [15](#advs1009-fig-0015){ref-type="fig"}e--g).[127](#advs1009-bib-0127){ref-type="ref"} The fabrication route involved photolithography processes accompanied with metal current collector coating (layered Ni/Pt/Ti) by sputtering deposition and chemical bath deposition of Ni(OH)~2~ layer to obtain interdigitated microelectrode fingers (Figure [15](#advs1009-fig-0015){ref-type="fig"}f). This micro‐supercapacitor when operating in KOH electrolyte delivered energy density of 21 mWh cm^−3^ at power density of 262.5 mW cm^−3^ (Figure [15](#advs1009-fig-0015){ref-type="fig"}h). Planar micro‐supercapacitors based on the asymmetric coupling of two redox‐active materials, as in the case of macro‐supercapacitors, have also been reported to enlarge the working potential window and to increase energy density.[128](#advs1009-bib-0128){ref-type="ref"} The materials used in the asymmetric cell were MnO~2~ as positive electrode and Fe~2~O~3~ as negative electrode (Figure [15](#advs1009-fig-0015){ref-type="fig"}i). The electrodes were fabricated on Si wafer by photolithography followed by physical vapor deposition of Cr/Ni layered current collectors and electrodeposition and thermal annealing to form MnO~2~ and Fe~2~O~3~ layers (Figure [15](#advs1009-fig-0015){ref-type="fig"}j,k). This on‐chip micro‐supercapacitor in KOH stored charge in a voltage window of 1.2 V and delivered energy densities of 12 mWh cm^−3^ and 35 µWh cm^−3^ at power densities of 1 and 14.8 W cm^−3^ (Figure [15](#advs1009-fig-0015){ref-type="fig"}l). Photolithography processes are currently employed for microfabrication of planar micro‐supercapacitors. However, the resolution of the pattern (interelectrode spacing) is low due to the light diffraction limit. Presently, photolithography processes are used to fabricate planar micro‐supercapacitors with interspacing size above 10 µm over footprint area of 1 cm^2^. For highly integrated planar micro‐supercapacitors, interelectrode spacing should be as small as possible; this issue is still limiting the use of photolithography. Recently, photolithography was combined with focus ion beam techniques to scribe a high‐resolution pattern for RuO*~x~*‐based micro‐supercapacitors (Figure [15](#advs1009-fig-0015){ref-type="fig"}m).[129](#advs1009-bib-0129){ref-type="ref"} This fabrication procedure involved evaporation deposition of Cr/Pt (40/200 nm) layers on Si/SiO~2~ and subsequent sputtering deposition of Ru film. Via optimal photolithography and ion beam etching processes, a high‐resolution pattern with interelectrode spacing down to 50 nm could be achieved (Figure [15](#advs1009-fig-0015){ref-type="fig"}n,o). Finally, RuO*~x~* layer was formed by thermal oxidation of the Ru film. This micro‐supercapacitor could operate in H~2~SO~4~ being stable over 10 000 cycles; it also showed very low equivalent series resistance of 8.2 µΩ cm^−2^ and areal capacitance of 18 mF cm^−2^, thanks to the narrow interspacing between the positive and negative microelectrodes. ### 4.2.2. High‐Frequency Micro‐Supercapacitors {#advs1009-sec-0250} Presently, supercapacitor studies are focusing on the reduction of the energy density gap between supercapacitors and batteries; yet, there is another gap, which is related to the frequency response between supercapacitors and Al electrolytic capacitors. Al electrolytic capacitors are currently being used for voltage ripple filtering when AC is converted to DC in electronic devices as well as for pulse power filtering produced by environmental energy harvesting. The voltage ripple can reduce or fasten the degradation of electronic devices and other energy storage devices. For example, it has been shown that the degradation of batteries is quicker when the charging sources contain voltage ripple.[130](#advs1009-bib-0130){ref-type="ref"} The standard AC line frequency is 60 Hz and the pulse powers are typically in the frequency range of tens to hundreds Hz, which requires capacitive response at frequencies of 120 Hz or tens to hundreds Hz, respectively. Commercial Al electrolytic capacitors display capacitive response at frequencies from tens to hundreds kHz with large phase angle up to −85.5°, fulfilling the requirement as voltage ripple filtering in most of the electronic devices and the pulse powers from environmental energy harvesting. However, Al electrolytic capacitors are too bulky, which are among the largest components occupying high volume and area in electronic circuits and, overall, they deliver very low energy density. Thus, smaller size AC lines or high‐frequency power filtering supercapacitors are being developed to integrate with emerging miniaturized electronic devices. Most of supercapacitors can only store charge at frequencies below 1 Hz with resistor--capacitor (RC) time constant of about 1 s, thus lacking voltage ripple filtering abilities to produce pure DC voltage. At high frequencies, most of supercapacitors behave like resistors with phase angles close to 0° due to their high electronic and ionic resistances, which does not comply with the phase angle requirement of −90° for AC line‐filtering supercapacitors. John Miller has first produced AC line‐filtering micro‐supercapacitors by chemical vapor deposition of graphene films, consisting of vertically aligned and interlinked graphene sheets on a metallic current collector.[131](#advs1009-bib-0131){ref-type="ref"} The symmetric supercapacitors when operating in 1 [m]{.smallcaps} KOH aqueous electrolyte showed phase angle at 120 Hz of −82°, achieving capacitance of 175 µF and RC time constant of less than 200 µs, with predominant graphene edge charge storage rather than their basal plane charge storage.[131](#advs1009-bib-0131){ref-type="ref"} Following this pioneering work, several carbon‐based materials and polymers such as electrochemically reduced graphene oxide,[132](#advs1009-bib-0132){ref-type="ref"} carbon nanotube,[133](#advs1009-bib-0133){ref-type="ref"} carbon nanofiber aerogel,[134](#advs1009-bib-0134){ref-type="ref"} N‐doped graphene,[135](#advs1009-bib-0135){ref-type="ref"} and coordination and conducting polymers have been reported for high‐frequency supercapacitors, displaying high phase angles in the frequency range of tens to hundreds Hz.[136](#advs1009-bib-0136){ref-type="ref"} High electric and ionic conductive electrodes are essentially required for high‐frequency supercapacitors. Most of metal oxides and hydroxides are wide‐bandgap materials with low conductivity, leading to high series resistances when operating as supercapacitor electrodes. Thus, engineering routes of their charge storage materials or their composites with highly conductive matrixes to reduce resistivity are expected to provide better electrochemical response at high frequency. Although metal oxide and hydroxide--based electrodes are still scarcely studied for high‐frequency supercapacitors, some results have been reported in the literature showing adequate frequency response. For example, symmetric sandwich micro‐supercapacitors based on composites of V~2~O~3~/VO~2~--V~2~O~5~ core--shell nanostructures grown on graphene conductive framework displayed phase angles of ≈−80° and −50° at frequencies of 10 and 100 Hz, respectively.[137](#advs1009-bib-0137){ref-type="ref"} Symmetric planar supercapacitors based on reduced graphene oxide--MnO~2~--Ag nanowire hybrid film showed phase angles of ≈−90° over the frequency range of 10--100 Hz.[138](#advs1009-bib-0138){ref-type="ref"} Graphene quantum dot\|\|MnO~2~ planar asymmetric micro‐supercapacitors showed phase angles of about −65° for frequencies up to 1 kHz.[139](#advs1009-bib-0139){ref-type="ref"} Although MnO~2~ single electrode studies were not reported in this work, the fast double‐layer charging at the graphene quantum dot negative electrode would enable the high‐frequency response of the whole asymmetric cell. 4.3. Flexible Supercapacitors {#advs1009-sec-0260} ----------------------------- Currently, flexible and wearable electronic devices are being extensively studied as new generation devices in different applications. Flexible devices such as roll‐up displays, flexible sensors, transistors, optoelectronics, and antennas would not only increase their resistance against deformations but also extend the ways they can be integrated into different systems and upgrade our interaction on these devices. Currently used rigid supercapacitor cells such as cylindrical, coin, prismatic, and pouch cells are not easy to integrate in flexible devices. Thus, flexible supercapacitors are crucial to power up those systems.[140](#advs1009-bib-0140){ref-type="ref"} Assembling of flexible supercapacitors generally requires development of flexible components, including flexible electrodes and flexible electrolytes. Quasi‐solid‐state gel electrolytes are being widely used, thanks to their flexibility and reduced liquid leakage. Therefore, the development of flexible electrode has become the main spotlight. Different types of flexibility such as bending, twisting, stretching, and compressing have been studied. While simple bendable and twistable flexible supercapacitors are commonly achieved, stretchability and compressibility properties, which are important mechanical response for wearable devices, still face many challenges. Metal oxides and hydroxides are fractal materials, which can only sustain very small deformations without breaking. Thus, electrode engineering routes have been proposed, such as coating of those materials on flexible substrates or forming composites with other flexible materials. Note that most of the engineering approaches proposed for flexible high energy density supercapacitors can also be exploited for micro‐supercapacitors. The essential compatibility of micro‐supercapacitors with fiber‐based microelectronic or wearable devices opened a designing route for fiber‐based flexible micro‐supercapacitors. The ultimate goal is to achieve flexible electrodes and devices exhibiting high charge storage performance under different deformations. Most of flexible substrates such as stainless steel, Cu, Al, and carbon derivatives can only support bending and twisting, rendering this flexibility in many metal oxide and hydroxide--based flexible supercapacitors. Using preformed flexible substrates, the active metal oxide and hydroxide layers can only be integrated by the deposition route and the adhesion of active materials on their flexible substrates, without and with deformations, is very important to maintain long‐term functioning. For example, MnO~2~ and Fe~2~O~3~ films were grown on flexible stainless steel sheet and assembled into Fe~2~O~3~\|\|MnO~2~ supercapacitors. These cells displayed good mechanical flexibility, operated in a wide working voltage of 2 V, and delivered energy density of 41.8 Wh kg^−1^ at power density of 1276 W kg^−1^.[141](#advs1009-bib-0141){ref-type="ref"} Compositing with carbon‐based materials enables the co‐growth of active materials and carbon into self‐supported flexible composite electrodes. The carbon networks enable the flexibility and conductivity of the electrodes and the redox‐active metal oxides and hydroxides enhance their charge storagecapacity. For example, MnO~2~--reduced graphene oxide composite bendable paper was prepared by vacuum filtration of graphene oxide solution containing Mn precursor followed by chemical reduction process.[142](#advs1009-bib-0142){ref-type="ref"} The asymmetric reduced graphene oxide paper\|\|MnO~2~--reduced graphene oxide paper cells displayed bendability and delivered energy densities of 35.1 and 11.5 µWh cm^−2^ at power densities of 37.5 µW cm^−2^ and 3.8 mW cm^−2^, respectively. 3D porous flexible substrates such as foam‐like substrates (graphene foams), textiles (carbon cloths), and carbon aerogels have also been exploited. Advantages over flexible planar current collectors include increased charge storage capability and macroporous architectures that enhance the dispersibility of the redox materials increasing their surface active area and diffusibility of electrolyte ions. Moreover, 3D flexible substrates can sustain a certain compression, favoring the assembly of compressible supercapacitors. For example, MnO~2~ was electrodeposited on 3D graphene foam for assembling flexible MnO~2~--graphene foam\|\|MnO~2~--graphene foam supercapacitors (**Figure** [**16**](#advs1009-fig-0016){ref-type="fig"}a,b), which showed good bendability without loss of capacitance and good charge storage performance.[143](#advs1009-bib-0143){ref-type="ref"} Asymmetric carbon nantube (CNT)--electrochemical reduced graphene oxide foam (ERGOF)\|\|MnO~2~--ERGOF cells displayed stable performance under different bending angles and times (Figure [16](#advs1009-fig-0016){ref-type="fig"}c,d).[144](#advs1009-bib-0144){ref-type="ref"} NiCo~2~O~4~ was grown on carbon cloth by a hydrothermal method and used as a positive electrode in flexible graphene paper\|\|NiCo~2~O~4~--carbon cloth supercapacitors operating in PVA--LiOH gel electrolyte.[145](#advs1009-bib-0145){ref-type="ref"} This cell worked and was stable, with 96.8% capacitance retention after 5000 cycles, under mechanical twisted and bent conditions. Moreover, it delivered energy densities of 60.9 and 37.56 Wh kg^−1^ at power densities of 568.2 W kg^−1^ and 11.36 kW kg^−1^ within the working voltage of 1.8 V. {ref-type="ref"}; h--m) stretchable symmetric MnO~2~--CNT\|\|MnO~2~--CNT supercapacitors based on editable route; and compressible Fe~2~O~3~--CNT supercapacitor electrodes. a,e,h,j,n) Schematic illustration of supercapacitor cells and the formation of electrodes; b,c,g,k,l,o) FEG‐SEM images of the supercapacitor electrodes; d) capacity retention of the flexible supercapacitor after bending at different angles and repetitions; g) capacity retention at 1 A g^−1^ during 10 000 stretching--releasing cycles; i,m) the honeycomb‐shaped supercapacitors under stretching at different strains and the capacity retention at 1.6 mA cm^−2^ during 10 000 stretching/releasing cycles; m,t) cyclic voltammograms before and after compressing the electrode for 1000 cycles; p) TEM image of Fe~2~O~3~--CNT. a,b) Reproduced with permission.[143](#advs1009-bib-0143){ref-type="ref"} Copyright 2013, American Chemical Society. c,d) Reproduced with permission.[144](#advs1009-bib-0144){ref-type="ref"} Copyright 2014, John Wiley and Sons. e,h--m) Reproduced with permission.[149](#advs1009-bib-0149){ref-type="ref"} Copyright 2017, John Wiley and Sons. o,p,t) Reproduced with permission.\[\[qv: 151b\]\] Copyright 2015, Royal Society of Chemistry.](ADVS-6-1801797-g016){#advs1009-fig-0016} Stretchable and compressible supercapacitors require more demanding architecture designs than bendable and twistable supercapacitors. Stretchable designs by using stretchable substrates such as polymers in combination with predesigned stretchability electrode materials such as wave‐like architectures or bridge--island architectures (designed for micro‐supercapacitors) and self‐supported electrodes with similar architectures and using stretchable textile are being carried out (Figure [16](#advs1009-fig-0016){ref-type="fig"}e).[146](#advs1009-bib-0146){ref-type="ref"} Stretchable carbon electrodes have been developed, including graphene papers and single‐walled carbon nanotube films, that would promote the fabrication of the stretchable metal oxide/hydroxide--carbon composite electrodes for high energy density flexible supercapacitors.\[\[qv: 146b,147\]\] For example, stretchable Fe~2~O~3~--carbon nanotube\|\|MnO~2~--carbon nanotube supercapacitors were reported.[148](#advs1009-bib-0148){ref-type="ref"} Fe~2~O~3~--carbon nanotube was prepared by chemical vapor deposition in the presence of Fe precursor and MnO~2~--carbon nanotube was prepared by redox deposition of MnO~2~ on etched Fe~2~O~3~--carbon nanotube (Figure [16](#advs1009-fig-0016){ref-type="fig"}f, bottom). These two self‐supported films were attached on prestretch polydimethylsiloxane (PDMS) and assembled with Na~2~SO~4~--PVA gel electrolyte, which was followed by release of the prestretch to form a wave‐like architecture and to enable stretchability (Figure [16](#advs1009-fig-0016){ref-type="fig"}f, top: MnO~2~--carbon nanotube, bottom: Fe~2~O~3~--carbon nanotube). These cells displayed excellent stretchability under tensile strains up to 100%, operated stably in the working voltage of 2 V (Figure [16](#advs1009-fig-0016){ref-type="fig"}g), and delivered energy density of 45.8 Wh kg^−1^ at power density of 406.6 W kg^−1^. Although predesigned strategies have enabled the development of stretchable supercapacitors, to enable compatibility with different devices, adaptable stretchability in different patterns is also an expected property. Moreover, mechanical mismatches between substrates, electrode materials, and gel electrolytes can lead to failure of supercapacitors assembled by predesigned approaches. Recently, stretchable supercapacitors with adaptability by cutting routes into different stretchable patterns were developed (Figure [16](#advs1009-fig-0016){ref-type="fig"}h).[149](#advs1009-bib-0149){ref-type="ref"} This supercapacitor was assembled in a symmetric design based on two MnO~2~ nanowire--carbon nanotube composite electrodes, which were separated and sealed by nanocellulose fiber films in LiCl--PVA electrolyte, forming a paper‐like cuttable supercapacitor (Figure [16](#advs1009-fig-0016){ref-type="fig"}j--l). By cutting route, different stretchable structures can be attained such as honeycomb‐like, pyramid pop‐up, and living‐hinge structures, displaying excellent stretchability and charge storage ability. For example, honeycomb‐like structures can be stretched up to 400% strain deformation and delivered stable charge storage capacity of 227.2 mF cm^−2^ during 10 000 stretching--releasing cycles (Figure [16](#advs1009-fig-0016){ref-type="fig"}i,m). Stretchability is an extremely important property for wearable supercapacitors. Thus, textile‐based stretchable substrates and fiber/yarn‐based supercapacitors, which can be then woven or knitted into textiles, are under considerable studied and will be discussed at the end of this section together with flexible micro‐supercapacitors. Compressible designs could be achieved by two different routes by employing compressible electrolytes or compressible substrates.[150](#advs1009-bib-0150){ref-type="ref"} Most of gel electrolytes currently used are compressible, favoring the assembly of compressible supercapacitors. Moreover, this route enables the use of noncompressible electrodes for compressible supercapacitors. Although the compressibility of different gel electrolytes is varied and can be optimized, a thin‐layer electrolyte is required to reduce their resistance; thus, compressibility is limited to millimeter scale. This limitation can be overcome by utilizing compressible substrates, which can display a compressible capability up to centimeter scale (Figure [16](#advs1009-fig-0016){ref-type="fig"}n). For example, composites of MnO~2~--carbon aerogels and α‐Fe~2~O~3~--carbon sponges displayed stable compressibility under 50% strain (Figure [16](#advs1009-fig-0016){ref-type="fig"}n,o,p,t).[151](#advs1009-bib-0151){ref-type="ref"} Electrode architecture designs could also form compressible electrodes, although this is rarely reported. MnO~2~--ultralong Ni nanowire grown on Ti foils displayed compressibility of 20% of their initial thickness, enabling an assembly of compressible supercapacitors.[84](#advs1009-bib-0084){ref-type="ref"} The compressible cells assembled with MnO~2~--ultralong Ni nanowire as positive electrode and polypyrrole--ultralong Ni nanowire as negative electrodes could be compressed about half of their thickness. Flexible micro‐supercapacitors have been proposed to integrate in flexible microelectronic devices. Planar micro‐supercapacitors are typically fabricated on flexible substrates such as plastic and paper to enable their flexibility.[152](#advs1009-bib-0152){ref-type="ref"} Moreover, different electrode architecture engineering approaches have been employed to enhance their charge storage performance. For example, planar symmetric Au/MnO~2~/Au flexible micro‐supercapacitors, fabricated on PET substrates, showed good bendability and delivered areal capacitance of 11.9 mF cm^−2^ at 0.05 mA cm^−2^.\[\[qv: 152a\]\] Stretchable planar micro‐supercapacitors were also designed on stretchable polymer substrates.[153](#advs1009-bib-0153){ref-type="ref"} The stretchable substrate was engineered into two different regions, in which stiff island arrays (PDMS) distributed over a soft polymer (mixture of Ecoflex and PDMS) as base. Micro‐supercapacitors were designed in planar configuration consisting of Mn~3~O~4~--multiwall carbon nanotube composite microelectrodes, which were fabricated on the top of stiff islands and connected by embedded liquid metal interconnections. This design enabled stable operation of the micro‐supercapacitor arrays under stretching deformation with uniaxial strain up to 40%, in which the deformation occurs mainly on the soft polymer substrate. Together with the development of wearable devices, the development of flexible fiber‐based microelectronics has led to the growth of wire/fiber/yarn‐based micro‐supercapacitors to integrate with their devices for power supplying. Their fiber‐based supercapacitors have been assembled by three different architectures, including twisting,[154](#advs1009-bib-0154){ref-type="ref"} paralleling of two fiber electrodes,[155](#advs1009-bib-0155){ref-type="ref"} or designing them in coaxial structures (**Figure** [**17**](#advs1009-fig-0017){ref-type="fig"}a,c,e).[156](#advs1009-bib-0156){ref-type="ref"} Currently, metal and carbon fibers/yarns are essentially employed as supports for fabrication of metal oxide and hydroxide--based fiber electrodes to enable flexibility.\[\[qv: 154a,157\]\] While metals can barely act as conductive supports, carbon fibers have been exploited as both conductive support and charge storage material via activation or oxidation of carbon materials to increase their double‐layer or pseudocapacitive charging.[158](#advs1009-bib-0158){ref-type="ref"} Note that for fiber‐shaped supercapacitors, capacity normalized by unit of length or area is the most important capacity metric among others. For example, twisted asymmetric fiber‐shaped and ordered mesoporous carbon--Ni fiber\|\|Ni(OH)~2~ nanowire--Ni fiber supercapacitors displayed capacity of 6.67 mF cm^−1^ (35.67 mF cm^−2^) at 0.1 mA, energy density of 0.01 mWh cm^−2^, and operated normally at different bending states (Figure [17](#advs1009-fig-0017){ref-type="fig"}b).\[\[qv: 154a\]\] Carbon fibers were also coated with a metallic conducting layer such as Ni to increase their support conductivity.\[\[qv: 154b\]\] Twisted asymmetric pen ink\|\|Ni--Co hydroxide supercapacitors based on Ni‐coated carbon fibers (Figure [17](#advs1009-fig-0017){ref-type="fig"}a) showed good charge storage performance with stretchable deformation. Carbon nanotubes were oxidized to increase their charge storage capacity, followed by a deposition of MnO~2~ to form MnO~2~--oxidized carbon nanotube fiber electrodes.[158](#advs1009-bib-0158){ref-type="ref"} The paralleled symmetric fiber cells constructed by the two paralleled fiber electrodes were assembled by a prestrained route to enable stretchability. The synergistic contribution to the charge storage of oxidized carbon nanotube and MnO~2~ resulted in high‐capacity stretchable fiber supercapacitors, displaying capacitance values of ≈409.4 F cm^−3^ (or 133 mF cm^−2^) at 0.75 A cm^−3^. The paralleled symmetric MnO~2~--CNT\|\|MnO~2~--CNT coiled yarn supercapacitor was assembled with MnO~2~‐coated CNT coiled yarn (Figure [17](#advs1009-fig-0017){ref-type="fig"}c).\[\[qv: 155b\]\] This microcell presented a static capacitance value of 2.72 mF cm^−1^, which retained 84% on stretching with 37.5% strain, and the static capacitance retained 96.3% under dynamic test at 20% strain deformation and strain rate of 6% s^−1^ (Figure [17](#advs1009-fig-0017){ref-type="fig"}d). Coaxial asymmetric polypyrrole--carbon nanotube\|\|MnO~2~--carbon nanotube fiber supercapacitors were assembled with MnO~2~--carbon nanotube as core, which was coated with a KOH--PVA gel electrolyte and the polypyrrole--carbon nanotube composite as an outer layer (Figure [17](#advs1009-fig-0017){ref-type="fig"}e).\[\[qv: 156b\]\] Moreover, this coaxial fiber supercapacitor was further twisted into a helical structure to enable stretchability. This cell displayed quite good reversible charge storage under stretching up to 20% (Figure [17](#advs1009-fig-0017){ref-type="fig"}f) and delivered energy density of 18.88 µWh cm^−2^. ![Wire/fiber/yarn‐based micro‐supercapacitors in twisting, paralleling, and coaxial designs of wire/fiber/yarn electrodes. a) Schematic representation of twisted asymmetric pen ink\|\|Ni--Co hydroxide supercapacitor and b) charge--discharge plots at 0.1 mA (bottom) under different bending of ordered mesoporous carbon--Ni fiber\|\|Ni(OH)~2~ nanowire--Ni fiber (top). Schematic representations and electrochemical response under different flexibility tests of c,d) paralleled symmetric MnO~2~--CNT\|\|MnO~2~--CNT coiled yarn supercapacitor and e,f) coaxial asymmetric polypyrrole--carbon nanotube\|\|MnO~2~--carbon nanotube fiber supercapacitor. d) Capacitance retention under different bending strain rates; f) capacitance values under different stretching strains and scan rates. a) Reproduced with permission.\[\[qv: 154b\]\] Copyright 2017, American Chemical Society. b) Reproduced with permission.\[\[qv: 154a\]\] Copyright 2014, John Wiley and Sons. c--f) Reproduced with permission.\[\[qv: 155b,156b\]\] Copyright 2016, John Wiley and Sons.](ADVS-6-1801797-g017){#advs1009-fig-0017} 4.4. Smart Supercapacitors {#advs1009-sec-0270} -------------------------- ### 4.4.1. Multipurpose Supercapacitors {#advs1009-sec-0280} Previous sections discussed supercapacitors as power devices for high energy density storage, microelectronics, and applications requiring flexibility. For electronic and portable devices, the integration area and volume of different electronic components are the key for miniaturization; thus, electronic components, displaying multiple functionalities or different properties, can advance miniaturized devices. Currently, multipurpose electronic components such as light‐emitting transistors, light‐emitting photodetector diodes, and electrical--optical transistors are widely used. Therefore, the development of multipurpose supercapacitors can further advance the next generations of miniaturized devices. Note that this section discusses supercapacitors displaying multipurpose functionalities; the integration of supercapacitors with other devices is also under development to achieve multipurpose systems, but this topic is out of scope of this section.[159](#advs1009-bib-0159){ref-type="ref"} Supercapacitors displaying properties such as optical transparent, electrochromic, sensing, lighting, and energy harvesting have been reported. Among those, transparent and electrochromic supercapacitors are currently successful multipurpose devices. Transparent supercapacitors are being developed via two main routes, including i) assembling on transparent supports[160](#advs1009-bib-0160){ref-type="ref"} and ii) as paper‐like freestanding supercapacitors.[161](#advs1009-bib-0161){ref-type="ref"} For example, transparent supercapacitors based on symmetric MnO~2~--Au nanonetworks on a separator were reported.\[\[qv: 161a\]\] The MnO~2~--Au nanonetwork architecture was prepared by evaporation of Au on a cracked separator followed by electrodeposition of MnO~2~ and enabled transparency with transmittance of 72% (λ = 550 nm) and charge storage capacity of 3 mF cm^−2^ at 5 µA cm^−2^ (**Figure** [**18**](#advs1009-fig-0018){ref-type="fig"}a,b). Symmetric paper‐like self‐standing transparent supercapacitors were assembled using transparent α‐MoO~3~ papers, which were prepared by vacuum filtration of ultralong α‐MoO~3~ (200 µm in length) and displayed transmittance of ≈90% in the visible region.\[\[qv: 161b\]\] This supercapacitor showed capacitance values of 257.6 F g^−1^ at 5 mV s^−1^ and 94.6 F g^−1^ at 200 mV s^−1^ and 96.5% capacitance retention after 20 000 cycles. ![Multipurpose supercapacitors. a,b) FEG‐SEM image and transmittance spectra of MnO~2~--Au network (black curve: Au network electrode; red curve: MnO~2~--Au network electrode; blue curve: assembled symmetric MnO~2~--Au network supercapacitor); inset: photo of MnO~2~--Au network on a separator. c,d) Transmittance spectra of symmetric WO~3~ supercapacitor upon assembling and charging and photos at bleached and colored states. e,f) Schematic illustration of the enhanced charge storage upon illuminating with solar light and cyclic voltammograms at 40 mV s^−1^ under dark and illumination. g) Schematic representations of the self‐charging supercapacitor and h) the charge storage processes and i) self‐charging plot under piezoelectric field on compressing. a,b) Reproduced with permission.\[\[qv: 161a\]\] Copyright 2017, John Wiley and Sons. c,d) Reproduced with permission.[165](#advs1009-bib-0165){ref-type="ref"} Copyright 2014, John Wiley and Sons. e,f) Reproduced with permission.\[\[qv: 169a\]\] Copyright 2016, John Wiley and Sons. g--i) Reproduced with permission.[170](#advs1009-bib-0170){ref-type="ref"} Copyright 2015, American Chemical Society.](ADVS-6-1801797-g018){#advs1009-fig-0018} Some redox‐active materials display color changes during charge and discharge processes, which is also known as electrochromic property. This property enables supercapacitor devices operating as both power supply devices and electrochromic devices. Moreover, color changes due to electrochromic effect depend upon charge states of supercapacitors, being a smart energy level indicator of supercapacitors.[162](#advs1009-bib-0162){ref-type="ref"} The bifunctional devices allow efficient use of energy, in which the devices can store charge during the electrochromic color changes and can function as power source during color bleaching processes. These advantages lead to the growing research in electrochromic supercapacitors. Several metal oxides/hydroxides and their composites have been recently reported for aqueous electrochromic supercapacitors such as NiO,[163](#advs1009-bib-0163){ref-type="ref"} W--Mo oxides,[164](#advs1009-bib-0164){ref-type="ref"} WO~3~,[165](#advs1009-bib-0165){ref-type="ref"} Ni(OH)~2~--MoO~3~,[166](#advs1009-bib-0166){ref-type="ref"} and Co~1−~ *~x~*Ni*~x~*(OH)~2~--rGO.[167](#advs1009-bib-0167){ref-type="ref"} For example, symmetric electrochromic supercapacitors consisting of thin‐film electrodes of WO~3~ nanoparticles delivered areal capacitance of 12.8 mF cm^−2^ at 0.4 mA cm^−2^ and showed optical transmittance of 78.8% in a bleached state and 15.1% in a colored state at a wavelength of 633 nm (Figure [18](#advs1009-fig-0018){ref-type="fig"}c,d).[165](#advs1009-bib-0165){ref-type="ref"} Asymmetric carbon nanotube\|\|Ni(OH)~2~--MoO~3~ electrochromic supercapacitors showed optical transmittance of 60% in the bleached state and 16% in the colored state at a wavelength of 500 nm.[166](#advs1009-bib-0166){ref-type="ref"} The charge storage and electrochromic processes were assigned to proton deintercalation/intercalation in layered WO~3~ or Ni(OH)~2~. Photoelectrochemical cells are among possible ways to convert solar energy into electrical energy. The presence of a space charge region upon photoexcitation immediately results in reduction reactions and oxidation reactions with generated electrons and holes, respectively. Thus, storing charge via photoelectrochemical processes has been a difficult task. Nevertheless, under certain conditions, electron--hole pairs can be separated and electrons could be stored, opening a concept of solar energy harvesting supercapacitors. For example, pseudocapacitive materials such as layered WO~3~ and MoO~3~ showed photocharging and storage ability, where the photoexcited electrons were stored via intercalation reactions with cations and photoexcited holes oxidize absorbed water to generate protons, thus balancing the total charge and suppressing electron--hole pair recombination to allow charge storing.[168](#advs1009-bib-0168){ref-type="ref"} In another approach, photoexcited electrons can be trapped by electrolyte cations and stored together with typical charge storage processes of supercapacitor electrodes, thanks to the applied voltage bias when charging supercapacitors.[169](#advs1009-bib-0169){ref-type="ref"} For example, symmetric Co(OH)~2~ supercapacitors showed enhanced areal capacity under blue light illumination, achieving capacity of 4.7 mF cm^−2^ at 0.1 mA cm^−2^, which is 2.4 times higher than a capacity obtained under dark conditions.\[\[qv: 169b\]\] Hexagonal WO~3~ showed ≈17% enhancement of charge storage capacity under solar light illumination compared to the value obtained under dark because its bandgapenergy is close to visible light energy (Figure [18](#advs1009-fig-0018){ref-type="fig"}e,f).\[\[qv: 169a\]\] The capacity of WO~3~‐based supercapacitors varied under light illumination with different wavelength and intensity, which corresponds to changes of discharge time and voltage drop. Thus, this response difference between dark and illumination environments was employed to detect light, being an indicator for the bifunctional photodetecting supercapacitors. By using piezoelectric materials as separator, when assembling supercapacitors, self‐chargeable supercapacitors have been proposed.[170](#advs1009-bib-0170){ref-type="ref"} The self‐charging supercapacitor (this concept represents supercapacitors that can be charged without using external power sources) was designed in symmetric configuration, using MnO~2~ nanowire electrodes and a piezoelectric polyvinylidene difluoride--ZnO separator, which were immersed in PVA--H~3~PO~4~ gel electrolyte (Figure [18](#advs1009-fig-0018){ref-type="fig"}g). Under vertical compression to the large dimension size of the supercapacitor, the piezoelectric separator generated a voltage causing positive and negative polarizations on opposite sides. This polarization drove electrolyte ions to positive and negative electrodes and created in‐equilibrium electrode--electrolyte interface, which led to proton intercalation/deintercalation redox reactions at negative/positive MnO~2~ electrodes (Figure [18](#advs1009-fig-0018){ref-type="fig"}h). These processes autonomously charged the supercapacitor, without need of external power sources. Under compression for 300 s (hand pressing), the voltage of the supercapacitor increased from 35 to 145 mV (Figure [18](#advs1009-fig-0018){ref-type="fig"}i). Its self‐charging capability was enhanced by increasing compression force, thanks to an enhanced piezoelectric potential polarization. Moreover, it also showed stable capacity under self‐charging and discharging at 10 µA. ### 4.4.2. Restorable and Degradable Supercapacitors {#advs1009-sec-0290} On practical use, supercapacitor devices can undergo different intentional and accidental deformations. Flexible supercapacitors using polymer substrates may fail due to irreversible plastic deformation. Thus, devices with restorable capabilities, i.e., able to recover their initial shape upon mechanical deformation, under external triggers are highly desirable. This sound concept has recently been achieved by employing shape‐memory alloys as current collectors.[171](#advs1009-bib-0171){ref-type="ref"} These alloys provide two important properties for restorable supercapacitors: i) the shape‐memory effect and ii) superelasticity, which can be restored to their initial shape under heating, thanks to the temperature‐induced reversible martensite--austenite phase change. Pioneering work demonstrated the use of NiTi shape‐memory alloy wires as current collectors, which were coated with pseudocapacitive layers consisting of MnO~2~ in the inner layers and polypyrrole in the outer layers (**Figure** [**19**](#advs1009-fig-0019){ref-type="fig"}b), and assembled in twisted symmetric wire‐shape supercapacitors.[171](#advs1009-bib-0171){ref-type="ref"} This device showed stable performance under bending, twisting, knotting, release, and repeated bending tests. Interestingly, by heating the supercapacitor to temperatures above the austenite phase transformation, the deformed supercapacitor was restored to its initial shape, without significant capacity loss, even under repeating deformation--restoration tests (Figure [19](#advs1009-fig-0019){ref-type="fig"}c). Knitting the shape‐memory supercapacitors into textile resulted in a restorable textile (Figure [19](#advs1009-fig-0019){ref-type="fig"}a). The heat‐induced restoring temperature was about 35 °C. This value is close to human body temperature; thus, the restorable textile can be used in smart clothes for autonomous heat dissipation (by shape changes) when the body temperature increases. Using a similar concept and making use of shape‐memory alloys, a smart watchband supercapacitor with automatic wrap ability in contact with skin has been reported (Figure [19](#advs1009-fig-0019){ref-type="fig"}d).[172](#advs1009-bib-0172){ref-type="ref"} The asymmetric supercapacitor consisted of reduced graphene oxide on a shape‐memory TiNi foil as negative electrode and MnO~2~ onto a Ni film as positive electrode, showing stable performance under static bending at different angles and dynamic shape‐memory restoration bending (Figure [19](#advs1009-fig-0019){ref-type="fig"}e). The shape‐memory alloy showed a phase transformation temperature at 15 °C, leading to a preformed round‐shape watchband supercapacitor, which under bending states at low temperature (0 °C) could be automatically restored to its initial shape and wrapped to our wrist upon touching (Figure [19](#advs1009-fig-0019){ref-type="fig"}d). {ref-type="ref"} Copyright 2015, Royal Society of Chemistry. d,e) Reproduced with permission.[172](#advs1009-bib-0172){ref-type="ref"} Copyright 2016, John Wiley and Sons. f,g) Reproduced with permission.[178](#advs1009-bib-0178){ref-type="ref"} Copyright 2017, John Wiley and Sons.](ADVS-6-1801797-g019){#advs1009-fig-0019} Deformations accumulated on supercapacitors during operation can generate mechanical failures, stopping their operation. In these cases, the shape restoration is not appropriate to repair failures. Thus, the capability to heal mechanical failures induced by the deformations would provide another innovative way to recover supercapacitor functionality. Thanks to healable materials, a self‐healing symmetric supercapacitor has been achieved by using healable materials as substrates to disperse carbon nanotubes working as double‐layer charging materials and current collectors.[173](#advs1009-bib-0173){ref-type="ref"} Under mechanical breaking, the supercapacitor could be healed by joining the cut sections under small pressure; the healable substrate could drag the displaced carbon nanotube layer together, thereby recovering conductivity and charge storage capability, with ≈82% of the initial capacitance restored after five cutting--healing cycles. Wire‐shape healable supercapacitors,[174](#advs1009-bib-0174){ref-type="ref"} using magnetic nanoparticles as internal trigger to join healable materials, were recently reported.[175](#advs1009-bib-0175){ref-type="ref"} Although metal oxide and hydroxide--based healable supercapacitors have not been reported yet, to the best of authors\' knowledge, from the above conceptual work, the coating of these active materials on conducting percolation networks such as carbon materials or metals followed by their dispersion on or wrapping with healable layers would enable self‐healing supercapacitor forms with enhanced capacity. Recently, a detailed review on healable materials was published,[176](#advs1009-bib-0176){ref-type="ref"} which would provide more insights into healing materials and mechanisms for further studies in healable energy storage devices. Integration of supercapacitors with biocompatible electronics to power up bioresorbable implanted devices and development of eco/bioresorbable supercapacitors (transient power devices) are presently under consideration. Bioresorbable supercapacitors can slowly degrade and dissolve in physiological media after working for a specified period, eliminating concerns about toxicological issues and additional surgeries for removal. Recently, bioresorbable supercapacitors were assembled using different types of biomaterials such as charcoal electrodes, egg white binders, polyelectrolyte drink, cheese segregation layer, and seaweed separator, forming an eatable device.[177](#advs1009-bib-0177){ref-type="ref"} However, the concept of eatable devices does not meet the principal requirement of bioresorbable devices, that is, to degrade after stable operation for a certain period. More recently, a biodegradable planar micro‐supercapacitor was reported, being composed of transition metals (W, Fe, or Mo) as electroactive materials and current collectors and biodegradable NaCl--agarose biopoly‐mer gel electrolytes. These materials were assembled together with a biodegradable polymer poly(lactic‐*co*‐glycolic acid) film and encapsulated with biodegradable hydrophobic polyanhydride.[178](#advs1009-bib-0178){ref-type="ref"} Metal oxide layers formed by metal corrosion and oxidation during charging/discharging process provided redox charge storage with enhanced capacity. This flexible device delivered a capacitance of 0.33 mF cm^−2^ at 0.15 mA cm^−2^, which is comparable to other micro‐supercapacitors and displayed stable performance, facilitating integration in different systems. Metal electrodes (Mo and W) dissolved in phosphate‐buffered saline (PBS) at body temperature as evidenced in the dissolution rate (by thickness change) plot (Figure [19](#advs1009-fig-0019){ref-type="fig"}g). A Mo micro‐supercapacitor, without encapsulation, could operate in PBS at physiological temperature for 6 h, being completely dissolved after 9 days (only the biodegradable substrate was left because of its longer degradation time) (Figure [19](#advs1009-fig-0019){ref-type="fig"}f). Encapsulation with hydrophobic polyanhydride expanded the working time up to 2 weeks. Several smart supercapacitors tailored for different applications have been reported. Although the concepts are all very exciting, some of those are not of practical use yet. For example, the self‐charging supercapacitor can only charge from 35 to 145 mV, which is far from application. Thus, supercapacitor components---including electrode materials, electrolytes, and separators---should be investigated further to enable the development of smart device assemblies. An interesting research path is the development of biocompatible supercapacitors, active materials, current collectors, and electrolytes to power up implantable systems. This is still at the concept level, but opens new routes to design smart devices tailored to serve a novel array of applications. 5. Concluding Remarks {#advs1009-sec-0300} ===================== This review comprehensively highlighted and discussed the most important advances from the authors\' viewpoints in metal oxide and hydroxide--based aqueous supercapacitors over the recent years, spanning from fundamental charge storage mechanisms, electrode materials engineering and functionalization, to tailored supercapacitor devices for multipurpose applications. Significant achievements have been attained during that last few years, including deeper understanding of the mechanisms governing charge storage, different electrode engineering routes to enhance electrochemical performance, and the design of flexible and/or smart devices. Despite considerable progress, there are still many research gaps to be filled and this paves the way for new exciting research streams in the near future. Concerning fundamental charge storage mechanisms, often contradictory results have been reported, probably because studies are based on a limited number of electrochemical studies and ex situ characterization. Therefore, collaborative studies based on different in situ/operando techniques are necessary to achieve deeper comprehension on the charge storage mechanisms of different electrode materials. Although some advances in electrode engineering have been made, energy density values are still modest. Often the reported values are generally based on laboratory‐based studies, using electrodes with low mass loadings. Normally these electrodes are assembled with few milligrams of active material, while commercial carbon‐based supercapacitors are typically assembled with mass loadings of one order of magnitude above. It has been shown that metal valences during the charge--discharge process are not fully oxidized or reduced, or only a part of the active materials is participating in the charge--discharge processes. Further electrode materials engineering routes that explore the full potential of charge storage by valence changes of metal ions are absolutely crucial to fully explore the potential of metal oxides and hydroxides. Moreover, important advances are still required to design electrodes with optimal properties (surface area, phase composition, doping/vacancies, electron conductivity, and architecture) for enhancing charge storage performance and cycling ability. Smart supercapacitors, tailored for different applications, can exhibit remarkable capabilities such as restorability and degradability. Although some initial results have been reported, this strategy is still at early research, but certainly future studies on this stream will pave the way toward many innovative results and hi‐tech applications. In conclusion, presently, aqueous supercapacitors based on metal compounds are a very dynamic research stream, in which understanding charge storage mechanisms, optimized electrode engineering, and tailored device assemblies are the main challenges to develop high‐performance devices and to meet the specific needs of the application. Conflict of Interest {#advs1009-sec-0320} ==================== The authors declare no conflict of interest. The authors would like to acknowledge Fundação para a Ciência e Tecnologia for the funding nos. UID/QUI/00100/2013 and M‐ERA.NET/0004/2014. **Tuyen Nguyen** obtained a double Ph.D. degree in materials engineering from Instituto Superior Técnico, Universidade de Lisboa, and Institut Polytechnique de Grenoble, Université Grenoble Alpes, in 2015. His research focuses on aqueous electrochemical energy storage materials and devices. {#nlm-graphic-1} **Maria de Fátima Montemor** is a Full Professor at Instituto Superior Técnico, Universidade de Lisboa. Her scientific activity focuses on the development of new multifunctional coatings for protection of surfaces and for energy conversion and storage. The most relevant research lines include multifunctional smart coatings for surface protection and new metal compound redox‐active coatings for energy storage. {#nlm-graphic-3}

**Specifications Table**TableSubject areaBiologyMore specific subject areaEndocrinologyType of dataText files, graphsHow data was acquiredAmino acid sequences were aligned using MEGA 6.0. Ligand selectivity assays were done using the CRE/Luciferase reporter assay [@bib2]. Luminescence was measured using a Bio-Tek Synergy HT plate reader (Bio Tek, Winooski, VT, USA), and the data were analyzed and graphed using Kaleidagraph software (Synergy Software, Reading, PA, USA)Data formatRawExperimental factorsMelanocortin DNAs were cloned from stingray genomic DNA or brain mRNA. Cloned DNA were expressed in Chinese Hamster Ovary cellsExperimental featuresSequence alignment was done using MEGA 6.0. The ligand selectivity assays were done as described in reference [@bib3].Data source locationKitasato University, Sagamihara, Kanagawa, Japan. University of Denver, Denver, Colorado, USAData accessibilityData is within this article **Value of the data**•These data are valuable for researchers participated in endocrinology of primitive fish and evolution of melanocortin systems.•These could be used as probes to explore orthologs in other cartilaginous fish such as skates, sharks and chimaeras.•The data on ligand selectivity could be useful tools for structure--function relationship studies in endocrinology and pharmacology. 1. Data {#s0005} ======= Data provided in this article show amino acid sequence comparison of melanocortin receptors (MCRs) in vertebrates and ligand selectivity of stingray MC peptides on these receptors. The amino acids sequences of MC1R ([Fig. 1](#f0005){ref-type="fig"}), MC2R ([Fig. 2](#f0010){ref-type="fig"}), MC3R ([Fig. 3](#f0015){ref-type="fig"}), MC4R ([Fig. 4](#f0020){ref-type="fig"}), and MC5R ([Fig. 5](#f0025){ref-type="fig"}) of stingray (*Squalus acanthias*) which determined by us [@bib1] were compared to corresponding sequences from two species of other cartilaginous fishes (i.e., *Callorhinchus milii*, elephant shark and *S. acanthias*, dogfish), a teleost (*Carassius auratus*, goldfish), and a mammal (*Mus musculus*, mouse). Data are also provided for ligand selectivity include effects of stingray Des-acetyl-α-MSH, β-MSH, γ-MSH, δ-MSH, ACTH(1-24) and β-endorphin on MC1R, MC3R, MC4R, and MC5R ([Fig. 6](#f0030){ref-type="fig"}, [Fig. 8](#f0040){ref-type="fig"}, [Fig. 9](#f0045){ref-type="fig"}, [Fig. 10](#f0050){ref-type="fig"}) and those of stingray Des-acetyl-α-MSH, ACTH(1-24), human ACTH(1-24) and NDP-MSH on stingray MC2R ([Fig. 7](#f0035){ref-type="fig"}). 2. Experimental design, materials and methods {#s0010} ============================================= In order to align the amino acid sequences of the melanocortin receptors for the Japanese stingray, *D. akajei*, the dogfish, *S. acanthias,* the elephant shark, *C. milii*, the goldfish, *C. auratus*, and the mouse, *M. musculus*, it was essential to identify putative transmembrane domains in each receptor sequence. To this end, the program ''MEMSAT3'' (<http://bioinf.cs.ucl.ac.uk/psipred/>) was used. The amino acid sequences where then aligned using the program MEGA 6.0. To functionally express and determine the ligand selectivity of the stingray (sr) MC1R, srMC2R, srMC3R, srMC4R, and srMC5R paralogs, the nucleotide sequences for the *srmcrs* were separately synthesized with a V-5 epitope tag at the N-terminal of the receptor, and inserted into a pcDNA3.1 expression vector (GenScript; Picataway, NJ, USA). Each *srmcr* cDNA was separately transiently transfected into Chinese Hamster Ovary (CHO) cells. The CHO cells were grown at 37 °C in a humidified 5% CO~2~ incubator in DMEM/F12 with 5% fetal calf serum. Each sr cDNA was co-expressed with a CRE/Luciferase reporter plasmid [@bib2] using the Solution T Cell Line Nucleofector Kit (Amaxa Inc., Gaithersburg, MD, USA) and program U-23 [@bib4]. The transiently transfected cells were seeded on a 96-well plate at a density of 1×10^−5^ cells/well. After 48 h in culture, the transfected cells were stimulated with either synthetic srACTH(1-24), srDes-acetyl-α-MSH, srβ-MSH, srγ-MSH, srδ-MSH, srβ-endorphin or hACTH(1-24), or NDP-MSH at concentrations ranging from 10^−6^ M to 10^−12^ M, in serum-free CHO media for four hours at 37 °C. At the end of the incubation period, 100 µl of Bright-Glo luciferase assay reagent (Promega Inc., Madison, WI, USA) was added to each well, and incubated for 5 min at room temperature. Luminescence was measured with a Bio-Tek Synergy HT plate reader (Bio Tek, Winooski, VT, USA), and the dose response curves were analyzed by using Kaleidagraph software (Synergy Software, Reading, PA, USA). All experimental treatments were performed in triplicate. Appendix A. Supplementary material {#s0020} ================================== Supplementary material We thank Mr. Kosuke Arai, Kitasato University, and Dr. Yasuhisa Kobayashi, Dr. Naoaki Tsutsui and Mr. Kazuhiro Saito, Okayama University, for technical assistance. Partial support for this study was provided by the Long Research Fund (RMD) and National Science Foundation, U.S.A. Grant IOB 0516958 Supplement (RMD). Supplementary data associated with this article can be found in the online version at [doi:10.1016/j.dib.2016.04.050](http://dx.doi.org/10.1016/j.dib.2016.04.050){#ir0010}. {#f0005} {#f0010} {#f0015} {#f0020} {#f0025} {#f0030} {ref-type="fig"}. Results are expressed as mean±S.E.M.; *n*=3.](gr7){#f0035} {ref-type="fig"}. Results are expressed as mean±S.E.M.; *n*=3.](gr8){#f0040} {ref-type="fig"}. Results are expressed as mean±S.E.M.; *n*=3.](gr9){#f0045} {ref-type="fig"}. Results are expressed as mean±S.E.M.; *n*=3.](gr10){#f0050}

Introduction ============ Since its introduction in 1991,[@b1-opth-12-849] optical coherence tomography (OCT) has become the reference standard imaging device for evaluating retinal and optic nerve disorders.[@b2-opth-12-849]--[@b4-opth-12-849] It uses low-coherence interferometry to generate in vivo, cross-sectional images of ocular tissues. Over the last 2 decades, technology has evolved to produce the spectral domain OCT (SD-OCT) with rapid image acquisition rate and high-resolution axial images of the retina and optic nerve.[@b5-opth-12-849] Thus, SD-OCT is commonly used in the monitoring of glaucoma and diabetic macula edema.[@b6-opth-12-849]--[@b10-opth-12-849] Topcon 3D OCT-1 Maestro is a noncontact SD-OCT system introduced in 2013. The instrument operates a fully automated "alignment, focus and capture" procedure, and captures 50,000 axial scans per second. The device simultaneously captures the macula and optic nerve head areas, giving both a digital fundus image and a 12×9 mm automated segmentation OCT scan ("wide scan" setting). The device also has the option for separate 6×6 mm optic disc and macula scans. It produces a 20 μm lateral and 6 μm axial resolution (Topcon; <http://www.top-conmedical.com/products/3doct1maestro-literature.htm>). One of the goals of ophthalmic imaging is to assist clinicians to discriminate between normality and pathology. In order to provide quantitative metrics for clinicians to determine retinal pathologies, we conducted a multicenter prospective study within the USA ([ClincialTrial.gov](http://ClincialTrial.gov) Identifier: NCT02447120) to collect OCT data of healthy eyes for the estimations of normal variation limits for various retinal and optic disc measurements using the Topcon 3D OCT-1 Maestro. The reference limits were estimated using non-crossing quantile regressions and 1st, 5th, 50th, 95th, and 99th percentiles were reported. Methods ======= The study was conducted at 7 investigational sites in the USA and was performed in accordance with Good Clinical Practices (as described by the International Conference on Harmonisation), the Code of Federal Regulations, the ethical principles in the Declaration of Helsinki, Health Insurance Portability and Accountability Act regulations, and other applicable local regulations. The study was registered at the US National Institutes of Health ([ClinicalTrials.gov](http://ClinicalTrials.gov) Identifier: NCT02447120). Between May and October 2015, 504 participants were enrolled. Institutional Review Board approval was provided by IntegReview IRB (Austin, TX, USA) for the following sites: Illinois College of Optometry, Marshall B. Ketchum University, State University of New York College of Optometry, Western University, and Valley Eye Care Center Medical Associates. Local IRB was used for the University of Alabama School of Optometry and New York Harbor Health Care System sites. All subjects, at all sites, provided written informed consent before the performance of any study procedure. Subjects were eligible for the study if both eyes were free of eye disease, with an intraocular pressure of ≤21 mmHg bilaterally and a best-corrected visual acuity of 20/40 or better in each eye. All subjects underwent Humphrey Field Analyzer (HFA; Carl Zeiss Meditech, Inc., Dublin, CA, USA) testing using the Swedish Interactive Threshold Algorithm (SITA) 24-2 test strategy. Subjects were excluded if they exhibited a field defect or if they had unreliable test result, (defined as fixation losses \>20%, or false positives \>33%, or false negatives \>33%). Subjects were also excluded if they were unable to tolerate ophthalmic imaging; had media opacities that inhibited OCT imaging; showed the presence of any ocular pathology except for cataract; had narrow angles on gonioscopy; had a history of leukemia, dementia, or multiple sclerosis; or were concomitantly using hydroxychloroquine and chloroquine. All ocular examinations to determine participant eligibility were performed within 2 calendar months of the scan acquisitions, and where possible, all OCT scans were performed in a single session. One eye of each study participant was randomly selected and scanned with the 3D OCT-1 Maestro (firmware version 1.27). A minimum of 9 scans were obtained under 3 different settings: 3 wide scans (12×9 mm), 3 disc scans (6×6 mm), and 3 macula scans (6×6 mm). Additional scans were taken at the operator's discretion if image quality was unacceptable. All study scans that were deemed acceptable by the clinical site operator were saved. Clinical sites were also allowed to make manual adjustments to correct automatic segmentation errors such as grid locations, boundary segmentations, and/or disc margin with Fastmap (version 8.27). Such modifications were performed only at the clinical sites by qualified investigators within the purview of typical clinical use. All manual adjustments were electronically documented. The fovea modification corresponded to relocation of the Early Treatment Diabetic Retinopathy Study (ETDRS) and Macula 6 grid center location, while the disc margin and disc center modifications corresponded to adjustments of the disc outline and location, respectively. The remaining modifications involved segmentation adjustments between boundaries. The scan adjustment rates were between 1% and 8%. The scan adjustment rates with respect to macula measurements ranged between 1% and 3%, and the adjustment rates with respect to optic disc and circumpapillary measurements fell within the 6%--8% range. OCT data were then exported to Topcon Reading Center (Oakland, NJ, USA), where image quality was checked by individuals with experience in OCT imaging. Scan exclusion criteria included image quality score, presence of eye blinks, eye motion, clipping, local weak signal, and feature centration. All scan exclusions were documented. No manual adjustments were made in the Topcon Reading Center. Approximately 1,200 scans were captured per scan mode (all subjects combined). Scan exclusion rates were 3% for wide scans, 3% for disc scans, and 2% for macula scans. All scans deemed acceptable by Topcon Reading Center qualified for data analysis. Measurement endpoints --------------------- Measurement endpoints by scan patterns are listed below: Macular full retinal thickness (FRT, [Figure 1](#f1-opth-12-849){ref-type="fig"}): ETDRS grid was placed automatically at the foveal center and sectors measured as per ETDRS chart specifications:[@b11-opth-12-849] central fovea, superior, nasal, inferior, and temporal inner quadrants (para-foveal); superior, nasal, inferior, and temporal outer quadrants (peri-foveal), measured with 12×9 wide and 6×6 macula modes; FRT consisted the distance between the inner limiting membrane (ILM) and the outer segments/retinal pigment epithelium junction boundary ([Figure 1](#f1-opth-12-849){ref-type="fig"}), the 2 green boundaries. An average value over the entire sector was reported. Macular ganglion cell + inner plexiform layer thickness (GCL + IPL, [Figure 1](#f1-opth-12-849){ref-type="fig"}): The Macula 6 sector circle was placed automatically at the foveal center ([Figure 1](#f1-opth-12-849){ref-type="fig"}). The center 1 mm region was excluded from measurement. Superior, superior nasal, superior temporal, inferior, inferior nasal, inferior temporal, and average thickness were measured with 12×9 wide and 6×6 macula modes; GCL + IPL thickness included the distance labeled in [Figure 1](#f1-opth-12-849){ref-type="fig"} (regions between 2 green boundaries). An average value of the entire sector was reported. Macular ganglion cell complex (GCC, [Figure 1](#f1-opth-12-849){ref-type="fig"}): GCC measurement was performed with the same Macula 6 sector circle as GCL + IPL measurements. Superior, superior nasal, superior temporal, inferior, inferior nasal, inferior temporal, and average thickness were measured with 12×9 wide and 6×6 macula modes; GCC thickness consisted of the regions labeled in [Figure 1](#f1-opth-12-849){ref-type="fig"}, which consisted of regions between the ILM (blue) and the green boundary vitreal to inner nuclear layer. An average value of the entire sector was reported. Circumpapillary retinal nerve fiber layer (cpRNFL) thickness: 3.4 mm diameter temporal-superior-nasal-inferior-temporal (TSNIT) circle was placed automatically at the optic disc center ([Figure 1](#f1-opth-12-849){ref-type="fig"}). Average RNFL thickness over TSNIT circle; 4 quadrants and 12 clock-hour sectors around TSNIT circle; measured with 12×9 wide and 6×6 disc modes; RNFL thickness consisted of regions labeled in [Figure 1](#f1-opth-12-849){ref-type="fig"} (between 2 green boundaries). Average values of each sector (excluding the optic disc contour and cyan circle) were reported. Optic disc measurements: Optic disc contour was automatically detected ([Figure 1](#f1-opth-12-849){ref-type="fig"}, cyan circle). Disc area, cup area, rim area, cup/disc (C/D) vertical diameter, C/D area, cup volume and rim volume were measured with 12×9 wide and 6×6 disc modes. Optic disc measurements were based on optic disc contour; an example is shown in [Figure 1](#f1-opth-12-849){ref-type="fig"}. Data analysis ------------- For each scan mode, the first acceptable scan by chronological order from each subject and scan mode was included in the analysis. The 99 percentiles (the 1st to the 99th) of each parameter were estimated by a simultaneous estimation of 99 non-crossing quantile regressions.[@b12-opth-12-849] Age was the regression covariate used in reference limits estimation for macula FRT, GCL + IPL, and GCC, whereas age and disc area were used as the regression covariates for RNFL thickness and optic disc parameters.[@b13-opth-12-849]--[@b17-opth-12-849] Estimation of the non-crossing quantile regressions was performed using an R program provided by Bondell (available at <http://www4.stat.ncsu.edu/~bondell/Software/NoCross/NoCrossQuant.R>). The 1st, 5th, 50th (median), 95th, and 99th percentile results were reported. The sample size was based on the nonparametric 95% CI for the percentiles of the scan parameters without any covariate adjustments. The goal was to find a minimum sample size such that the nonparametric 95% CI for the 1st and the 5th percentiles did not overlap. For a sample size of 315 eyes, the nonparametric 95% CI values are (X~(1)~, X~(9)~) and (X~(10)~, X~(27)~) for the 1st and the 5th percentiles, respectively, where X~(k)~ is the kth smallest value in a sample of 315 measurements without ties.[@b18-opth-12-849] Therefore, at least 315 evaluable eyes were needed to achieve the sample size goal. A study eye was evaluable if the subject met all eligibility criteria and had at least 1 acceptable scan. The final sample size was increased by about 10% of the minimum number of evaluable eyes to 350 evaluable eyes to ensure that all scan parameters would have at least 315 eyes for analysis. Assuming 30% of the subjects would not meet eligibility criteria, the study planned to enroll 500 subjects. Results ======= Five hundred and four subjects were screened for the study. One hundred and two did not meet the eligibility criteria and were excluded from imaging. Reasons for screen failure included visual fields with an apparent defect or artifact per study criteria (n=73), unreliable HFA visual field (n=26), and other ocular pathology/presence of eye disease (n=33). Some subjects failed multiple eligibility criteria. Two subjects who met the eligibility criteria did not have acceptable scans and 1 subject with unknown eligibility status did not have any scans. Therefore, 399 eyes of the 504 enrolled subjects were included in the analysis. The mean (SD) age was 46.3 (16.3) years (ranged 18--88 years). The most prominent age group was 18--30 years (22%) followed by 51--60 years (20%). Fifty-seven percent (n=226) of the subjects were female and 18% (n=71) of the subjects were Hispanic or Latino. Fifty-nine percent of the subject were Caucasians, followed by Black/African American (20%), Asian (13%), Native American/Pacific Islander (2%), and American Indian/Alaskan Native (1%); 6% reported their race as "other". The study eyes had a mean sphere of −1.441 diopters (D)±2.537 (ranged −12.5D to 4.0D), a cylinder of 0.565D±0.691 (ranged 0--5.0D), and a manifest refractive spherical equivalent of −1.159D±2.418 (ranged −11.00 to 4.50D). Subjects had at least 3 acceptable scans per scan mode. Images were reviewed for acceptability prior to calculating results. The first accepted scan of each scan parameter in each scan mode from each eligible subject was included in the analysis. Scan rejection rates ranged from 2% to 5%. Specifically, the scan rejection rates were 2%--4% with respect to macula measurements and 4%--5% with respect to optic disc and circumpapillary measurements. Macular FRT ----------- Summary statistic of foveal FRT measured with 2 scan modes are shown in [Table 1](#t1-opth-12-849){ref-type="table"}. Four patients were excluded from this analysis, as all their macula scans were determined to be unacceptable; thus, 395 of 399 subjects were included. The 1st, 5th, 50th, 95th, and 99th percentiles of foveal FRT by age are shown in [Figure 2](#f2-opth-12-849){ref-type="fig"}. The median intercepts and coefficients were highly similar between 12×9 wide and 6×6 macula scan modes. Overall, there was a reduction in FRT with age in the outer ETDRS quadrants. GCL + IPL thickness ------------------- The 1st, 5th, 50th, 95th, and 99th percentiles of average macula GCL + IPL thickness are shown in [Figure 2](#f2-opth-12-849){ref-type="fig"}. The median intercepts and coefficients were highly similar between 12×9 wide and 6×6 macula scan modes, and overall, there was a reduction in GCL + IPL thickness with age. GCC thickness ------------- Summary statistics of average GCC thickness with 2 scan modes are shown in [Table 1](#t1-opth-12-849){ref-type="table"}. The 1st, 5th, 50th, 95th, and 99th percentiles of average macula GCC thickness are shown in [Figure 2](#f2-opth-12-849){ref-type="fig"}, bottom panels. The median intercepts and slopes were highly similar between 12×9 wide and 6×6 macula scan modes, and overall, there was a reduction in GCC thickness with age. cpRNFL thickness ---------------- Summary statistics of average cpRNFL thickness with 2 scan modes are shown in [Table 1](#t1-opth-12-849){ref-type="table"}. The disc areas ranged from 1.25 to 3.97 mm^2^ (2.26±0.41 mm^2^, mean ± SD) for 12×9 wide scan and 1.03--3.85 mm^2^ (2.10±0.41 mm^2^) for 6×6 disc scan. The median intercepts and slopes were highly similar between 12×9 wide and 6×6 disc scan modes. Age had little or no effect, whereas disc area showed large degree of influence on the cpRNFL thickness ([Figure 3A](#f3-opth-12-849){ref-type="fig"}). Reference limits of the circle profile around the perimeter of the 3.4 mm TNSIT circle is shown in [Figure 3B](#f3-opth-12-849){ref-type="fig"}. Optic disc parameters --------------------- Summary statistics of disc area, cup area, rim area, cup volume, and rim volume with both 12×9 wide and 6×6 disc scan modes are reported in [Table 1](#t1-opth-12-849){ref-type="table"}. Age and disc area were the covariates used in the quartile regression analyses. Overall, optic disc parameters increased with disc area, except rim volume, and the effect of aging was not significant. Discussion ========== The purpose of this paper was to report the normal reference limits for macula, optic nerve, and nerve fiber layer thickness measurements from Topcon 3D OCT-1 Maestro. The measurements included various parameters of full retinal thickness, RNFL thickness, ganglion cell layer plus the IPL layer thickness, ganglion cell complex, and the optic disc for healthy eyes. Across all parameters, the measurements obtained by the 12×9 wide, the 6×6 macula, and 6×6 disc scan modes were generally similar. FRT measurements were consistent with reported OCT measured thickness ranges for healthy eyes.[@b19-opth-12-849]--[@b23-opth-12-849] We found a negative association between peri-foveal (outer retinal) thickness with age, but there were no significant age associations with central foveal or inner ETDRS quadrants. There are conflicting reports in the literature regarding whether retinal thickness, as measured with digital imaging techniques, decreases with age. Some authors found no association between overall macula thickness and age.[@b19-opth-12-849]--[@b21-opth-12-849] However, like us, others have found a decreased FRT in both the inner and outer quadrants with age.[@b23-opth-12-849]--[@b26-opth-12-849] Histological studies suggest that retinal thickness reduces with age, but that the changes in the foveal region are much less than those in the outer retinal areas.[@b27-opth-12-849] As with others' reports, we recommend that due to differences in image acquisition and processing methods, imaging devices should not be interchanged when evaluating retinal thickness measurements.[@b21-opth-12-849]--[@b28-opth-12-849] Our reported reference values for ganglion cell and inner limiting plexiform layer thickness were thinner than those reported by others using the Cirrus SD-OCT, but similar to measurements found using swept source OCT.[@b29-opth-12-849]--[@b33-opth-12-849] Furthermore, we found a thicker GCC average measurement than previous reports.[@b34-opth-12-849]--[@b36-opth-12-849] Differences may be primarily due to size and location of grid sectors used by the different devices, and add further evidence that imaging devices should not be interchanged when monitoring ophthalmic disease in an individual. We found that both the GCL + IPL thickness and GCC thickness decreased with advancing age, which is consistent with some published data,[@b37-opth-12-849],[@b38-opth-12-849] but not with other published data.[@b39-opth-12-849],[@b40-opth-12-849] An age-related reduction in the ganglion cell layer has been noted in histological studies;[@b27-opth-12-849] thus, it might be expected that in vivo imaging of the area in a wide age range would show similar findings. In addition to age-related trends in loss of tissue, individual variation in the location of the major RNFL arcuates (since the GCC includes the nerve fiber layer, the ganglion cell layer, and the inner plexiform layer) is also likely factoring in our results. cpRNFL thickness parameters included average, superior quadrant, nasal quadrant, inferior quadrant, temporal quadrant, and 12 clock-hour sections around the TSNIT circle. Overall, the measurements were consistent with those in the literature,[@b41-opth-12-849]--[@b43-opth-12-849] although there is some variation in the values reported by different devices.[@b44-opth-12-849] Some sectors showed the trend of cpRNFL thickness decreasing with age, which can be explained by 2 competing factors: a neuronal tissue loss and a non-neuronal tissue gain that typically occurs to a smaller extent.[@b43-opth-12-849],[@b45-opth-12-849]--[@b47-opth-12-849] However, our data showed that disc area had a more profound effect on cpRNFL thickness. This is mainly due to the following reasons: The measurement region was a circle with a fixed diameter of 3.4 mm centering at the geographical center of the optic disc. As it has been proposed in previous studies, there could be greater amount of retinal neural fibers present with eyes having large optic disc, or it could be an artifact with fixed diameter scans.[@b2-opth-12-849],[@b48-opth-12-849],[@b49-opth-12-849]Individual variation in the location of blood vessels and RNFL arcuates could contribute to variations in RNFL layer segmentations. To encompasses individual variations, the 3D OCT-1 Maestro's cpRNFL reference variation limits were estimated based on the influence of both age and disc area. In summary, the measurements and trends observed for the various parameters were largely consistent with those in the literature. Thus, the measurements presented in this report can be considered as a reference database for the measurements of healthy eyes. This study collected measurements of healthy eyes for full retinal thickness, RNFL thickness, ganglion cell layer plus the IPL layer thickness, GCC, and the optic disc in addition to small and large super pixel grid and TSNIT circle profile measurements. The reference limits at the 1st, 5th, 95th, and 99th percentile points establish thresholds to provide for the quantitative comparison of the RNFL, optic nerve head, and the macula in the human retina to a database of known healthy eyes. Consistent with the device's proposed indications for use, this quantitative comparison can aid in the diagnosis, documentation, and management of ocular health and diseases in the adult population. Additional investigators and data collection sites included: Jeffrey Chou, New York Harbor Health Care System, Brooklyn, NY; George Comer, Marshall B Ketchum University, Southern California College of Optometry Fullerton, CA; Mitchell Dul, State University of New York College of Optometry, New York, NY; Michael Gagnon, Valley EyeCare Center Medical Associates, Pleasanton, CA; Michael D Twa, University of Alabama School of Optometry, Birmingham, AL. **Disclosure** MC, MF, and PGD received financial support from Topcon Medical Systems. WCH, DL, and CAR are employees of Topcon Medical Systems. EN is a consultant of Topcon for this work. The authors report no other conflicts of interest in this work. {#f1-opth-12-849} {#f2-opth-12-849} {#f3-opth-12-849} ###### Summary of selected scan parameters Parameter Scan size 12 ×9 mm Scan size 6× 6 mm ------------------------ -------------------- ------------------- ------------------ --------- ----- ------------------ ------------------ --------- Central fovea FRT (μm) 395 237.079 (20.899) 235.012--239.146 235.520 395 234.000 (20.657) 231.956--236.043 232.290 Average GCL + IPL (μm) 398 71.363 (5.924) 70.779--71.946 71.450 397 71.726 (5.880) 71.146--72.306 71.700 Average GCC (μm) 398 105.949 (8.533) 105.108--106.789 105.850 397 106.268 (8.602) 105.419--107.116 105.800 Average RNFL (μm) 398 104.720 (11.829) 103.555--105.886 105.225 398 104.036 (11.341) 102.918--105.153 103.990 C/D vertical 398 0.489 (0.200) 0.469--0.508 0.520 398 0.480 (0.190) 0.462--0.499 0.510 C/D area (mm^2^) 398 0.278 (0.179) 0.261--0.296 0.260 398 0.280 (0.169) 0.263--0.297 0.270 Disc area (mm^2^) 398 2.264 (0.409) 2.223--2.304 2.230 398 2.102 (0.414) 2.061--2.142 2.065 Cup area (mm^2^) 398 0.655 (0.485) 0.608--0.703 0.570 398 0.613 (0.432) 0.570--0.655 0.545 Rim area (mm^2^) 398 1.608 (0.415) 1.568--1.649 1.580 398 1.489 (0.383) 1.452--1.527 1.460 Cup volume (mm^3^) 398 0.127 (0.143) 0.112--0.141 0.080 398 0.125 (0.135) 0.112--0.139 0.080 Rim volume (mm^3^) 398 0.287 (0.153) 0.272--0.302 0.260 398 0.264 (0.140) 0.250--0.278 0.240 **Note:** 95% CI=95% CI for mean based on t-distribution. **Abbreviations:** C/D, cup/disc; CL, ganglion cell; FRT, full retinal thickness; GCC, ganglion cell complex; GCL, ganglion cell; IPL, inner plexiform layer; RNFL, retinal nerve fiber layer.

Data files are available from the University of New England cloud drive: <http://dx.doi.org/10.4226/95/590bbd3954504>. Introduction {#sec001} ============ As humans go through daily life, memory for the context or location in which objects or events were experienced can be important. For example, it can be important for us to remember where we last saw objects such as our car keys so we can retrieve them when necessary. Or, it can be important for us to remember the contexts in which we previously experienced people we meet so we can respond appropriately to them. This type of memory is commonly referred to as source memory, and is often contrasted with recognition of the object itself \[[@pone.0188727.ref001]\]. The objective of this study was to examine attentional correlates of source memory encoding and retrieval using eye tracking technology. Source memory tests are considered by some investigators to assess the same construct underlying tests of episodic memory (e.g. \[[@pone.0188727.ref002]\]). Episodic memory is often assessed using participant self-reports of the subjective experience of remembering as recollection versus familiarity. For example participants may be asked whether they can replay the prior episode in their minds or whether they know the information without episodic recall \[[@pone.0188727.ref003]--[@pone.0188727.ref005]\]. It is also possible to use reduced or simplified experimental procedures to test encoding and retrieval of information without a verbal report of subject experience \[[@pone.0188727.ref006]\]. These procedures are more suitable for studies where it is not feasible to obtain a verbal report, such as during functional imaging \[[@pone.0188727.ref007]\], during tests of episodic memory in young children \[[@pone.0188727.ref008]\], or during tests of episodic-like memory in animals \[[@pone.0188727.ref009]\]. Source memory tests are one class of reduced procedures, in which both recognition of a previously experienced object or event and identification of an aspect of the original context can be assessed \[[@pone.0188727.ref007], [@pone.0188727.ref010]--[@pone.0188727.ref013]\]. Source memory depends on binding and retention of all the features in the experienced context during an encoding episode. Then at test, memory of one or more contextual features is assessed along with recognition of a specific object that occurred during encoding. In one common source memory procedure \[[@pone.0188727.ref007], [@pone.0188727.ref010]--[@pone.0188727.ref013]\] participants are asked to attend to images of objects that are incidentally presented at different quadrants on a screen. Then in a later test phase, the images are presented at the screen center and participants are asked to respond whether the object is new or previously seen (item recognition), and if it was previously seen, to indicate the original screen location (source memory test). In other studies, subjects have been asked to indicate in what color font recognized words were originally presented \[[@pone.0188727.ref014]\], whether recognized sentences were originally presented in a male or female voice \[[@pone.0188727.ref015]\], or where remembered trivia items were originally learned \[[@pone.0188727.ref016]\]. In such studies, correct answers on the source memory component of the test could then be based on either recollection of the prior episode or familiarity with and recognition of the correct source possibility. Dual-process models have been proposed in which familiarity and recollection are main cognitive processes involved in remembering \[[@pone.0188727.ref001], [@pone.0188727.ref017]\]. According to these models, familiarity involves a fast and automatic process that allows for recognition of a previous experience without retrieval of contextual details from the encoding experience, while recollection is a slower process that involves retrieval of details about the surrounding context of the encoding episode. A wide range of experimental evidence supports models for a dual-process memory systems (for review, see \[[@pone.0188727.ref017]\]). Recollection has been considered as an essential component for source memory \[[@pone.0188727.ref018]\], but models of familiarity-based recognition can also account for source memory, and a variety of empirical evidence has suggested that familiarity contributes to source recognition in studies in which the test of source requires participants to recognise the correct source from a set of possibilities \[[@pone.0188727.ref001], [@pone.0188727.ref017]\]. This means that measures of source memory that do not rely on verbal reports of subjective recollection cannot be unambiguously interpreted as recall rather than recognition of, or familiarity with, the source possibilities. Models of the neural activity associated with memory encoding and retrieval have suggested that retrieval is associated with activation of the same patterns of neural activity that occurred as a result of sensory experience during the time of encoding. Several studies have supported this type of model by demonstrating that the neural systems that are active during encoding are also active during retrieval \[[@pone.0188727.ref019]--[@pone.0188727.ref021]\]. Interaction between the stored memory traces and environmental cues present during retrieval is thought to be key, in that the pattern of activity elicited by the environmental cues overlaps the stored memory trace and completion of activation of the stored pattern occurs \[[@pone.0188727.ref022]--[@pone.0188727.ref024]\]. Moreover, pattern separation must occur with accurate episodic memory retrieval in that multiple patterns of neural reactivation are possible during memory retrieval but one pattern resolves into a distinct representation \[[@pone.0188727.ref025], [@pone.0188727.ref026]\]. Incorrect pattern completion or pattern separation may thus lead to memory failure or to false memories. There has been recent discussion about whether source memory retrieval is a thresholded all-or-nothing process or whether it can be graded \[[@pone.0188727.ref027]--[@pone.0188727.ref029]\]. It is possible the process is both thresholded and graded. Recent experimental evidence suggests a threshold for retrieval, but also that, when successful, retrieval can have varying levels of precision or accuracy \[[@pone.0188727.ref030]\]. An increasing number of studies have demonstrated that eye movements can reveal aspects of memory for the spatial positions of elements within observed scenes even in the absence of a verbal report about memory for those elements. For example, Ryan et al. \[[@pone.0188727.ref031]\] presented participants with pictures containing scene elements with different relationships relative to each other. At a subsequent test, eye movements were monitored as participants viewed scenes that were either novel, repeated, or repeated with one of the elements transposed to a new location. The participants had more fixations to the critical regions of scenes when they contained transposed elements even when they reported being unaware that the picture had changed. Other studies have demonstrated that information about the locations of particular faces in arrays can be expressed through gaze directions \[[@pone.0188727.ref032], [@pone.0188727.ref033]\]. This gaze direction effect has been found to correlate with recall of the original location of faces as assessed through verbal responses, but in some cases can occur even when participants are unable to make correct verbal responses \[[@pone.0188727.ref034]\]. Here we studied the temporal dynamics of eye movements during a source memory procedure. One goal was to see whether attention to an object during encoding would correlate with later recognition of the object and of its source location. A second goal was to see whether eye movements at test reveal attention to correct source locations as subjects successfully retrieve a source memory, cannot retrieve a source memory, or retrieve a false source memory. Method {#sec002} ====== Participants {#sec003} ------------ Twenty four healthy participants volunteered to participate in the experiment. The participants were all undergraduate students at the University of New England, Australia. There were 11 female participants and 13 male participants whose ages ranged from 19--24 years (*M* = 20.33). The experiment was approved by the Human Research Ethics Committee of the University of New England. Materials {#sec004} --------- A total of 96 colored images of common objects were employed. The images were developed and described by Cansino \[[@pone.0188727.ref012]\]. From the pool of images, a set of 64 images were selected to be displayed during the encoding phase. During the retrieval phase, 32 images from the encoding phase were used again, along with 32 new images. The stimuli were presented on the screen of a laptop computer (1366 × 768 pixels; 60 Hz) using e-Prime software version 2.0. A Tobii Technology X2-30 eye tracker was positioned below the screen to track eye movements. The subjects were seated so that their eyes were 50--80 cm from the screen. Procedure {#sec005} --------- The experiment was conducted in a single session that consisted of an encoding phase, a retention interval, and a retrieval test phase. Eye movements were tracked during the encoding phase and the test phase. Before beginning the study, each subject read and signed an informed consent form and filled out a computerized demographic questionnaire. Participants started the experiment by calibrating their eyes to the eye-tracking software. Once the calibrations were satisfactory, the encoding phase began, which lasted for approximately 6 minutes. The participants were presented with an illustrative encoding slide and instructed to study the objects that appeared on screen for a later test. After the illustration slide was shown, a series of 32 additional slides was presented. Each encoding slide was divided into four quadrants by a red cross. In two of the quadrants randomly selected pairs of objects were presented. Participants were not instructed to remember image locations or image pairings. Unknown to them, one of the objects from each slide would be part of the retrieval test (the target) and the other would not be seen again (the distractor). Quadrants that contained stimuli were always diagonally opposed: either top left and bottom right, or top right and bottom left ([Fig 1](#pone.0188727.g001){ref-type="fig"}, top panel). These screen positions, as well as the position of the object that would be presented again later, were counterbalanced across the session and presented in individually randomized orders. Before each new slide was displayed, the participants were presented with a fixation cross for 1000 ms to center their focus. Each slide was presented for 3000 ms, after which the images and the quadrants disappeared and were replaced with the message "press space to continue". By pressing spacebar, the participant could then choose when they would see the next slide. On average, participants took 1439 ms to continue onto the next slide. {#pone.0188727.g001} Immediately after the encoding phase there was a ten minute retention interval. During this interval participants completed a distractor task that involved solving simple arithmetic problems. The participants were asked to select the correct count-down series by threes out of 3 options. They had to do this for 10 different numbers ranging from 17--32. For the test phase, participants were presented with test screens divided into four quadrants by a red cross. The same object was displayed in each quadrant ([Fig 1](#pone.0188727.g001){ref-type="fig"}, bottom panel). The subjects were first presented with a sequence of three illustrative test trials and advised that after viewing the objects in each trial they would be asked to either point with their hand to the quadrant that they had previously seen the image in, say "new" if they had not seen the image previously, or say "don't know where" if they had seen the image before but could not recall where its previous location was. For each test trial, a fixation cross in the center of the screen appeared for 1000 ms before each slide was presented to enable the participant to center their focus. The objects appeared for 3000 ms, then they were removed while the red dividing cross remained. At that point, the verbal responses from the subject were collected. These verbal reports were recorded by the experimenter. The subjects were presented with 64 test slides, 32 of which contained objects that were previously shown in the encoding phase and the other 32 contained new objects. The sequence of new images and previously presented images in the test screens was individually randomized. Coding of responses {#sec006} ------------------- The third author noted participants' response as they were being given and later coded these responses according to the following guidelines: - Incorrect item recognition---previously seen objects which were labelled as new by the participant. - Correct item recognition---previously seen objects which were not labelled as new by the participant. - Correct source location---previously seen objects which were recognized and for which the participant had pointed to the correct quadrant. - Incorrect source location---previously seen objects which were recognized but for which the participant had pointed to an incorrect quadrant. - Don't know where---previously seen objects which were recognized but of which the participant said they did not know the location. New objects could also be correctly identified as new or falsely considered to have been seen before. These were not analyzed further. Data analysis {#sec007} ------------- The eye tracker sampled gaze direction at 30 Hz, or about once every 33.33 ms. The data were then coded as gaze directed within the target, in one of the other filled quadrants, or as no gaze detection, which was assumed to reflect gaze outside the areas of interest. For data collected during encoding, the averaged amount of time (expressed as number of samples in which the participant's gaze was detected) looking at the target or distractor quadrant across the entire trial was calculated. For data collected during the test, the time looking at the target or one of the competitor quadrants was calculated for each 100 ms time bin. Two types of analyses were carried out. To see the effect that initial time spent looking at an object during encoding had on the likelihood that the object was later recognized and the source correctly indicated, the number of samples in which gaze was detected as falling on a given quadrant during a trial's encoding phase was modelled with linear mixed-effects logistic regression \[[@pone.0188727.ref035]\] using the lme4 package in R version 3.1.3 \[[@pone.0188727.ref036], [@pone.0188727.ref037]\], and lmerTest to calculate the resulting coefficients' degrees of freedom (Satterthwaite approximation) and associated p-values \[[@pone.0188727.ref038]\]. The models included the fixed factors (predictors) of quadrant content (target or distractor) and response accuracy (correct or incorrect item recognition, or source memory). A maximal random effect structure including random intercepts and slopes over subjects and items was always attempted first \[[@pone.0188727.ref039]\]. However, non-convergence issues in the case of the model analyzing source memory forced a simplification of the random effects which was implemented by eliminating the higher order factors \[[@pone.0188727.ref039]\]. In contrast with the encoding phase, where we were only interested in whether increased attention to the target object would result in increased memory accuracy, in the test phase it was important to consider eye movement patterns across time. The total number of samples in which gaze was detected in a quadrant could mask a different temporal pattern (e.g., the target may be preferentially fixated first and, once identified, participants could turn their attention to other quadrants). To this end, growth curve analyses (GCA; \[[@pone.0188727.ref040]\]) were carried out on the number of samples in which gaze was detected as falling on a given quadrant within 100 ms time bins starting from the time the test slide came into view. The analysis contrasted the number of samples in which gaze was detected as falling on at different quadrants during the test phase as a function of whether the quadrant had originally contained the target or was one of the competitor quadrants, and whether it was the quadrant selected by the participant for their verbal response (predictors). The overall time course of such gaze-detections was modelled with a first (linear), a second (quadratic), and a third (cubic) orthogonal polynomial; and always a maximal random effect structure consisting of random effects of time terms over subjects and over subject-by-quadrant content. In these analyses, a significant intercept for a given main effect or interaction indicates an overall difference of total gaze-detections associated with different quadrants due to that main effect or interaction---i.e., whether there were differences in the total number of gaze-detections for different quadrants regardless of how these gaze-detections were distributed across time. An effect of the linear term gives an indication of whether the number of gaze-detections rose or fell differently according to the main effect or interaction---that is, whether there were differences in the slopes associated with looks to different quadrants. Finally, an effect of the quadratic or cubic terms indicates differences in the shape of the curve across the time window suggesting more complex differences in the dynamics of eye movements associated with different quadrants as a result of the main effect or interaction. Results {#sec008} ======= Task performance {#sec009} ---------------- The percent of previously seen objects that were correctly recognized was 83.2 ± 2.6 (mean ± s.e.), leaving 16.8 ± 2.6 of previously seen objects not recognized. Of previously seen objects correctly recognized, 66.4 ± 2.8 locations were accurately indicated, 22.1 ± 3.0 locations were incorrectly indicated, and 11.4 ± 2.7 locations could not be remembered. The percent of new objects correctly identified as new was 86.3 ± 2.2. When participants indicated an incorrect source location, they were more likely to choose a quadrant adjacent to the correct quadrant than choose the opposite quadrant. In trials where there was an incorrect source location, the adjacent quadrants in the horizontal direction were chosen 33% of the time, adjacent quadrants in the vertical direction were chosen 43%, and the opposite quadrant was chosen 24% of the time. This result suggests that the mistaken quadrant choice was probably not based on an overall recollection of the configuration of objects displayed at the encoding session (i.e. in opposite quadrants). Eye movements during the encoding phase {#sec010} --------------------------------------- [Fig 2](#pone.0188727.g002){ref-type="fig"} shows the average number of samples in which gaze was detected as falling on the target and distractor quadrants (i.e., the quadrant containing the object that would be presented during the later test phase and the quadrant that would not) per trial per person during the complete 3 seconds of the encoding phase trials. The top panel shows number of such samples according to whether the object was later correctly recognized, and the bottom panel shows number of such samples according to whether the source location of the object was later correctly remembered. {#pone.0188727.g002} A comparison of number of samples for which gaze was detected within different quadrants per trial (*N* = 768) according to their content (target vs distractor) and whether the target object was later recognized did not result in statistically reliable differences between targets and distractors overall (*Quadrant content estimate* = -3.27, *SE* = 3.98, *p* = .418), or between trials in which the object was later correctly recognized vs when it was not recognized (*Item recognition estimate* = 0.91, *SE* = 2.39, *p* = .708). The interaction between these two factors was also not significant (*Interaction between quadrant content and item recognition estimate* = 0.97, *SE* = 4.23, *p* = .821). Analyzing number of samples for which gaze was detected within different quadrants as a function of quadrant content and whether the source had been correctly indicated or not (excluding trials where the object had not been correctly recognized, *N* = 639) again failed to show an effect of quadrant content (*Quadrant content estimate* = -4.02, *SE* = 2.79, *p* = .154), an effect of source memory (*Source memory estimate* = -0.71, *SE* = 1.58, *p* = .657), or an interaction between the two (*Interaction between quadrant content and source memory estimate* = 2.58, *SE* = 1.99, *p* = .194). Eye movements during the test phase {#sec011} ----------------------------------- Figs [3](#pone.0188727.g003){ref-type="fig"} to [6](#pone.0188727.g006){ref-type="fig"} show the number of samples in which gaze was detected as falling on the target and competitor quadrants within 100 ms time bins per trial per person in the test phase. Each figure shows the dynamics of the eye movements for different subsets of the trials depending on correct or incorrect verbal responses. There were no a priori predictions regarding the specific time course of eye movements. Therefore, we chose a time window of interest on the basis of the observable patterns for the default case: when participants correctly recognize an object and remember its source location. Visual inspection of eye movement patterns in these conditions ([Fig 3](#pone.0188727.g003){ref-type="fig"}) showed a window from 500 ms until the slide disappeared (3000 ms) in which participants preferentially looked at the quadrant containing the target which will be selected. In consequence, we focused our analyses on the interval from 500 ms to 3000 ms. {#pone.0188727.g003} {#pone.0188727.g004} {#pone.0188727.g005} {#pone.0188727.g006} For trials in which the object source had been correctly indicated (*N* = 431; [Fig 3](#pone.0188727.g003){ref-type="fig"}), the GCA showed the number of samples with gaze detections within different quadrants to be dependent on quadrant content (target vs. competitor) both with respect to total number of gaze detections, greater for target quadrants (*Intercept estimate* = 469.04, *SE* = 32.36, *p* \< .001), and with respect to the dynamics of eye movements across time (*Linear term estimate* = 487.57, *SE* = 87.26, *p* \< .001; *Quadratic term estimate* = -469.72, *SE* = 79.12, *p* \< .001; *Cubic term estimate* = 385.42, *SE* = 26.54, *p* \< .001). This indicates that the target quadrant that the participant intended to name in their response was looked at more often than the other quadrants on the slide. We also analyzed the eye movement patterns during the test phase for trials where one of the competitors had been wrongly selected (i.e. false memory trials; *N* = 139; [Fig 4](#pone.0188727.g004){ref-type="fig"}). The target was looked at more often than the competitor not selected (*Intercept estimate* = 60.01, *SE* = 19.05, *p* = .002) and less often than the competitor selected (*Intercept estimate* = -88.19, *SE* = 19.04, *p* \< .001), which itself was also looked at more often than the other competitors, the ones not selected (*Intercept estimate* = 148.20, *SE* = 19.04, *p* \< .001). As for the temporal dynamics of eye movements, the target was significantly different from the competitor not selected on the quadratic term (*Linear term estimate* = -1.36, *SE* = 75.44, *p* = .986; *Quadratic term estimate* = -131.88, *SE* = 59.50, *p* = .027; *Cubic term estimate* = 2.59, *SE* = 23.07, *p* = .910), and from the competitor selected in the cubic term and marginally on the quadratic term (*Linear term estimate* = -40.69, *SE* = 75.44, *p* = .590; *Quadratic term estimate* = -114.02, *SE* = 59.50, *p* = .055; *Cubic term estimate* = -78.53, *SE* = 23.07, *p* \< .001). The two types of competitors were significantly different from each other on the cubic term (*Linear term estimate* = 39.33, *SE* = 75.45, *p* = .602; *Quadratic term estimate* = -17.86, *SE* = 59.50, *p* = .764; *Cubic term estimate* = 81.13, *SE* = 23.07, *p* \< .001). We analyzed the eye movement patterns during the test phase for trials where source memory failed and the participant did not select any quadrant after having recognized the object (i.e. the participants responded with "I don't know" or similar when asked to indicate the source information; N = 69; see [Fig 5](#pone.0188727.g005){ref-type="fig"}). The GCA on those trials showed a significant difference on the linear term (*Linear term estimate* = 40.91, *SE* = 14.93, *p* = .006), which stems from a late tendency to predominantly fixate within the correct target quadrant over the incorrect competitor quadrants. This tendency appears starting at 2000 ms after slide onset. No other effects were significant (all *p*'s \> .127). Finally, we analyzed the time course of gaze detections within target and competitor quadrants for those trials in which the previously viewed object was not recognized and was incorrectly declared new (*N* = 129; [Fig 6](#pone.0188727.g006){ref-type="fig"}). In this case, no significant differences were found (all *p*'s \> .330). Discussion {#sec012} ========== Our objectives in this study were to determine whether attention to an object during encoding would correlate with later recognition of the object and retrieval of its source location, and to determine whether attention to the correct source location at test occurs even when verbal expression of that source location is incorrect or has failed. Consistent with prior studies \[[@pone.0188727.ref007], [@pone.0188727.ref010]--[@pone.0188727.ref013]\], our participants were able to recognize previously seen objects and indicate the screen location where the objects originally occurred. However, the subjects also made errors in failure to indicate the original source locations of objects, in indicating incorrect locations, or in failure to recognize previously seen objects. Regarding whether visual attention to objects during the encoding session is related to later recognition of those objects and later retrieval of source information about the objects, analysis of gaze direction revealed that response accuracy was not determined by how much an item was initially attended to during the encoding phase. There were no statistically reliable overall differences between how often targets and distractors were looked at during encoding, and no dependency was found of response accuracy on the amount of time spent looking at particular quadrants during encoding. Other studies have demonstrated an effect of attention on source encoding by dividing attention during the encoding session \[[@pone.0188727.ref041]\] or by using emotionally valenced items \[[@pone.0188727.ref014]\]. Also, the fact that source memory is sensitive to frontal cortical lesions may be evidence that attention during encoding is important for memory, as frontal cortical dysfunction impairs attention \[[@pone.0188727.ref015], [@pone.0188727.ref042]\]. One possible reason for the discrepancy in the literature is variation across studies in amount of time the participants are afforded to encode the items. In our study the subjects could scan the objects for three seconds, which may have been sufficient to encode both objects. Therefore, during encoding, subjects divided the time similarly between the two objects in each slide. Given all objects were similarly attended to, it is not surprising that attention was not found to be a critical factor in which objects were later remembered. Regarding the evidence that we collected from eye movements during the source memory test, it clearly shows attention to the original source location on those trials where the subjects provided incorrect explicit location responses or provided a response indicating they didn't remember the location. During trials in which the object was recognized but an incorrect source location was indicated, the most time was spent looking at the quadrant the subjects would incorrectly select. However, significantly more time was spent looking at the correct source location than at incorrect, non-selected locations. In fact, the distribution of looking across the different quadrants and the dynamics of the eye movements during the retrieval phase suggests competition between the correct location and a wrong alternative (see [Fig 4](#pone.0188727.g004){ref-type="fig"}). In these cases, the target quadrant may have attracted eye movements either because it was explicitly considered an alternative to the quadrant eventually chosen or because, despite a decision to report another quadrant, it was recognized as a possible source. We collected further evidence from eye movements which shows attention to the original source location on trials where the participants failed to explicitly indicate any source location. On trials in which the object was recognized but participants responded with "I don't remember" or similar when asked to point to the original location of the object, significantly more time was spent looking at the correct source location than at incorrect competitor locations starting 2000 ms after slide onset. These two results indicate that accurate source information can be expressed through gaze direction even when verbal expression of that information is incorrect or has failed. These findings related to eye movements during source memory retrieval are consistent with models suggesting that even though the verbal response associated with retrieval of source memory may be dichotomized as correct or incorrect, underlying processes of source memory are graded. It is possible our subjects were experiencing explicit and conscious partial source recollections and then screening them out in verbal reports or providing incorrect responses depending on their confidence (see \[[@pone.0188727.ref043],[@pone.0188727.ref044]\]). Eye gaze cannot then be assumed to reflect memory processes wholly outside awareness in those trials, but only memory processes (whether conscious or not) that the subject withholds from verbal responding. If participants were basing responses on as sense of familiarity with a particular source location then that also would be graded. Source memory has been defined as the retrieval of contextual details that were incidentally acquired during the prior observation of a remembered item, and is contrasted with item memory which reflects recognition of the item itself \[[@pone.0188727.ref016]\]. Item recognition and source memory have been widely thought of as separate processes that involve different brain systems \[[@pone.0188727.ref007], [@pone.0188727.ref011], [@pone.0188727.ref042]\] and degrade at different rates across ageing \[[@pone.0188727.ref010]\]. The neural processes underlying recognition of items has been proposed as continuous and underlying weak to strong familiarity, while neural processes underlying source memory have been thought to operate in a threshold manner in which subjects either recall or fail to recall a past event \[[@pone.0188727.ref027]\]. However, a growing body of behavioral evidence indicates that source memory retrieval can be revealed as a continuous process if participants are allowed to make responses along a graded scale of memory strength \[[@pone.0188727.ref045]--[@pone.0188727.ref048]\]. In a typical study \[[@pone.0188727.ref049]\], participants initially heard a male or a female voice saying words, and then during the test phase saw words printed. When the participants made a seven-point confidence rating from "very sure female" to "very sure male" as a source memory measure, the results fit a model that suggests source memory as a continuous process rather than a thresholded process. Other studies have used similar procedures to show that hippocampal BOLD activation likewise best fits a continuous rather than thresholded model for source memory \[[@pone.0188727.ref050]\]. Nevertheless, no consensus on a wholly continuous model for source memory retrieval has been reached. Studies with similar procedures have resulted in data consistent with a threshold model for source retrieval, but also showed that, when successful, it can have varying levels of precision or accuracy \[[@pone.0188727.ref030], [@pone.0188727.ref051]\]. Continuous models for source memory retrieval are also supported by studies that show the source judgments at test can be based on either familiarity or recall in certain conditions \[[@pone.0188727.ref001], [@pone.0188727.ref017]\]. In particular, familiarity, a graded process, can be used to support memory for the source of the cues when the test is a recognition test, as in this study when there can be recognition of the correct quadrant. Our data support graded or continuous models for source memory by showing that even when verbal report of object source has failed, source information can be revealed by study of gaze direction. It is not clear from the current data that the graded source memory retrieval processes revealed by our gaze direction data are outside the awareness of the subjects. It is entirely possible it is outside awareness, particularly considering recent data showing subjects are unable to recognize or report on their own eye movements \[[@pone.0188727.ref052]\]. However, there are also studies showing that people withhold responses about their explicit recollections depending on their confidence levels or other criteria \[[@pone.0188727.ref053]\]. Thus, eye gazes towards the correct target area when source responses are inaccurate or when participants say they have no location memory could be due to attention related to explicit partial or false source information retrieval as well as to source information processing that occurs outside conscious awareness. What is unambiguous about our data is the usefulness of eye movements in their capacity to show attentional processes during memory of source information. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: ‡ These authors are joint senior authors on this work.

Introduction {#s0001} ============ The positive effects of physical activity for individual and population health are well documented \[[@CIT0001]\]. Rheumatoid arthritis (RA) is an inflammatory auto-immune disease, and physical activity forms a crucial part of its management \[[@CIT0002]\]. However, the RA population performs less physical activity than recommended for a healthy lifestyle, and has difficulties in maintaining health-enhancing physical activity (HEPA) levels over time \[[@CIT0005]\]. People with RA experience higher levels of activity limitation, pain and fatigue, and lower levels of perceived health than the general population \[[@CIT0008]\], and these factors are associated with physical inactivity in the RA population \[[@CIT0005],[@CIT0011]\]. To be able to understand why some people regularly participate in activities adequate to improve physical fitness and health, and some do not, it is crucial to identify determinants relevant for an individual's decision to initiate, adopt and maintain physical activity \[[@CIT0012],[@CIT0013]\]. One psychological mediator of physical activity is self-efficacy \[[@CIT0014],[@CIT0015]\], defined as peoples' judgments of their capabilities to organize and apply courses of action that are required to produce given attainments \[[@CIT0016]\]. It operates to regulate human motivation, behavior and well-being. Furthermore, self-efficacy influences other determinants such as goals and aspirations \[[@CIT0017]\], and is associated with psychological determinants of physical activity such as fear-avoidance \[[@CIT0018]\], outcome expectations \[[@CIT0017],[@CIT0019],[@CIT0020]\] and anxiety/depressive states \[[@CIT0021]\]. Another important aspect of self-efficacy is that it is not general in nature but related to specific situations, and also temporary, task-related, and relatively easy to influence \[[@CIT0016]\]. One of several ways to measure self-efficacy is the Exercise Self-Efficacy Scale (ESES), a self-administered questionnaire developed in English by Dzewaltowski \[[@CIT0012]\], and adapted to Swedish (ESES-S) by Denison et al. \[[@CIT0022],[@CIT0023]\]. The ESES evaluates self-efficacy to perform exercise despite a number of common barriers. As the ESES was originally developed to assess self-efficacy in a general population \[[@CIT0012]\], one crucial issue is whether this scale suits the characteristics of different subpopulations \[[@CIT0024]\], such as people with RA. In addition, versions of the scale in different languages have to be assessed for measurement properties. Internal consistency and test--retest reliability have previously been reported with satisfactory results in other subpopulations for the ESES, in both the English \[[@CIT0012],[@CIT0025]\] and Swedish versions \[[@CIT0023]\]. However, to our knowledge the ESES-S has not previously been tested for reliability and validity in people with RA. Aim {#s0002} --- The aim of the present study was to investigate aspects of reliability and validity of the ESES-S in an RA population. Materials and methods {#s0003} ===================== Participants {#s0004} ------------ A convenience sample of 244 people with RA participating in the Physical Activity in RA (PARA) 2010 study was included \[[@CIT0026]\]. The inclusion criteria were: RA according to the American College of Rheumatology (ACR) criteria \[[@CIT0027]\], 18--75 years and independent in daily activities measured by Stanford Health Assessment Questionnaire Disability Index (HAQ) scores ≤2 \[[@CIT0026]\]. The participants were identified from six rheumatology clinics in Sweden. They did not reach the recommendations on physical activity for a healthy lifestyle, and did not have any other health condition that prevented HEPA. They were also speaking and understanding the Swedish language. Data collection {#s0005} --------------- All data were collected by questionnaires at two measurement occasions before a planned physical activity intervention. More information on the PARA 2010 study is described elsewhere \[[@CIT0026]\]. The first measurement was performed in the recruitment phase of the PARA 2010 study, and the second measurement was performed at baseline immediately before the start of the intervention. The time interval between the first and second measurements varied between four and six months, as the intervention started at different times at the participating study sites. Assessment methods {#s0006} ------------------ Exercise self-efficacy was measured with the self-administered ESES-S. The main question "How confident are you to exercise ..." is followed by six items describing common barriers for exercise: "in spite of your work schedule", "when physically fatigued", "when exercise is boring", "with minor injuries", "in spite of other time demands", "in spite of family responsibilities" \[[@CIT0012]\]. The original version of the ESES employs a 0--100 scale, but the Swedish version ranges from "not certain" (=1) to "very certain" (=10), thus retaining a similar scale structure as the original. A total score (6--60) for the six items is calculated. The modified Fear Avoidance Belief Questionnaire (mFABQ) focuses on peoples' beliefs about how physical activity affects their current pain, and is mainly based on fear theory and fear-avoidance cognitions \[[@CIT0028]\]. The mFABQ comprises four items scored on 0--6 scales, where 0 signifies absence of beliefs in relation between pain and physical activity, and 6 signifies a strong belief. A total score (0--24) is calculated. Both HEPA and exercise were measured using the International Physical Activity Questionnaire (IPAQ) short version \[[@CIT0029],[@CIT0030]\]. HEPA was assessed with an aggregated dichotomized score indicating whether HEPA levels were reached or not, and exercise measured with the total minutes per day of estimated vigorous physical activity during the previous 7 days. Outcome expectations for physical activity were measured with two questions: "How certain are you that HEPA is beneficial for your health in the long run?" and "How certain are you that HEPA has a positive impact on your RA-related difficulties?". The questions are measured on scales 1--10 where 1 signifies "not at all sure" and 10 "totally sure" \[[@CIT0026]\]. General health perception, pain and fatigue were rated on 0--100 mm visual analogue scale (VAS) where 0 signifies the best condition and 100 the worst. Activity limitation was assessed with the HAQ \[[@CIT0031]\] employing a 0--3 scale, where 0 signifies performing tasks without any problems and 3 that it is impossible to perform tasks. The HAQ has eight items, and an average (0--3) for all items is calculated. Perception of a depressive state was measured by one item in the EQ-5D questionnaire \[[@CIT0032],[@CIT0033]\] using an 1--3 scale with 1 being indicative of not worried or depressed, and 3 of worried or depressed to a high degree. Data management and statistical procedure {#s0007} ----------------------------------------- Due to the interval between the two measurement occasions in the test--retest investigation, participants were excluded if perceived general health, pain or fatigue differed more than 20 mm on a 100 mm VAS, or if EQ-5D differed 1 step or more on a 1--3 scale. The exclusions were made to protect from bias in terms of health changes between measurement occasions that were likely to affect the ESES scores. The intraclass correlation coefficient (ICC) was used to assess test--retest reliability, comparing the results of the first and second measurement occasion. A two-way mixed calculation with absolute agreement, and variability presented as 95% confidence interval, was used. Agreement was classified as follows: ICC ≤ 0.4 = poor; 0.4 to ≤0.8 = moderate; and ≥0.8 = good \[[@CIT0034]\]. Internal consistency was calculated with Cronbach's alpha to measure the degree to which single ESES-S items measured a common construct, and to assess the correlation between each item and the sum of the other items (corrected item-total correlation). For measuring common construct, alpha coefficients between 0.7 and 0.8 would be considered as minimally acceptable, 0.8--0.9 as respectable and over 0.9 as very good \[[@CIT0035]\]. For the corrected item-total correlation alpha coefficients over 0.4 are considered good \[[@CIT0038]\]. In order to assess validity of the ESES-S in an RA population, we formulated a number of hypotheses based on previous research on self-efficacy. For construct convergent validity, it was thus hypothesized that the ESES-S should correlate positively with HEPA, exercise \[[@CIT0039],[@CIT0040]\] and outcome expectations on physical activity \[[@CIT0040]\]. ESES-S should also correlate negatively with pain, activity limitation \[[@CIT0041],[@CIT0042]\] and fear avoidance beliefs \[[@CIT0043]\]. For construct divergent validity, the ESES-S should have no correlation with age or gender. To calculate validity, the Pearson correlation coefficient (*r*) was used and categorized as follows: 0.0--0.2 is a very weak relationship, 0.2--0.4 is weak, 0.4--0.6 is moderate, 0.6--0.8 is strong and 0.8--1.0 is very strong \[[@CIT0044]\]. Point-biserial correlation is a special case of the Pearson correlation coefficient, and was used to calculate the relation between dichotomized and interval variables \[[@CIT0045]\]. Ceiling and floor effects were examined for the ESES-S single items, as the percentage of individuals selecting the highest and lowest scores respectively. For the ESES-S total score, the percentage of individuals assigned to any of the 10 highest and 10 lowest scores respectively were used. Over 15% responses on the highest or lowest scores were considered to represent ceiling or floor effects \[[@CIT0024]\]. All calculations, except that of test--retest reliability, were based on the data from the first measurement occasion. The Statistical Package for the Social Sciences (SPSS) version 22 (Armonk, NY) was used for the statistical analysis. Ethics {#s0008} ------ Ethical approvals were obtained from the Stockholm regional ethical review board (2010/1232-31/1, 2011/1241-32). The participants gave their informed written consent by filling out and returning their postal questionnaires. Results {#s0009} ======= Ninety-two participants had stable health conditions between the two measurement occasions, and were thus included in the test--retest sample. They were overall exhibiting similar characteristics to the total study sample, with some differences on perceived health, pain and fatigue ([Table 1](#t0001){ref-type="table"}). Table 1.Demographic data for the total study sample (*n* = 244) and the test--retest subsample (*n* = 92). Total study sampleMissing, *n*Test--retest subsampleMissing, *n*Age, years, mean (SD)59 (9.0) 59 (8.7) Females, *n* (%)198 (81.1) 74 (80.4) Income below average, *n* (%)75 (30.9)223 (25.0) Other diagnoses, *n* (%)141 (57.8)249 (53.3) Perceived health, 1--100, mean (SD)31 (33.8)122 (22.8) Pain, 1--100, mean (SD)29 (25.0) 19 (20.9) Fatigue, 1--100, mean (SD)36 (39.9) 26 (24.1) ESES total, 6--60, mean (SD)32 (12.3)1232 (12.3)2mFABQ total, 0--24, mean (SD)6.3 (4.9)15.3 (4.5)1HEPA obtained, *n* (%)188 (77.0) 74 (80.4) Outcome expectations on health, 1--10, mean (SD)9.4 (1.3) 9.6 (1.3) Outcome expectations on RA-related difficulties, 1--10, mean (SD)7.9 (2.4) 7.8 (2.5) HAQ total score, 0--3, mean (SD)0.54 (0.55)20.44 (0.52)1EQ-5D, 1--3, mean (SD)1.4 (0.5) 1.2 (0.4)  In the test--retest investigation, eight of the 92 individuals had missing data in the first or second measurement occasion, and thus calculations of test--retest reliability for the ESES-S total score was performed for the remaining 84 participants. The ICC was 0.59 (95% CI, 0.37--0.73) for the ESES total score, and 0.45 (CI 0.17--0.64, *n* = 86), 0.42 (CI 0.13--0.62, *n* = 89), 0.43 (CI 0.13--0.62, *n* = 88), 0.67 (CI 0.50--0.79, *n* = 89), 0.61 (CI 0.40--0.74, *n* = 87), 0.56 (CI 0.33--0.71, *n* = 87) for items 1--6 respectively. The results thus indicated "moderate" agreement for ESES-S between measurement occasions 1 and 2. Cronbach's alpha was 0.87 at the first measurement occasion and 0.89 at the second, indicating that the ESES-S had "respectable" inter-relatedness of items. Item-total correlations for the ESES-S single items are presented in [Table 2](#t0002){ref-type="table"}, indicating a "good" corrected item-total correlation. The ESES-S total score for the 84 participants had a mean of 32 (CI, 29--34) at the first measurement occasion, and 35 (CI, 32--37) at the second. Table 2.Corrected item-total correlations for the six items in ESES-S.How confident are you to exerciseCronbach's *α*1. In spite of your work schedule0.692. When physically fatigued0.733. When exercise is boring0.534. With minor injuries0.645. In spite of other time demands0.716. In spite of family responsibilities0.68 In preparation for the calculation of construct validity, an initial screening revealed skewed distributions of the two questions on outcome expectation ([Table 1](#t0001){ref-type="table"}). Subsequently they were dichotomized into "not certain" (ratings = 1--9), and "certain" (rating = 10). The results of the construct validity investigation are shown in [Table 3](#t0003){ref-type="table"}. Construct convergent validity was partly supported through significant, although weak, correlation between the ESES-S and HEPA, as well as outcome expectations regarding positive impact on RA-related difficulties. Divergent construct validity was supported by the absence of correlations with age and gender. Table 3.Correlations between the ESES-S total score and other study variables. Age^a^Gender^b^General pain (VAS)HAQ total scoreHEPA^a,b^ExerciseOutcome expectations on health^b^Outcome expectations on RA-related difficulties^a,b^mFABQ total scoreESES-S Pearson correlation0.05−0.06−0.07−0.080.18\*0.030.120.18\*−0.07 Sig. (2-tailed)0.460.360.310.250.010.620.080.010.29 *N*232232232230232232232232231[^1] No floor or ceiling effects for the ESES-S were found. Discussion {#s0010} ========== This study provides new knowledge on the measurement properties of the ESES-S in people with RA. The ESES-S demonstrated moderate test--retest reliability. Internal consistency was respectable, as was item-total correlation. Construct convergent and divergent validity were only partially supported by correlations in line with our pre-set hypotheses. Furthermore, the ESES-S showed no ceiling or floor effects. Test--retest reliability investigation provided an ICC of *r* = 0.59, which was lower than those previously reported by Yordy (*r* = 0.76) and Johansson (*r* = 0.64) for the original English and Swedish ESES respectively \[[@CIT0023],[@CIT0025]\]. Nevertheless, these results were all moderate according to the adapted ICC classification of agreement \[[@CIT0034]\]. Despite excluding people with major changes in health status from the test--retest analysis, the present results may have been influenced by the long time interval of four to six months between the two measurement occasions. A time interval of 2--14 days is a common recommendation \[[@CIT0046]\], even though there are no absolute limits for time intervals between measurement occasions \[[@CIT0047]\]. Hence, it should be prudent to expect changes in the individual and the environment over time \[[@CIT0046]\]. On the other hand, due to the dynamic nature of self-efficacy, changes may also occur over hours, days or weeks \[[@CIT0048]\]. One possible advantage of a long test--retest interval is that sensitization to the questions may be reduced \[[@CIT0046]\]. This may lead to less risk of remembering the questions and responses from the previous measurement occasion. Another possible bias is that, at the second measurement occasion, the participants had committed to participate in a physical activity intervention. Even though the results did not yield any significant differences in ESES-S total score means between the measurement occasions, such commitment may lead to changes in self-efficacy. These changes can occur through participants inquiring about more information on exercise, observing others exercising or performing exercise to a higher extent than if not committed to an intervention. Internal consistency was respectable with alpha coefficients of 0.87 and 0.89, indicating acceptable interrelatedness between the ESES-S items. This is in line with previous results for the ESES in other populations. Yordy tested the internal consistency of the ESES in a sample of college students, which yielded an alpha coefficient of 0.83 \[[@CIT0025]\] and Johansson found an alpha coefficient for the ESES-S of 0.85 in patients with low back pain \[[@CIT0023]\]. Construct convergent and divergent validity were only partially supported, which may question the validity of the ESES-S in the present sample. However, it may also indicate that our hypotheses, although founded in theory and previous research, could be incorrect or incomplete. For example, other variables, such as fatigue, are also known to correlate with self-efficacy \[[@CIT0049]\] and could have been used to test the ESES-S validity in the present study. Furthermore, previous research is inconclusive as to correlations between ESES and other variables. For instance, even though there is a substantial body of research indicating a correlation between self-efficacy and physical activity \[[@CIT0039]\], some studies report absence of such correlation \[[@CIT0050]\]. Interestingly, HEPA was found in the present study to have a weak, but statistically significant, positive correlation with the ESES-S, while planned and structured exercise was not. This could indicate that people with RA perceive the concept of exercise more in line with the broader concept of physical activity. It should, however, also be noted that the HEPA variable was dichotomized, which may result in loss of strength in the correlation \[[@CIT0051]\]. One reason for the hypothesized correlations of the present study not being supported, could be that self-reports may contain systematic bias. If all variables share a common data collection method, in this case self-reports, a potential contamination of the correlations between the variables may occur \[[@CIT0052]\]. Another possible bias is response distortion, such as response styles biased in some direction, for example overly positive responses. A third bias could be distorted response sets, where the respondent tries to make an impression, for example to respond in a socially desirable way \[[@CIT0046],[@CIT0053]\]. In spite of the limitations of self-reports, they are relevant to measure many constructs, and self-report instruments are common in social and behavioral sciences \[[@CIT0054]\]. However, since all variables in the present study were self-reports, it could be useful to complement them with other methods such as objective measures, for example physical activity monitoring with accelerometers, to reduce potential bias \[[@CIT0052]\]. The external validity extends to people with RA fulfilling the PARA 2010 criteria \[[@CIT0026]\], rather than to the RA population as a whole. Hence, the participants were not reaching HEPA at the inclusion of the study, and they had no diseases preventing them from reaching HEPA. Moreover, they had expressed interest in attending an intervention with organized exercise, which is likely to indicate a more positive attitude toward HEPA compared to the RA population as a whole. In conclusion, our results suggest that the ESES-S has moderate test--retest reliability and respectable internal consistency. Construct convergent and divergent validity were only partially supported. Further research on the concurrent construct validity, as well as on the predictive validity, of the ESES-S is recommended. Moreover, using complementing methods such as objective measures and adjusting the hypotheses for self-efficacy correlations by including other variables associated with self-efficacy, may be appropriate before using it in research and clinical environments. The authors would like to thank the participants in the PARA 2010 study for their contribution to the study. Declaration of interest {#s0011} ======================= Financial support was received from the Swedish Research Council, Combine Sweden, the Swedish Rheumatism Foundation, the Stig Thune Foundation, the Strategic Research Program in Health Care Research at Karolinska Institutet/Umeå University, Karolinska Institutet part time financing of doctoral students. The authors report no conflicts of interest. [^1]: ^a^Results in line with pre-set hypotheses. ^b^Point-biserial calculations were applied. \*Correlation is significant at the 0.01 level (2-tailed).

Pregnancy constitutes a major challenge to the maternal immune system, which must tolerate fetal alloantigen encoded by paternal genes.^[@bib1],\ [@bib2]^ The disturbance of maternal--fetal immune regulation is associated with several complications of human pregnancy, including spontaneous abortion (SA), intrauterine growth restriction (IUGR) and preeclampsia.^[@bib3],\ [@bib4],\ [@bib5]^ Accumulating evidence indicates that decidual macrophages (dM*φ*), the second largest decidual leukocyte population during the first trimester (\~20%) following decidual NK cells (dNKs, 50--70%), are involved in several processes required for a successful pregnancy, including trophoblast invasion, as well as tissue and vascular remodeling.^[@bib6],\ [@bib7]^ However, the mechanisms responsible for dM*φ* differentiation and polarization at the maternal--fetal interface remain largely unexplored. Of note, two distinct states of polarized activation of macrophages have been recognized: the classically activated (M1) macrophage phenotype and the alternatively activated (M2) macrophage phenotype.^[@bib8],\ [@bib9],\ [@bib10]^ Bacterial moieties such as LPS and TH1 cytokine interferon-*γ* (IFN-*γ*) polarize macrophages toward the M1 phenotype. These M1 macrophages are characterized by high interleukin (IL)-12 and IL-23 and low IL-10 production, and accordingly can kill intracellular microorganisms and induce Th1 immunity. In contrast, M2 polarization was originally discovered as a response to the Th2 cytokines IL-4 and IL-13, the anti-inflammatory cytokine IL-10, M-CSF, glucocorticoids and immune complexes. They generally share characteristics such as high IL-10 and low IL-12 and IL-23 production, anti-inflammatory and tissue remodeling properties, and scavenging of apoptotic cells and debris, and therefore have been considered to be important regulators of the immune response. The dM*φ* were classified as resembling an M2 phenotype.^[@bib11]^ However, there is still ambiguity with regard to the distinct functions of the dM*φ* subset.^[@bib4],\ [@bib6],\ [@bib12]^ Recent research has revealed that first-trimester dM*φ* can be divided into two distinct subsets, CD209^+^ and CD209^−^dM*φ*.^[@bib6],\ [@bib12]^ In comparison with CD209^−^dM*φ*, CD209^+^dM*φ* express high levels of the scavenger receptor CD163, the phagocytic receptors CD206 and CD304, and the CD209 ligand ICAM-3, and low levels of CD11c, which are associated with spiral arteriole remodeling.^[@bib6]^ Receptor activator of NF-*κ*B ligand (TNFSF11, also known as RANKL) and its tumor necrosis factor (TNF)-family receptor RANK are essential regulators of osteoclast differentiation and thereby fundamental aspects of bone physiology, bone remodeling,^[@bib13],\ [@bib14]^ lymph node formation,^[@bib15]^ establishment of thymic microenvironment,^[@bib16]^ mammary gland development during pregnancy^[@bib17],\ [@bib18]^ and bone metastasis of cancer.^[@bib19]^ Osteoprotegerin (OPG) is a decoy receptor for RANKL. By binding RANKL, OPG blocks the RANKL--RANK interaction. Osteoclasts are derived from monocyte/macrophage precursors.^[@bib13]^ However, the role of RANKL in inducing macrophage differentiation and functional regulation at the maternal--fetal interface is largely unknown. In this study, we investigated the effect of RANKL from human embryonic trophoblasts and maternal DSCs on dM*φ* differentiation and maternal--fetal immune tolerance, and we analyzed the relationship of RANKL production at the interface with miscarriage. Results ======= The crosstalk between fetus and mothers leads to high levels of RANKL/RANK expression at the maternal--fetal interface ---------------------------------------------------------------------------------------------------------------------- To investigate the role of RANKL/RANK signaling at the maternal--fetal interface, we first analyzed the expression of RANKL and found that both embryonic trophoblasts from villi and maternal DSCs from decidua are positive for RANKL in human first-trimester pregnancy ([Figures 1a and b](#fig1){ref-type="fig"}). As observed by immunohistochemistry, RANKL expression was located both in cell membrane and cytoplasm ([Figure 1a](#fig1){ref-type="fig"}). Similar results for RANKL expression levels were obtained by ELISA and flow cytometry (FCM) of the isolated trophoblasts and DSCs. In comparison with DSCs, trophoblasts secreted more soluble RANKL (sRANKL) and expressed higher level of membrane RANKL (mRANKL), and the co-culture of trophoblasts and DSCs produced higher levels of sRANKL ([Figure 1b](#fig1){ref-type="fig"}). To identify target cells of RANKL at the maternal--fetal interface, we analyzed the expression of RANK in decidual leukocyte cells (DLCs) primarily isolated from human decidual tissue. Among DLCs, the percentage of RANK^+^ cells in CD45^+^CD14^+^dM*φ*, as well as decidual NKT and CD3^+^T cells was \~80% ([Supplementary Figure 1a](#sup1){ref-type="supplementary-material"}), which was 4.45-fold higher than that of CD14^+^monocyte cells from peripheral blood (pMo) ([Figure 1c](#fig1){ref-type="fig"}). Interestingly, further phenotypic analysis showed that both RANK^+^pMo and RANK^+^dM*φ* expressed higher levels of M2-polarized macrophages markers (10) (the scavenger receptor CD163, the phagocytic receptors CD206 and CD209) and M1 co-stimulatory molecules CD80, CD86, HLA-DR, CD11c and cytokine IL-12p40, compared with RANK^−^cells ([Figure 1d](#fig1){ref-type="fig"} and [Supplementary Figure 1b](#sup1){ref-type="supplementary-material"}). These significant differences in M2 phenotype between RANK^+^pMo and RANK^−^pMo was maintained and even augmented in dM*φ*. Conversely, the M1 advantages possessed by RANK^+^pMo gradually weakened in RANK^+^dM*φ*. In comparison with RANK^−^dM*φ*, the expression pattern of RANK^+^ in dM*φ* suggests that RANK signaling may regulate the differentiation and function of dM*φ*. Embryonic extravillous trophoblasts (EVT) are in direct contact with maternal DSCs and DLCs, and the interaction between these cell subsets has an important role in maintaining maternal--fetal tolerance, which is required for a successful pregnancy.^[@bib3]^ To investigate this intercellular crosstalk, we co-cultured primary human trophoblasts, DSCs and dM*φ* (T+D+M). FCM analysis revealed markedly increased expression of RANKL in trophoblasts and DSCs and RANK on dM*φ* in the T+D+M co-culture ([Figures 1e and f](#fig1){ref-type="fig"}). The percentage of RANK^+^dM*φ* was reduced to 60% after culture alone for 48h. The presence of embryonic trophoblasts in the absence of DSCs led to a frequency of 80% RANK^+^dM*φ* ([Figures 1e and f](#fig1){ref-type="fig"}). Taken together, these data suggest that embryonic trophoblasts may have a crucial role in the regulation of maternal dM*φ* differentiation and function through the RANKL--RANK interaction. RANKL from trophoblasts and DSCs triggers M2 differentiation of dM*φ* and Th2 bias ---------------------------------------------------------------------------------- To investigate the potential effect of RANKL on dM*φ*, we directly co-cultured the purified CD14^+^dM*φ* with RANKL-overexpressed DSCs and JEG-3 cells (human placental choriocarcinoma cell line) (RANKL^+^D+J). Compared with control DSCs and JEG-3 cells (Ctrl-D+J), RANKL^+^D+J resulted in the elevation of CD206, CD209, CD163 and IL-10 and the decline of IFN-*γ*, IL-12/23p40, CD80 and CD86 in CD14^+^dM*φ* ([Figures 2a and b](#fig2){ref-type="fig"}). In contrast, blocking the RANKL--RANK interaction with a neutralizing antibody against RANKL (*α*-RANKL) or OPG protein could reverse the expression of CD80 and CD86 and the release of IL-10, IL-12p40 and IL-23 induced by RANKL of trophoblasts and DSCs ([Supplementary Figure 2](#sup1){ref-type="supplementary-material"}). RANKL participates in the regulation of monocyte migration by inducing the secretion of chemokine, such as CCL22 and CCL2.^[@bib20],\ [@bib21]^ However, current results show that RANKL was not involved in the regulation of the M2-recruited chemokines CCL17, CCL22 and CCL24 and the M1-recruited chemokines CXCL9 and CXCL10 of dM*φ* (data not shown). We have previously reported that RANKL stimulates DSCs to secrete the chemokine CCL2.^[@bib22]^ Our study suggests that RANKL may recruit peripheral monocyte to decidua, and further polarize them toward M2 dM*φ*. Evidence suggests that the RANKL--RANK interaction increases macrophage/dendritic cell (DC) survival and enhances the induction of T-cell response.^[@bib23],\ [@bib24],\ [@bib25]^ However, published evidence shows that RANKL-mediated modulation of DCs in mucosal tissues increases the number of CD4^+^CD25^+^ regulatory T (Treg) cells and promotes immunosuppressive activity toward foreign antigens, such as food or commensal bacteria in the intestines.^[@bib26],\ [@bib27],\ [@bib28]^ However, the molecular mechanism underlying RANKL on M*φ*s and DCs remains unclear. We further investigated the regulation of these RANKL-instructed dM*φ* on the differentiation of decidual naive T cells. After pre-culture with trophoblasts and DSCs, we collected dM*φ*, and then co-cultured them with decidual naive T cells for 5 days ([Figure 2c](#fig2){ref-type="fig"}). We observed that either *α*-RANKL or OPG abolished the stimulatory effect on the IL-10 and Th2 transcription factor GATA-3 and the inhibitory impact on tumor necrosis factor-*α* (TNF-*α*) and Th1 transcription factor T-bet in CD4^+^ T cells mediated by dM*φ* pre-treated with trophoblasts and DSCs (D+T-dM*φ*) ([Figures 2d and g](#fig2){ref-type="fig"}). In addition, blocking RANKL in the T+D+M co-culture further inhibited IL-4 secretion but stimulated IFN-*γ* production of CD4^+^T cells ([Figures 2d and g](#fig2){ref-type="fig"}). However, RANKL-instructed dM*φ* had no effect on decidual Treg cell differentiation (data not shown). As a potent inducer of decidual M2 M*φ*,^[@bib6]^ the increased IL-10 in dM*φ* and decidual CD4^+^T cells induced by RANKL may further amplify the impact of RANKL on M2 polarization of dM*φ*. These data provide strong evidence that RANKL is expressed at the maternal--fetal interface, and support the presence of a positive regulatory loop between trophoblasts and dM*φ* to induce maternal--fetal immune tolerance during pregnancy. The effect of RANKL on dM*φ* is dependent on the activation of the Akt/STAT6-Jmjd3/IRF4 signaling pathway --------------------------------------------------------------------------------------------------------- Of note, mRANKL or sRANKL cleaved by matrix metalloproteinases (MMPs) or a disintegrin and metalloproteases (ADAMs) binds RANK and then mainly activates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-*κ*B) pathway to control osteoclastogenesis through adaptor molecules such as TRAFs and Gab2,^[@bib29],\ [@bib30],\ [@bib31]^ thus regulating osteoimmunology by pro- and anti-inflammatory effects on the immune system.^[@bib32]^ To identify the downstream pathway through which RANKL drives M2 polarization during pregnancy, we assessed the impact of RANKL on different signaling pathways in dM*φ*. T+D+M led to changes in the phosphorylation of several signaling pathways such as Akt, NF-*κ*Bp65 and c-Jun N-terminal kinases (JNK) (data not shown), suggesting that several pathways should be involved in the functional regulation on dM*φ*. Notably, *α*-RANKL or OPG specifically inhibited the activation of Akt and STAT6 (a master regulator of M2 genes in mice downstream of IL-4R)^[@bib33],\ [@bib34],\ [@bib35]^ in dM*φ* (data not shown). Culture with RANKL^+^-D+J gave rise to the increased level of Akt and STAT6 phosphorylation in dM*φ* compared with the culture with Ctrl-D+J ([Figures 3a and b](#fig3){ref-type="fig"}). The Akt signal inhibitor (Akti) LY294002 partly reversed the level of STAT6 phosphorylation ([Figure 3c](#fig3){ref-type="fig"}). Compared with the control, the release of IL-10 and IL-12p40 and IL-23 by dM*φ* increased and decreased, respectively, under co-culture with RANKL^+^-D+J, and these effects could be clearly impaired by Akti or STAT6 inhibitor (STAT6i) AS1517499^[@bib35]^ treatment ([Figure 3d](#fig3){ref-type="fig"}). Interestingly, treatment with STAT6i resulted in M1 differentiation and Th1 bias in the mice uterus *in vivo* ([Figure 3e](#fig3){ref-type="fig"}). Therefore, these data suggest that RANKL induces M2 differentiation by activating the Akt/STAT6 signaling pathway. It has been reported that Jmjd3 regulates M2 polarization by inhibiting the transcription of typical M1-associated genes and inducing IRF4.^[@bib36],\ [@bib37],\ [@bib38]^ Unlike IRF5,^[@bib39]^ IRF4 has been recognized as an essential transcription factor for M2 polarization and the expression of M2 signature genes such as *Arg1, Ym1 and Fizz1* in mice.^[@bib40]^ We investigated the effects of RANKL on these transcription factors. Blocking RANKL resulted in a marked decrease in *Jmjd3* and *IRF4* but not *IRF5* transcription in the co-culture of D+T-dM*φ* ([Supplementary Figure 3](#sup1){ref-type="supplementary-material"}). Conversely, there was an increase in the transcription of *Jmjd3* and *IRF4* in RANKL^+^-D+J-dM*φ*, and both Akti and STAT6i treatment could partially abrogate these effects on *Jmjd3* and *IRF4* induced by RANKL *in vitro* ([Figure 3f](#fig3){ref-type="fig"}). Similarly, both Jmjd3 selective inhibitor (JMJD3i) GSK J4 HCl and STAT6i treatment could downregulate IRF4 expression in uM*φ* ([Figure 3g](#fig3){ref-type="fig"}). Furthermore, treatment with JMJD3i also led to M1 differentiation ([Figure 3h](#fig3){ref-type="fig"}) and a Th1 bias ([Figure 3i](#fig3){ref-type="fig"}) in mouse uterus. Taken together, these results place RANKL upstream in the Akt/STAT6-Jmjd3/IRF4 signaling cascade involved in M2 polarization of dM*φ*. Downregulation of RANKL expression leads to murine decidual M*φ* dysfunction and fetal loss ------------------------------------------------------------------------------------------- In comparison with non-pregnant mice, uterine M*φ* (uM*φ*) from pregnant mice had a high level of RANK ([Supplementary Figure 4](#sup1){ref-type="supplementary-material"}). To further explore the role of absent RANKL at the maternal--fetal interface during the differentiation of M*φ in vivo*, we first evaluated RANKL/RANK expression in the uterus in normal pregnancy and abortion-prone matings. In CBA/J × DBA/2 matings as an abortion-prone model, decreased RANK expression was detected in F4/80^+^uM*φ* on gestational days 5 and 9, compared with CBA/J × BALB/c matings as a mouse model of normal pregnancy ([Figure 4a](#fig4){ref-type="fig"}). Interestingly, uM*φ* in mice are divided into two subsets: F4/80^+^MHCII^hi^ and F4/80^+^MHCII^lo^. These two subsets differentially express *CD163* and *Mrc1*, which may represent mouse analogs of CD209^−^ and CD209^+^dM*φ*.^[@bib4],\ [@bib41]^ Here, a characteristic M1 rather than M2 phenotype was also observed in uM*φ* from CBA/J × DBA/2 abortion-prone matings ([Figure 4a](#fig4){ref-type="fig"}). Similarly, uM*φ* in RANKL knockout (RANKL^−/−^) pregnant mice had low levels of CD206, CD209 and IL-10, and high levels of CD80 and CD86 compared with wild-type (WT) pregnant mice ([Figure 4b](#fig4){ref-type="fig"}). In comparison with WT group, there was a Th1 bias in the uterus of RANKL^−/−^pregnant mice ([Figure 4c](#fig4){ref-type="fig"}). Furthermore, there were low levels of Akt and STAT6 phosphorylation, Jmjd3 and IRF4 in uM*φ* of RANKL^−/−^pregnant mice compared with WT group ([Figures 4d--f](#fig4){ref-type="fig"}). To investigate the influence of RANKL/RANK signaling on outcome of pregnancy *in vivo*, we evaluated embryo-absorbing level between RANKL^−/−^ and WT pregnant mice. The embryo-absorbing site could be macroscopically distinguished as hemorrhagic spots and necrosis at late gestation (gestational day 14, [Figure 4g](#fig4){ref-type="fig"}). The RANKL^−/−^mice were more susceptible to fetal loss than WT mice ([Figures 4g and h](#fig4){ref-type="fig"}). These findings provide evidences of a key role of RANKL in the regulation of dM*φ* differentiation and function, promoting maternal--fetal tolerance to support normal pregnancy. Abnormal suppression of RANKL expression contributes to uM*φ* dysfunction and fetal loss *in vivo*. Adoptive transfer of RANK^+^M*φ* relieves murine embryo absorption induced by M*φ* depletion -------------------------------------------------------------------------------------------- The depletion of M*φ* in pregnant mice using Clodronate Liposomes ([Supplementary Figures 5a and b](#sup1){ref-type="supplementary-material"}) led to a significant decrease in RANKL in uterine DSCs (uDSCs) and placental trophoblasts (pTros) ([Supplementary Figure 5c](#sup1){ref-type="supplementary-material"}), suggesting that dM*φ*s are involved in the maintenance of high levels of RANKL at the maternal--fetal interface. To provide insight into the role of RANKL-instructed dM*φ* in maternal--fetal immune regulation and pregnancy outcome *in vivo*, we evaluated the effect of dM*φ* depletion and adoptive transfer of RANK^+^M*φ* on these processes. Next, we isolated RANK^+^ and RANK^−^M*φ*s from mouse spleen and observed that these RANK^+^M*φ*s, like human RANK^+^pMo, had high levels of M1 and M2 phenotype molecules ([Supplementary Figure 6a](#sup1){ref-type="supplementary-material"}). To investigate the process of RANK^+^M*φ* differentiation in the uterus *in vivo*, we labeled these RANK^+^ and RANK^−^M*φ*s with PKH-67 and transferred them to M*φ*-deleted pregnant mice ([Figure 5a](#fig5){ref-type="fig"} and [Supplementary Figure 6b](#sup1){ref-type="supplementary-material"}). RANK^+^M*φ*s with high CCR2 were preferentially recruited to the uterus ([Supplementary Figure 6c](#sup1){ref-type="supplementary-material"}). In comparison with the PKH-67-RANK^−^M*φ* transferred group, PKH-67-RANK^+^M*φ* recruited to uterus presented high levels of CD206 and CD209, and low levels of CD86 and similar levels of CD80 ([Figure 5b](#fig5){ref-type="fig"}). In addition, the transfer of PKH-67-RANK^+^M*φ* led to a Th2 bias in mouse uterus ([Figure 5c](#fig5){ref-type="fig"}), an increase in Akt and STAT6 activation ([Figure 5d](#fig5){ref-type="fig"}), and a high level of IRF4 ([Figure 5e](#fig5){ref-type="fig"}) in uM*φ*. Subsequently, we observed that M*φ* depletion caused a significant increase in fetal loss ([Figures 5f and g](#fig5){ref-type="fig"}). To further identify the role of RANKL-instructed M*φ* in ameliorating fetal loss, we transferred RANK^+^ or RANK^−^M*φ* to M*φ*-deleted pregnant mice and found that adoptive transfer of RANK^+^M*φ* could significantly relieve the murine embryo absorption induced by M*φ* depletion ([Figures 5h and i](#fig5){ref-type="fig"}). The suppression of RANKL/RANK expression and dM*φ* dysfunction in human miscarriage ----------------------------------------------------------------------------------- The imbalance of maternal--fetal immunoregulation has been previously reported in pregnancy complications^[@bib3],\ [@bib4],\ [@bib5]^ such as miscarriage, preeclampsia and IUGR. Therefore, we evaluated RANKL/RANK expression in the tissue from patients with miscarriage during the first trimester. In comparison with normal pregnancy, we observed a decrease in RANKL expression in trophoblasts and DSCs ([Figure 6a](#fig6){ref-type="fig"}), as well as reduced RANK expression on CD14^+^dM*φ* ([Figures 6b and c](#fig6){ref-type="fig"}) in miscarriage, accompanied by a decreased frequency of dM*φ* with an M2 phenotype and an increase in the M1 phenotype ([Figures 6d and e](#fig6){ref-type="fig"}). Taken together, the suppression of RANKL/RANK signaling may result in dM*φ* dysfunction and further trigger miscarriage during the first trimester. Discussion ========== Collectively, we have demonstrated that RANKL derived from embryonic trophoblasts and maternal DSCs drives dM*φ* polarization toward an M2 phenotype by activating Akt/STAT6 signaling and enhancing the transcription of *IRF4* and *Jmjd3*; finally, it contributes to the formation and maintenance of maternal--fetal tolerance by further inducing Th2 bias ([Figure 7](#fig7){ref-type="fig"}). This maternal--fetal dialog mediated by RANKL guarantees a smooth gestation by creating a tolerant microenvironment. The process of maternal--fetal tolerance formation is not only derived from a maternal behavior for immune adaptation to pregnancy but also, most importantly, the fetus can instruct the mother's immune system to adapt to it. In comparison with the peripheral, the M2 phenotype advantage of RANK^+^dM*φ* becomes more prominent in the decidua. The levels of M1 phenotype markers in RANK^+^dM*φ* are also higher than those in RANK^−^dM*φ*, but still very low, similar to the level of RANK^−^ pMo. Similarly, it has been reported that both CD209^+^ and CD209^−^dM*φ* stimulate the release of proinflammatory cytokines such as IL-6 and TNF-*α* after LPS stimulation *in vitro*.^[@bib12]^ These data emphasize the complexity of dM*φ* biology. During normal pregnancy, the M2 advantage of dM*φ* at the maternal--fetal interface is relative and mainly depends on the local microenvironment. This advantage may be disrupted by intrauterine infection and lead to an M1 advantage to limit infection. The expression of proinflammatory molecules in dM*φ* may align more with the theory that immune activation is required to facilitate trophoblast invasion and implantation, as well as the establishment of fetal--maternal tolerance during the first trimester. The human maternal--fetal interface is characterized by intimate contact between the maternal decidua and extravillous cytotrophoblast cells that invade the decidua. Trophoblasts can influence the maternal immune system during pregnancy by expressing soluble and cell surface molecules, such as HLA-G,^[@bib42],\ [@bib43]^ IDO^[@bib44]^ and anti-inflammatory cytokines.^[@bib45],\ [@bib46],\ [@bib47]^ These molecules limit the proliferation and activation of T cells, antigen-presenting cells and NK cells in decidua. In our present study, we found that the crosstalk between embryonic trophoblasts and maternal DSCs and dM*φ* contributes to the accumulation of RANKL expression at the maternal--fetal interface. RANKL expressed by trophoblasts and DSCs induces M2 differentiation of dM*φ* and further drives the Th2 bias, suggesting that RANKL/RANK signaling has a critical role in dM*φ* differentiation and maternal--fetal tolerance. It is noteworthy that trophoblasts upregulate RANK expression specifically on dM*φ*. These findings further highlight the core role of trophoblasts in dM*φ* differentiation regulation. Rather than traditional NF-*κ*B signaling under the RANKL/RANK axis, we found that Akt/STAT6-Jmjd3/IRF4 signaling is required for M2 differentiation of dM*φ* induced by RANKL at the maternal--fetal interface *in vitro* and *in vivo*. Further studies should clarify the molecular mechanisms by which RANKL specifically activates Akt/STAT6 signaling in dM*φ*. In human pregnancy, embryo implantation in the receptive endometrium triggers a series of responses collectively called decidualization. During decidualization, endometrial stromal cells (ESCs) undergo steroid hormone-dependent proliferation and differentiation into decidual cells.^[@bib48]^ Interestingly, pregnancy-associated hormones (PAHs, such as estrogen and prolactin) upregulate RANKL and RANK levels, downregulate OPG expression, and further affect osteoclastogenesis at distinct stages of development.^[@bib49],\ [@bib50]^ RANKL/RANK system also controls the incidence and onset of progestin-driven breast cancer and physiological thermoregulation in females under the control of sex hormones.^[@bib51],\ [@bib52]^ Therefore, high levels of PAH during pregnancy may also be one of the important factors leading to such high levels of RANKL/RANK at the maternal--fetal interface. The decidua has been considered a specialized mucosal wall of the uterus. Research examining other mucosal tissues (skin and intestine) shows that epidermal and Peyer's patch-derived DCs stimulated with RANKL induce immunosuppressive activity by modulating surface barrier DCs and increasing the expansion and function of Treg cells.^[@bib26],\ [@bib27],\ [@bib28]^ Our results partially echo the immunosuppressive effect of RANKL in the mucosa. This function in the decidua is independent of the regulation of Treg differentiation. In comparison with normal pregnancy, we observed that RANKL in trophoblasts and DSCs and RANK on dM*φ* in patients with miscarriage were greatly decreased. Moreover, the dM*φ* phenotype during human and mouse pregnancy wastage shows an M1 predominance. RANKL^−/−^mice presented uM*φ* dysfunction and increased fetal loss. This deregulation of uM*φ* supports an inflammatory environment that further triggers abortive processes.^[@bib53]^ Therefore, our study reveals a novel pathogenic role of abnormal RANKL/RANK signaling at the maternal--fetal interface during SA in humans and mice. Trials conducted *in vivo* also showed that RANKL^−/−^mice had no significant influence on the total number of embryo implantations (data not shown). However, our unpublished data show that either endogenous or exogenous RANKL directly stimulates the proliferation and enhances the invasiveness of human trophoblasts, partially echoing its role in tumor cells.^[@bib19]^ We propose that the lack of RANKL *in vivo* may result in a decrease in trophoblast proliferation and invasion, but to a certain extent, it will also create a proinflammatory microenvironment. This inflammatory pattern during the initial stage of pregnancy may be conducive to the invasion and implantation of trophoblasts. Therefore, the overall effect of absent RANKL signaling *in vivo* may not affect embryo implantation. However, with advancing pregnancy, abnormally low levels of RANKL will result in miscarriage via the M1 dM*φ*-triggered disorder of maternal--fetal immune tolerance. Therefore, further research is needed to elucidate the cause of low RANKL/RANK expression in miscarriage patients. In conclusion, as shown in [Figure 7](#fig7){ref-type="fig"}, accompanied by the implantation of blastocyst during normal pregnancy, PAH trigger ESCs to differentiate into DSCs and further induce high levels of RANKL expression and CCL2 release. The latter allows the recruitment of peripheral monocytes into decidua. Embryonic trophoblasts that are deeply implanted in decidua are in close contact with DSCs and DLCs. The dialog of these cells not only further increases RANKL expression in trophoblasts and DSCs but also enhances the sensitivity of RANK on dM*φ* to RANKL by upregulating RANK expression. Subsequently, the RANKL--RANK interaction drives dM*φ* to M2 differentiation. After being instructed, these dM*φ* will create and maintain a maternal--fetal tolerant microenvironment. Once the maternal--fetal interface presents abnormal low level of RANKL, dysfunction of dM*φ* and then miscarriage will occur. Therefore, our study provides a potential target molecule, RANKL, for the identification of new strategies to prevent and treat pregnancy complications. Materials and methods ===================== Patient and sample collection ----------------------------- All procedures involving participants in this study were approved by the Human Research Ethics Committee of Obstetrics and Gynecology Hospital, Fudan University (Shanghai, China), and all subjects completed an informed consent to collect tissue samples. First-trimester human peripheral blood was obtained from 41 women with clinically normal pregnancies (age: 27.45±7.21 years; gestational age at sampling: 48.35±7.6 days (mean±S.D.)\]), which were terminated for nonmedical reasons. Human villi tissues were obtained from 172 women with clinically normal pregnancies (age: 29.88±6.91 years; gestational age at sampling: 49.17±9.34 days \[mean±S.D.\]) (Termination for nonmedical reasons), or from 12 women with SA (age: 31.09±4.28 years; gestational age at sampling: 47.95±10.1 days (mean±S.D.)). Decidual tissues were obtained from 135 women with clinically normal pregnancies (age: 27.19±5.61 years; gestational age at sampling: 51.09±8.72 days (mean±S.D.)) (termination for nonmedical reasons) and 23 women with spontaneous miscarriage (age: 31.54±5.71 years; gestational age at sampling: 53.06±5.8 days (mean±S.D.)). All pregnant women were confirmed by ultrasound and blood tests, and the women with spontaneous miscarriage because of endocrine, anatomical, and genetic abnormalities, as well as infection were excluded. Cell line --------- The human placental choriocarcinoma cell line (JEG-3 cells) was purchased from Bank of Cell, Chinese Academy of Sciences, Shanghai, China. Mice ---- RANKL heterozygote mice were obtained from the Jackson Laboratories (Sacramento, CA, USA) and subsequently maintained in the Laboratory Animal Facility of Fudan University (Shanghai, China). RANKL knockout (RANKL^−/−^) mice and wild-type littermates (WT) were obtained by mating of male and female RANKL heterozygote mice. A group of adult female C57BL/6 mice were purchased from the Laboratory Animal Facility of Fudan University and used for this study. They were usually maintained for 2 weeks in the animal facility before use. The Animal Care and Use Committee of Obstetrics and Gynecology Hospital, Fudan University approved all animal protocols. Immunohistochemistry -------------------- Immunohistological staining was performed as previously described.^[@bib46]^ Human villi and deciduas were labeled with mouse anti-RANKL Abs (15 *μ*g/ml, MAB626, R&D Systems, Abingdon, UK). Antibodies for FCM ------------------ For the identification of cell purity, primary trophoblasts and DSCs were stained with phycoerythrin (PE)-conjugated anti-human vimentin (562337; BD Biosciences) and fluorescein isothiocyanate (FITC)-conjugated anti-human cytokeratin (347653; BD Biosciences, San Jose, CA, USA); DLCs were stained with allophycocyanin (APC)-conjugated anti-human CD45 antibody (304012; Biolegend); dM*φ* was stained with FITC-conjugated anti-human CD14 antibody (301804; Biolegend); decidual naive T cells were stained with FITC-conjugated anti-human CD4 antibody (300506; Biolegend) and PE-conjugated anti-human CD45RA antibody (304107; Biolegend, San Diego, CA, USA). PE-conjugated anti-human RANKL antibody (347504; Biolegend) and PE-conjugated anti-human RANK antibody (FAB683P; R&D Systems) were used to analyze the expression of RANKL on trophoblasts and DSCs, and the expression of RANK on dM*φ*; pMo and or dM*φ* were stained with FITC-conjugated anti-human CD14 antibody (301804), phycoerythrin -cyanine 7 (PE-Cy7)-conjugated CD80 antibody (305218), APC-conjugated CD86 antibody (305412), PE-Cy7-conjugated IL-10 antibody (501420), PE-conjugated IL-12/23p40 antibody (501807), APC-conjugated IFN-*γ* (502511), APC-conjugated CD206 (321110), allophycocyanin-cyanine 5.5 (PerCP/Cy5.5)-conjugated CD209 antibody (330110) and PE-conjugated CD163 antibody (333606) (all from Biolegend). Decidual naive T cells were stained with PE-Cy7-conjugated CD4 antibody (303718), Alexa Fluor 488-conjugated anti-GATA-3 (653808) and PE-conjugated anti-T-bet (644810) (all from Biolegend). The uSC and pTros were stained with PE anti-mouse RANKL antibody (510005); the uM*φ* was stained with PE anti-mouse RANK antibody (119805), Alexa Fluor 647 anti-mouse F4/80 antibody (122610), PE/Cy7 anti-mouse CD45 antibody (103114), FITC anti-mouse CD11b antibody (101206), FITC anti-mouse CD80 antibody (104706), PE anti-mouse CD86 antibody (105008), FITC anti-mouse CD206 (MMR) antibody (141704), Brilliant Violet 421 (BV421) anti-mouse IL-10 antibody (505021) (all from Biolegend), PE anti-mouse CD209 (DC-SIGN) antibody (12-2091; ebioscience; Thermo Fisher Scientific, Inc., Waltham, MA, USA), PE anti-mouse MHCII antibody (12-5322; ebioscience), and PE/Cy7 anti-IRF4 antibody (25-9858; ebioscience); uCD4^+^T cells were stained with PE/Cy7 anti-mouse CD45 antibody (103114), FITC anti-mouse CD4 antibody (100405), PE anti-mouse IL-4 antibody (504103), BV421 anti-mouse IL-10 antibody (505021), APC anti-mouse IFN-*γ* antibody (505810), PE anti-mouse TNF-*α* antibody (506305) (all from Biolegend), BV421 anti-mouse GATA-3 antibody (563349; BD Biosciences), and PE anti-mouse T-bet antibody (561268; BD Biosciences). For intracellular staining of signaling molecules in dM*φ* or uM*φ*, cells were permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and incubated with Alexa Fluor 647-conjugated Akt (pS473) antibody (561670) and Alexa Fluor 488-conjugated Stat6 (pY641) antibody (612600 for dM*φ*; 558243 for uM*φ*) (Phosflow antibodies were from BD Biosciences). Isolation and culture of human trophoblasts, DSCs and DLCs ---------------------------------------------------------- The villi and decidua tissues from the first-trimester pregnancy were put immediately into ice-cold Dulbecco's modified Eagle's medium (DMEM high [d]{.smallcaps}-glucose; Gibco, Grand Island, NY, USA), transported to the laboratory within 30 min after surgery and washed in Hank's balanced salt solution for isolation of human trophoblasts from villi, and DSCs and DLCs from deciduas according to a previously described method.^[@bib46]^ This method supplies a 95% purity of vimentin^−^cytokeratin (CK)7^+^ trophoblast cells and greater than 98% vimentin^+^CK7^−^ DSCs and CD45^+^ DLCs, which was confirmed by FCM analysis. Enzyme-linked immunosorbent assay (ELISA) ----------------------------------------- Cytokine concentrations were measured using ELISA kits (R&D Systems). Detection of RANK expression on peripheral blood mononuclear cells (PBMCs) and DLCs ----------------------------------------------------------------------------------- The PBMCs were isolated from the peripheral blood of pregnant women who were terminated for nonmedical reasons. Next, FCM was performed to analyze the expression of RANK on pMo and dM*φ* by labeling anti-human CD14, RANK and CD45 antibodies. In addition, we further evaluated the phenotype of RANK^+^ and RANK^−^ pMos and dM*φ* purified from PBMC and DLC (*n*=24) by FCM. Purification of dM*φ* and decidual naive T cells ------------------------------------------------ We isolated dM*φ* and decidual naive T cells from DLCs by MACS (Miltenyi Biotec, Bergisch Gladbach, Germany) and performed FCM analysis with standard protocols. RANKL-overexpressed JEG-3 cells and DSCs ---------------------------------------- We obtained RANKL-overexpressed JEG-3 cells and DSCs by transfection with pcDNA(+)-RANKL plasmid, and the results were confirmed by FCM analysis. The pcDNA(+)-RANKL plasmid and pcDNA(+)-vector plasmid were from GeneChem Co., Ltd (Shanghai, China). Co-culture of trophoblasts/JEG-3 cells, DSCs and dM*φ* ------------------------------------------------------ The dM*φ* were cultured in culture medium, directly contacted with primary trophoblasts/JEG-3 cells and or DSCs at a 1 : 1 : 1 ratio. In addition, 5 *μ*g/ml anti-human RANKL neutralizing antibody (AB626, R&D Systems), 100 ng/ml rhOPG protein (185-OS-025, R&D Systems), 10 *μ*M LY294002 (Cells Signal Technology, Danvers, MA, USA) or 21 nM STAT6 signal inhibitor (STAT6i) AS1517499 (Axon Medchem, Groningen, The Netherlands) was added to the co-culture unit. After 48 h, the expression of RANKL on trophoblasts and DSCs, the expression of RANK, and the M1 phenotype and M2 phenotype on dM*φ* were analyzed by FCM, and the concentration of IL-10, IL-12p40 and IL-23 in the supernatants was detected by ELISA (R&D Systems). The intracellular phosphorylation level of Akt and STAT6 -------------------------------------------------------- The intracellular phosphorylation level of Akt and STAT6 in dM*φ* after 24-h culture with JEG-3 and DSCs was analyzed using BD Phosflow antibodies, according to standard protocols. The transcription of *Jmjd3, IRF4* and *IRF5* --------------------------------------------- After 24-h co-culture, the transcription level of *Jmjd3, IRF4* and *IRF5* in dM*φ* was analyzed by real-time PCR, according to standard protocols. The primer sequences were designed and synthesized by TaKaRa Biotechnology Co., Ltd (Tokyo, Japan) as described in [Supplementary Table 1](#sup1){ref-type="supplementary-material"}. Co-culture of dM*φ* and decidual naive T cells ---------------------------------------------- After 48 h of culture with trophoblasts/JEG-3 cells and DSCs, CD14^+^ dM*φ* were collected and washed three times with phosphate-buffered saline to remove excess cytokines. The remaining cells were co-cultured with autologous decidual naive T cells at a 1 : 1 ratio. The decidual naive T cells were pre-activated with 5 *μ*g/ml anti-CD3 (OKT3; 16-0037; ebioscience), 1 *μ*g/ml anti-CD28 (CD28.2; 16-0289; ebioscience), and 20 U/ml rhIL-2 (202-IL-010, R&D Systems) for 2 days, and collected, washed, and then incubated with culture medium only. After 5 days of co-culture, the naive T cells were reactivated with anti-CD3 and anti-CD28 for 24 h before the supernatants were collected. The expression of GATA-3 and T-bet in decidual naive T cells, and the secretion level of IL-4, IL-10, and TNF-*α* and IFN-*γ* were analyzed by FCM and ELISA (R&D Systems), respectively. Animals and experimental design ------------------------------- We divided female C57BL/6 mice (age: 8 weeks old, weight: 20--23 g) into two groups by using a random number table by body weight, age and family: the adoptive transfer of RANK^+^ M*φ* group and the adoptive transfer of RANK^−^ M*φ* group. This was an unblinded trial. The differentiation of uM*φ* and uCD4^+^T cells, the activation of Akt and STAT6, and the level of IRF4 in uM*φ* were analyzed by FCM, and the level of fetal loss was counted at day 14 of gestation in WT and RANKL^−/−^ pregnant mice. The day of appearance of a copulatory plug was arbitrarily designated as day 0 of gestation. The embryo absorption rate and implantation number were counted on day 14 of gestation. The percentage of fetal loss (the embryo absorption rate) was calculated as follows: percentage fetal loss=R/(R+V) × 100, where R represents the number of hemorrhagic implantatio (sites of fetal loss) and V stands for the number of viable, surviving fetuses. In addition, mouse uterine tissues were removed, minced on ice and digested with an enzyme mix of Liberase and Dispase (Invitrogen, Carlsbad, CA, USA). uM*φ* were isolated from mouse uterus by MACS (Miltenyi Biotec) to analyze the transcription of *Jmid3* and *IRF4* (Takara Bio Inc.,Tokyo, Japan) by RT-PCR at day 10 of gestation. The primer sequences were described in [Supplementary Table 1](#sup1){ref-type="supplementary-material"}. For M*φ* depletion and M*φ* adoptive transfer in pregnant C57BL/6 mice, and Clodronate Liposomes were injected intraperitoneally at day 1 (200 *μ*l) and day 4 (100 *μ*l) of gestation, and then the expression of RANKL in Vimentin^+^ uSC and CK7^+^ pTros were analyzed at day 7 by FCM. RANK^+^ and RANK^−^ M*φ*s were isolated from mouse spleen and labeled with PKH-67, and then they were transferred to M*φ*-depleted pregnant mice at day 5 of gestation. Subsequently, the differentiation of uM*φ* and uCD4^+^T cells, the activation of Akt and STAT6, and the IRF4 level in uM*φ* were analyzed by FCM at day 10 of gestation, and the level of fetal loss was counted at day 14 of gestation in the RANK^+^ transfer group and RANK^−^ transfer group. To investigate the role of STAT6 and Jmjd3 signals in uM*φ* and uCD4^+^T cells, the pregnant C57BL/6 mice were intraperitoneally injected with STAT6i AS1517499 (200 *μ*l at the concentration of 2 mg/kg) or JMJD3i GSK J4 HCl (200 *μ*l at a concentration of 4.48 mg/kg) in (*n*=5 mice per group) at day 4, and then the differentiation of uM*φ* and uCD4^+^T cells and the IRF4 level in uM*φ* cells at day 10 were determined by FCM. Statistics ---------- The continuous variable is shown as the mean±S.E.M. Continuous variables were analyzed by Student's *t*-test in case of two groups and by one-way ANOVA using Tukey's post-hoc test in multiple groups. The embryo resorbing rate was analyzed using an adjusted *t*-test. All analyses were conducted with SPSS 16.0 Statistical Package for the Social Sciences software (IBM SPSS, Armonk, NY, USA). *P*\<0.05 was considered statistically significant. Publisher's Note ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. We thank Professor Petra Clara Arck from Hamburg University (Hamburg, Germany) for her kind suggestions regarding the design of this study and language editing of the manuscript. We thank Assistant Professor Yi-Qin Wang from the Department of Pathology, Hospital of Obstetrics and Gynecology, Fudan University for helping with the histological analysis. This study was supported by the National Basic Research Program of China (2015CB943300), and Major Program of National Natural Science Foundation of China (NSFC) (31671200, 81490744, 91542108, 81471513, 81571275 and 81671460), the Oriented Project of Science and Technology Innovation from Key Lab. of Reproduction Regulation of NPFPC (CX2017-2), the Shanghai Rising-Star Program 16QA1400800, the Training Program for young talents of Shanghai Health System XYQ2013104 and the Program for Zhuoxue of Fudan University, the Program for Shanghai leaders, the Program of Shanghai Outstanding Academic Leader (15XD1500900), and the Postdoctoral Science Foundation of China No.2014M550573. **Author contributions** Y-HM and W-JZ conducted all experiments and prepared the figures and the manuscript. L-PJ and JW edited the manuscript. L-BL and K-KC assisted with the flow cytometry assay and animal trials. HL assisted with the sample collection. JM assisted with the IHC assay. M-QL and D-JL designed the research, supervised the project and edited the manuscript. All authors were involved in writing the manuscript. [Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis) Edited by H-U Simon The authors declare no conflict of interest. Supplementary Material {#sup1} ====================== ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. {#fig1} {#fig2} {ref-type="fig"}. (One-way ANOVA). (**g**) After intraperitoneal injection of STAT6i or the Jmjd3 selective inhibitor (JMJD3i, 200 *μ*l at a concentration of 4.48 mg/kg) GSK J4 HCl in pregnant C57BL/6 mice (*n*=5 mice per group) at day 4, the IRF4 level in uM*φ* cells at day 10 was detected. (**h** and **i**) After intraperitoneal injection of JMJD3i in pregnant C57BL/6 mice (*n*=5 mice per group) at day 4, the levels of CD206, CD209, IL-10, CD80 and CD86 in uM*φ*, and the ratios of GATA-3 to T-bet and IL-4 to IFN-*γ* in uCD4^+^T cells at day 10 were detected by FCM. (Student's *t*-test). dM*φ*: human dM*φ*; uM*φ*: mouse uterus macrophages. Data are expressed as the mean±S.E.M. \**P*\<0.05, \*\**P*\<0.01 and \*\*\**P*\<0.001](cddis2017505f3){#fig3} {#fig4} {#fig5} {#fig6} {#fig7} [^1]: These authors contributed equally to this work.

Background {#Sec1} ========== Several researches focused on the influence of the dietary supplementation of different fat sources such as animal oil or plant oilseeds \[[@CR1]--[@CR3]\] on performances, digestibility, health status, meat and fatty acid (FA) composition of rabbit meat. The 60%--70% increase of animal products consumption expected by 2050 has pushed research to investigate new raw materials for animal feeds \[[@CR4]\]. In this context, insects are very interesting as innovative feed for fish and terrestrial animals due to their valuable chemical composition and their claimed sustainability \[[@CR5], [@CR6]\]. So far, research has been mainly addressed to the protein content of insect larvae meals in fish, poultry and pigs feeds to replace conventional protein sources (mainly fishmeal and soybean meals) \[[@CR7]--[@CR9]\]. The insect species most investigated were black soldier fly (H, *Hermetia illucens* L.) and yellow mealworm (T, *Tenebrio molitor* L.) \[[@CR9], [@CR10]\]. Nevertheless, insects have different lipid content (ether extract from 5% to 40% of dry matter, DM) and FA profile related to the species and the rearing substrate \[[@CR6], [@CR11], [@CR12]\]. When processing larvae and in order to increase the protein content and the storability of the insect processed meals, some authors underlined the importance of defatting larvae meal \[[@CR13], [@CR14]\]. Unlike protein meals, which use in farm animal feeding is restricted (except for fish) in Europe (Reg. EC 999/2001), there are no prohibitions on the use of insect fats as a raw material for animal compound feeds \[[@CR15]--[@CR17]\]. Concerning the insect species mentioned above, *Hermetia illucens* is a worldwide distributed Diptera which is reared for its ability to convert low value substrates into valuable products \[[@CR12], [@CR18]\], and whose fat is characterised by high levels of saturated fatty acids (SFA), mainly lauric (C12:0) acid \[[@CR19]\]. *Tenebrio molitor* is a Coleoptera which larvae, considered as pests of stored grains, contain from 15% to 45% DM of fat, mostly palmitic (C14:0), oleic (C18:1 *n*-9) and linoleic (C18:2 *n*-6) acids \[[@CR6], [@CR19]\]. So far, the use of insect lipids in animal feeding has received little attention. Researchers investigated the effects of the dietary inclusion of H fat in partial or total replacement of soybean oil (S) without negative effects on performance and carcass traits of broiler chickens \[[@CR17], [@CR20]\]. Li et al. \[[@CR15]\] evaluated the effect of H fat in substitution of up 100% S in diets for juvenile carps and did not find negative effects on growth, feed efficiency or chemical composition of fish fillets. Recently, Kierończyk et al. \[[@CR16]\] completely substituted soybean oil with *Tenebrio molitor* or *Zophobas morio* fat (5% of dietary inclusion) in broiler diets without adverse consequences on growth performance and nutrient digestibility. As far as rabbits are concerned, to the best of our knowledge only two old researches have been carried out using insect meal (silkworm) replacing soybean meal \[[@CR21], [@CR22]\] and only two research has been performed using insect fat \[[@CR23], [@CR24]\] in diets for growing rabbits. The aim of this research was therefore to evaluate the effect of H and T fats used as partial or total substitute of soybean oil on growth performances, digestibility, blood metabolites, intestinal morphology and histological traits of growing rabbits. Methods {#Sec2} ======= The Ethical Committee of the University of Turin (Italy) approved the experimental protocol (Ref. 386638, 4/12/2017). The trial was carried out at the experimental rabbit facility of the Department of Agricultural, Forest, and Food Sciences (DISAFA; University of Turin, Turin, Italy). Diets {#Sec3} ----- An experimental control diet (C) containing 1.5% soybean oil and formulated to satisfy growing rabbits requirements \[[@CR25]\] was tested against four experimental diets where S was partially (50%) or totally (100%) substituted by H (H50 and H100) or T (T50 and T100) fats (Table [1](#Tab1){ref-type="table"}). All diets were pelleted at the feed mill of the experimental facility at the beginning of the trial and stored in darkness and fresh to protect against lipid oxidation and degradative processes.Table 1Ingredients (g/kg as fed) and chemical composition (% DM) of experimental dietsIngredientsExperimental dietsCH50H100T50T100Dehydrated alfalfa meal (17 g CP/100 g)320320320320320Alfalfa hay7575757575Wheat bran235235235235235Barley meal100100100100100Dried sugar beet pulp160160160160160Soybean meal (44 g CP/100 g)7070707070Soybean oil157.5--7.5--*Hermetia illucens* fat^a^--7.515----*Tenebrio molitor* fat^b^------7.515Cane molasses1212121212Dicalcium phosphate33333Sodium chloride44444*L*--methionine (98 g methionine/100 g)11111Vitamin--mineral premix^c^55555Chemical composition Dry matter, %89.490.089.289.589.6 Ash, % DM8.587.677.778.187.75 Crude protein, % DM17.016.416.816.816.3 Ether extract, % DM4.224.073.924.133.87 Neutral detergent fibre (aNDF), % DM40.242.541.739.840.5 Acid detergent fibre (ADF), % DM21.723.823.021.422.8 Acid detergent lignin (ADL), % DM4.815.095.094.875.02 Gross Energy, MJ/kg DM18.5018.6318.5018.7518.62*C*, control diet; *H50* and *H100*, diets with *Hermetia illucens* fat; *T50* and *T100*, diets with *Tenebrio molitor* fat; *DM*, dry matter; *CP*, crude protein^a^*Hermetia illucens* fat was provided by Hermetia Deutschland GmbH & Co. KG (Baruth / Mark, Germany)^b^*Tenebrio molitor* fat was provided by Ynsect (Evry, France)^c^Premix provided per kg of complete diet: vitamin A 16,000 IU; vitamin D~3~ 1,600 IU; vitamin E acetate 30 mg; vitamin B~1~ 0.8 mg; vitamin B~6~ 1.65 mg; niacin 40 mg; folic acid 1 mg; Mn 30 mg; Fe 116 mg; Cu 12.5 mg; Zn 60 mg; Co 0.45 g; Ca 1.3 mg; Se 0.3 mg Animals and experimental conditions {#Sec4} ----------------------------------- After weaning at 36 days of age, 200 commercial crossbred rabbits (Hycole, France) purchased from a commercial farm (Verzuolo, Cuneo, Italy), 1051 ± 138 g live weight (LW), were randomly allocated in individual wire-net cages (41 cm × 30 cm × 28 cm height). Forty rabbits, homogeneous by LW and variability, were assigned to each dietary treatment. The rabbitry had automatic control of environmental temperature (22 ± 2 °C) and photoperiod (16L:8D). The rabbits were fed ad libitum and had free access to clean drinking water during the whole trial that lasted 41 d, until 77 days of age. At 78 days of age, rabbits were slaughtered in an experimental slaughterhouse. Data of dressing percentage, carcass traits and meat quality, and chemical composition were determined but not reported in this paper. Growth performance and health status {#Sec5} ------------------------------------ During the trial, mortality and morbidity were controlled daily by the same observer according to Gidenne et al. \[[@CR26]\]. LW and feed intake were recorded per every rabbit on a weekly basis. At the end of the trial, the averages of LW, daily feed intake (ADFI), daily weight gain (ADG) and feed conversion ratio (FCR) were calculated. Digestibility trial {#Sec6} ------------------- The apparent digestibility coefficient (ADC) and the nutritive value of experimental diets were measured during an in vivo digestibility assay performed according to the European standardised method \[[@CR27]\]. The trial involved 60 rabbits (12 rabbits per dietary treatment) among those monitored for the fattening trial and started at 50 days of age. Faeces were daily collected per each rabbit and four consecutive days (09:00 h) and stored at − 20 °C until analyses. Chemical analyses and diet fatty acid profile {#Sec7} --------------------------------------------- Diets and faeces were analysed to determine the contents of DM (method 934.01), ash (method 967.05), crude protein (CP - method 2001.11), and starch (amyloglucosidase α amylase method, 996.11) using AOAC methods \[[@CR28]\] following harmonised procedures \[[@CR29]\]. Ether extract (EE) was analysed after acid hydrolysis \[[@CR30]\]. The sequential procedure and the filter bag system (Ankom Technology, Macedon, NY, USA) were used in the analyses of fibre fractions: neutral detergent fibre (aNDF) was analysed according to Mertens \[[@CR31]\], assayed with a heat stable alfa-amylase and expressed inclusive of residual ash, without sodium sulphite; acid detergent fibre (ADF), expressed inclusive of residual ash, was analysed according to AOAC \[[@CR28]\] (method 973.187); lignin was analysed with sulphuric acid according to Van Soest et al. \[[@CR32]\]. Gross energy (GE) contents of diets and faeces were measured by adiabatic bomb calorimeter (IKA C200, Staufen, Germany). The FA composition of insect lipids and feeds was determined according to Trocino et al. \[[@CR33]\]. The fat was extracted by accelerated solvent extraction (Application Note 334; ASE®, Dionex, Sunnyvale, CA, USA) using two extraction cycles. The extracted lipids were initially trans-methylated as fatty acid methyl esters (FAMEs). An internal standard (13: 1 methyl ester) was added to the extracts before methylation. After centrifugation, the surnatant was submitted to two-dimensional gas chromatography (GC × GC) by using an Agilent 7890 A Gas Chromatograph (Agilent Technologies, Santa Clara, CA, USA). Supelco SP 2560 (Sigma-Aldrich, St. Louis, MO, USA) was used as the first capillary column (75 m × 0.18 mm internal diameter, 0.14 μm film thickness), with hydrogen as carrier. J&W HP 5 ms (Agilent Technologies) was used as the second capillary column (3.8 m × 0.5 mm internal diameter, 0.25 μm film thickness), with hydrogen as carrier. The FA were identified by comparing the retention time of standard FAMEs mixture (Supelco 37 -- component FAME Mix, 47,885 -- U). Individual FAMEs were expressed as the percentage of the total area of eluted FAMEs. Serum biochemistry parameters {#Sec8} ----------------------------- At slaughtering, blood samples were collected from 15 rabbits per treatment at the moment of jugular exsanguination and put into 2.5 mL serum-separating tubes. The tubes without anticoagulant were left to clot in a standing position at room temperature for approximately two hours to obtain serum. The serum was separated by means of centrifugation at 700×*g* for 15 min and frozen at − 80 °C until analysis. The total proteins (TP) were quantified by the "biuret method" (Bio Group Medical System kit; Bio Group Medical System, Talamello, Italy). Alanino-aminotransferase (ALT), aspartate-aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), triglycerides, cholesterol, calcium, phosphorus, iron, uric acid, urea, lactate dehydrogenase (LDH) and creatinine serum concentrations were analysed with enzymatic methods using the Screen Master Touch automated instrument (Hospitex 130 Diagnostics S.r.l., Sesto Fiorentino, Italy). Histomorphological variables {#Sec9} ---------------------------- The same 15 rabbits per treatment used for serum biochemistry determinations were used for anatomo-pathological investigations. Segments (approximately 5 cm in length) of duodenum (after the pylorus), jejunum (middle portion) and ileum (before ileo-caecal junction) were excised and flushed with 0.9% saline to remove all the intestinal content. Samples of liver, spleen and kidney were also collected. The collected samples were fixed in 10% buffered formalin solution and submitted to morphometric analysis (intestine tracts) and histopathological examination (other organs). The tissues were routinely embedded in paraffin wax blocks, sectioned at 5 μm thickness and mounted on glass slides. Intestinal sections were submitted to haematoxylin-eosin (HE) staining, with a total of five serial sections prepared for each intestinal segment. The same slide among the serial sections was examined by light microscopy and captured with a Nikon DS-Fi1 digital camera coupled to a Zeiss Axiophot microscope using a 2.5× objective lens. NIS-Elements F software was used for image capturing. Morphometric analysis was performed by Image®-Pro Plus software. The evaluated morphometric indices were: villus height (Vh, from the villous tip to the crypt bottom), crypt depth (Cd, from the crypt bottom to the submucosa) and the villus height to crypt depth ratio (Vh/Cd; Additional file [1](#MOESM1){ref-type="media"}) \[[@CR34], [@CR35]\]. Morphometric measurements were performed on 10 well-oriented and intact villi and 10 crypts chosen from each gut segment. Organ sections were submitted to HE staining and examined by light microscopy. The observed histopathological findings were evaluated using a semi-quantitative scoring system as follows: absent (score = 0), mild (score = 1), moderate (score = 2) and severe (score = 3). The following histopathological alterations were evaluated: inflammatory infiltrates and degenerative changes in liver and kidney and white pulp hyperplasia and depletion in spleen. All slides were assessed blinded by three observers and the discordant cases were reviewed at a multi-head microscope until a consensus was reached. Statistical analyses {#Sec10} -------------------- The statistical analyses were performed using the IBM SPSS software package (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. IBM Corp, Armonk, NY). Shapiro-Wilk's test established normality or non-normality of distribution. One-way ANOVA was used to evaluate the effect of experimental diets on growth performance, apparent digestibility coefficients, serum biochemical parameters and intestinal morphometric indices. The assumption of equal variances was assessed by Levene's homogeneity of variance test. Mortality rate was analysed by Chi-square test, using the C group as the reference. Differences amongst groups were evaluated by the Duncan's test. Histopathological scores were analysed by Kruskal-Wallis test (post-hoc test: Dunn's Multiple Comparison test). For all statistical analyses significance was declared at *P* ≤ 0.05 and a statistical trend was considered for 0.05 \< *P* ≤ 0.10. The results are presented as the means and standard error of the means (SEM). Results {#Sec11} ======= Diet composition and fatty acid profile {#Sec12} --------------------------------------- Diets were comparable in terms of main crude protein (on average 16.6% DM) and gross energy contents (18.6 MJ/kg DM), despite some differences in aNDF and ADF contents (Table [1](#Tab1){ref-type="table"}). The substitution of S with insect fats modified the FA profile of the lipids in the diets (Table [2](#Tab2){ref-type="table"}). In fact, lauric acid (C12:0) and, thus, total SFA rate were the highest in H fat and H diets; oleic acid (C18:1 *n*-9) and MUFA rates were the highest in T fat and T diets; whereas, linoleic acid (C18:2 *n*-6) and PUFA dominated the FA profile of S and C diets.Table 2Fatty acids profile (% of total FA) of dietary fats and experimental dietsFatty acidsDietary fatsExperimental dietsSHTCH50H100T50T100C12:00.0248.00.230.059.1220.30.750.30C14:00.0810.32.220.092.114.470.971.33C16:010.412.717.612.115.716.117.318.4C18:04.431.902.312.842.622.082.652.22C16:1 *n*-70.093.201.660.121.311.990.701.04C18:1 *n*-923.09.1137.820.117.312.724.627.3C18:2 *n*-651.59.0033.252.140.931.042. 938.9C18:3 *n*-37.031.011.807.436.796.285.295.51SFA^1^15.874.823.116.531.545.423.424.0UFA^1^84.225.276.983.568.554.676.676.0MUFA^1^25.414.141.123.620.516.927.930.9PUFA^1^58.811.135.859.948.037.748.745.1^1^Included minor FAs*S* soybean oil, *H Hermetia illucens* fat, *T Tenebrio molitor* fat, *C* control diet; *H50* and *H100* diets with *Hermetia illucens* fat; *T50* and *T100* diets with *Tenebrio molitor* fat, *MUFA* monounsaturated fatty acid, *PUFA* polyunsaturated fatty acid, *SFA* saturated fatty acid, *UFA* unsaturated fatty acid Growth performance {#Sec13} ------------------ Table [3](#Tab3){ref-type="table"} reports the effects of the experimental diets on growth performance. After 41 d on trial, no differences were observed among the control and the experimental groups in terms of performance, morbidity and mortality. The final LW ranged from 2811 g (diet T50) to 2917 g (diet T100). The ADFI, ADG and FCR were 152 g/d, 44.5 g/d and 3.44, respectively, on average for the five groups.Table 3Growth performance of rabbits fed the experimental diets (*n* = 40 rabbits/group)ItemsExperimental dietsSEM*P*-valueCH50H100T50T100Number of rabbits4040403938Initial live weight, g103910591068103010579.70.74Final live weight, g2907285928802811291721.30.54ADG, g/d45.643.944.243.545.10.430.53ADFI, g/d1541491531491541.20.55FCR3.403.443.523.453.410.020.38Mortality, %0002.55.0--0.99Morbidity, %27.522.527.525.022.5--0.86*C* control diet, *H50* and *H100* diets with *Hermetia illucens* fat, *T50* and *T100* diets with *Tenebrio molitor* fat, *SEM* standard error of the means, *ADG*, daily weight gain, *ADFI* daily feed intake, *FCR* feed conversion ratio Digestibility trial {#Sec14} ------------------- The ADC are reported in Table [4](#Tab4){ref-type="table"}. The addition of H and T fats did not influence the digestibility of nutrients and energy. Only the digestibility of ADF tended to be lower in T diets compared to the control diet (22.5% on average vs. 26.4%; *P* = 0.08).Table 4Rabbit feed intake and apparent digestibility coefficients during the digestibility trial (*n* = 12 rabbits/group)ItemsExperimental dietsSEM*P*-valueCH50H100T50T100Feed intake, g/d1581511591551552.550.87Dry matter, %61.660.860.660.961.60.300.78Organic matter, %61.260.660.560.861.40.310.84Crude protein, %71.972.172.971.072.50.290.35Ether extract, %82.682.782.280.981.90.380.57aNDF, %33.532.032.232.030.20.500.44Hemicelluloses, %41.739.742.442.640.60.490.27ADF, %26.426.024.022.922.20.540.08Energy, %60.760.459.960.861.00.300.83Nutritive value DP, g/kg109.3105.5109.7106.6105.5---- DE, MJ/kg10.010.19.910.210.2---- DP/DE ratio, g/MJ10.910.511.110.510.4----*C* control diet, *H50* and *H100* diets with *Hermetia illucens* fat, *T50* and *T100* diets with *Tenebrio molitor* fat, *SEM* standard error of the means, *aNDF* Neutral detergent fibre, *ADF* Acid detergent fibre, *DP* digestible protein, *DE* digestible energy; *DP/DE ratio,* digestible protein to digestible energy ratio Serum biochemistry parameters {#Sec15} ----------------------------- The different dietary H or T fat inclusion levels did not influence (*P* \> 0.10) the serum biochemical parameters of the rabbits (Table [5](#Tab5){ref-type="table"}). Only serum TP, cholesterol, LDH and phosphorus tended to differ among diets (*P* ≤ 0.10).Table 5Effect of lipid sources on serum biochemical traits of rabbits fed experimental diets (*n* = 15 rabbits/group)ItemsExperimental dietsSEM*P*-valueCH50H100T50T100TP, g/dL5.45.15.15.15.10.040.08AST, IU/L8.912.29.88.810.20.510.20ALT, IU/L42.543.145.247.240.31.130.38ALP, IU/L34.743.732.245.639.72.830.53GGT, IU/L49.038.757.950.556.62.750.19Uric acid, mg/dL1.51.41.51.51.50.030.99Urea, mg/dL18.017.214.715.917.70.870.76Creatinine, mg/dL0.60.70.80.80.80.040.53Triglycerides, mg/dL123.0122.8120.4126.1103.16.790.84Cholesterol, mg/dL47.452.645.738.351.41.820.10LDH, IU/L143.5146.9120.7199.9117.410.40.09Iron, μg/dL293.060.8242.1289.3312.213.10.47Calcium, mg/dL9.511.210.09.910.20.390.73Phosphorous, mg/dL6.87.69.37.76.60.340.08*C* control diet, *H50* and *H100* diets with *Hermetia illucens* fat, *T50* and *T100* diets with *Tenebrio molitor* fat, *SEM* standard error of the means, *TP* total proteins, *AST* aspartate-aminotransferase, *ALT* alanine-aminotransferase, *ALP* alkaline phosphatase, *GGT* Gamma-glutamyl transferase, *LDH* lactic acid dehydrogenase Histomorphological investigations {#Sec16} --------------------------------- Villi height, crypt depth and their ratio were not affected by the experimental treatment (Table [6](#Tab6){ref-type="table"}).Table 6Effects of lipid sources on the gut morphometric indices of rabbits (*n* = 15 rabbits/group)ItemsExperimental dietsSEM*P*-valueCH50H100T50T100Duodenum Vh, mm1.691.431.581.671.590.080.56 Cd, mm0.090.090.080.090.090.0040.92 Vh/Cd18.5516.9518.6020.7318.140.640.49Jejunum Vh, mm1.531.501.691.681.630.070.51 Cd, mm0.090.080.080.080.080.0030.99 Vh/Cd18.7618.0322.2920.2420.150.770.51Ileum Vh, mm1.021.351.241.401.250.080.55 Cd, mm0.080.090.090.080.090.0040.80 Vh/Cd13.4715.7714.8517.2715.170.460.12*C* control diet, *H50* and *H100* diets with *Hermetia illucens* fat, *T50* and *T100* diets with *Tenebrio molitor* fat, *SEM* standard error of the means, *Vh* villi height, *Cd* crypt depth, *Vh/Cd* villus height to crypt depth ratio Histopathological alterations were present in all the organs of all rabbits regardless from the dietary treatments (Table [7](#Tab7){ref-type="table"}). Multifocal to diffuse (especially centrilobular) vacuolar degeneration of the hepatocytes and focal to multifocal periportal and/or interstitial lymphoplasmacytic inflammation were identified in liver (Additional file [2](#MOESM2){ref-type="media"}). The spleen showed multifocal to diffuse white pulp hyperplasia or depletion. Finally, focal to multifocal interstitial lymphoplasmacytic inflammation was observed in kidney, with no degenerative changes being identified. Dietary inclusion of H or T fats did not affect the severity of the histopathological alterations (*P* \> 0.05).Table 7Effects of lipid sources on the histopathological scores of rabbits (*n* = 15 rabbits/group)ItemsExperimental dietsSEM*P*-valueCH50H100T50T100Liver Inflammation score1.211.210.880.670.790.110.42 Degeneration score1.461.791.331.171.640.120.35Kidney Inflammation score0.640.790.670.500.570.110.91 Degeneration scoreAbsence of degenerative changesSpleen White pulp hyperplasia0.140.140.000.000.140.050.79 White pulp depletion0.290.140.000.170.290.070.71*C* control diet, *H50* and *H100* diets with *Hermetia illucens* fat, *T50* and *T100* diets with *Tenebrio molitor* fat, *SEM* standard error of the means Discussion {#Sec17} ========== Only few papers investigated the effects of insect oils in poultry feeds \[[@CR16], [@CR17], [@CR20]\] and only two deals with the inclusion of insect fat in growing rabbit diets \[[@CR23], [@CR24]\]. The results obtained in this study are consistent with the hypothesis enunciated in the background and showed how the dietary insect oil inclusion did not negatively affected performances, digestibility, gut mucosa and rabbit health. Growth performance and nutrient digestibility {#Sec18} --------------------------------------------- At the end of the growing period, no statistical differences among groups were reported for the growth performance traits. During the trial, the rabbits were not treated with antibiotics and the mortality was low and not influenced by dietary treatment. The similar feed intake indicated that 0.75% and 1.50% inclusion levels of both insect fats were acceptable to growing rabbits. A preference test where poultry were free to choose between diets containing H or S fats did not reveal any difference, confirming a good acceptability of both insect lipid sources \[[@CR20]\]. Our data are fully consistent with those reported by Martins et al. \[[@CR25]\] who observed unaffected growth performances in growing rabbits fed H fat with two inclusion level of supplementation in comparison to two inclusion levels of extruded linseed. Consistently with our results, in poultry, Schiavone et al. \[[@CR17], [@CR20]\] reported that partial or full replacement of S with H fat had no effect on birds growth performance or health. Similarly, the use of 5% of T or *Zophobas morio* fats in total substitution of S in a 28-day trial did not affect the growth and feed efficiency of broiler chickens \[[@CR16]\]. As far as fish are concerned, Li et al. \[[@CR15]\] studied the effect of increasing the replacement rate of S with H fat on juvenile Jian carp (*Cyprinus carpio* var. Jian) and reported no differences on growth performance and nutrient utilization. Recently, Dumas et al. \[[@CR36]\] evaluated up to 10% of H fat dietary inclusion in partial or total substitution of fish oil in diets for rainbow trout and reported no significant effect on final body weight, weight gain, FCR and mortality. Other researches using different (animal and vegetable) oils and fats in rabbit diets did not find significant differences in growth performance, indicating that rabbit can digest and metabolize different lipid sources, at least until 3% of inclusion level \[[@CR3]\]. For instance, both Rodríguez et al. \[[@CR37]\] and Kowalska and Bielanski \[[@CR38]\] found similar growth performance and feed conversion efficiency when feeding rabbits with lard fat, fish or soybean oils. Also, Trebušak et al. \[[@CR39]\] showed similar growth performance in rabbits fed diets containing palm fat (rich in SFA) or linseed oil (rich in PUFA). Peiretti et al. \[[@CR40]\] did not find differences in growth performance of rabbits fed diets supplemented with maize or palm oil. The lack of an effect of the lipid source on growth performance was also reported by Chen and Li \[[@CR41]\] when lard was added to rabbit diets instead of S, as well as by Casado et al. \[[@CR42]\] feeding fattening rabbits with diets containing 3% of animal fat (lard), vegetable sources (sunflower or linseed oils) or a 1:1 mixture of sunflower oil and lard or linseed oil and lard. Conversely, Djakalia et al. \[[@CR43]\] reported higher performance in growing rabbits fed S diet compared to those fed palm oil diet and ascribed this effect to the higher PUFA and lower SFA levels of the former. Differences among studies could depend more on the different fat levels tested \[[@CR2]\] than the lipid source \[[@CR3]\]. Indeed, in our trial, dietary fat level was maintained around standard values for growing rabbits \[[@CR25]\]. Likely, at higher inclusion levels, differences in FA profile among the different added fat could have caused large differences in fat digestive utilization and thus in growth \[[@CR2], [@CR3]\]. To go in deep on the evaluation of dietary treatments on nutrient digestibility, in the present trial DM and energy digestibility coefficients were high and similar in all treatments indicating that both insect lipid sources are suitable for rabbit feeding. As the digestibility of EE is concerned, the mean value was consistent with literature data. Differences may depend on the type of fat source, their unsaturation degree as well as the level of structural lipids linked to cell walls \[[@CR3], [@CR44], [@CR45]\]. In fact, the digestibility of more saturated fats, like tallow and lard, may be lower than that of unsaturated fat, like sunflower and soybean oils \[[@CR46]\]. However, in the current study, the higher levels of SFA of H diets, did not determine EE digestibility lower than C or T diets. Similarly, in diets for rainbow trout, Dumas et al. \[[@CR36]\] did not find significant differences for EE digestibility when 20% H fat was added to diets. Also, Chen and Li \[[@CR41]\] did not observe significant difference in the EE digestibility depending on the FA profile of the lipid source (lard vs soybean oil) in diets for growing rabbits. In the contrary, Martins et al. \[[@CR24]\] reported that H fat with two inclusion level in growing rabbit's diet decreased the ADC of DM, OM, EE and GE when compared to linseed fat source. The authors found that increasing the level of H fat in the diets led to a significant increase in the ADC of EE and a significant decrease in the ADC of the cellulose. Serum biochemistry traits {#Sec19} ------------------------- In the current trial, the values of the different traits lay within the normal range for rabbits \[[@CR47]--[@CR49]\]. This suggests that the rabbits were within physiological conditions. In our trial, only the type of fat included were different and diets had the same nutrient levels. This could justify the lack of significant differences in TP values between groups. Indeed, Iyayi and Tewe \[[@CR50]\]. reported that serum TP synthesis is generally related to the content of available protein in the diet. Including insect lipids in rabbit diets did not influence AST, ALT and ALP enzyme activities, which are considered to be important indicators of liver functions \[[@CR51]\]. Indeed, also in broiler chickens, Schiavone et al. \[[@CR17], [@CR20]\] showed that dietary partial or total replacement of S with H fat did not affect serum biochemistry parameters. The same finding was reported with Li et al. \[[@CR15]\] in juvenile Jian carp fed diets with different substitution levels of S with H fat. Histomorphological investigations {#Sec20} --------------------------------- Since both gut morphology and growth performance were unaffected in H and T groups of the current research, insect lipids utilization did not negatively influence gut development and functionality. Consistently with the results of the present study, Schiavone et al. \[[@CR17]\] observed unaffected intestinal morphometric indices in broiler chickens fed with H fat replacing 50% or 100% of S. Dietary H or T fat inclusion did not affect either the development or the severity of the histopathological alterations in the rabbits of the current research. Degenerative and inflammatory changes of liver and kidney are usually observed in toxic diseases \[[@CR52]\] or toxicity-induced oxidative stress conditions \[[@CR53]\]. Hepatic and renal oxidative stress development has also been reported in rabbits fed with hypercholesterolemic diets \[[@CR54]\], thus underlying the impact of dietary lipid modifications on liver and kidney functionality. However, no hypercholesterolemia was observed in H and T rabbits of the present study. Furthermore, the majority of the histopathological alterations varied from absent to moderate in all the dietary treatments and they were also identified in the animals fed with C diet, thus allowing to reliably exclude a potential influence of insect lipids utilization in their development. Conclusions {#Sec21} =========== The obtained results showed that H and T fats can be used as partial or total substitute of soybean oil until 1.5% of the diet in rabbit feeding without impacting growth performance, nutrient digestibility, serum biochemical traits and gut development. These findings suggest that H and T fats is a suitable ingredient for rabbit diets. However, further studies should be performed to establish the most effective source and to determine the optimal inclusion level of insect lipids for rabbit feeding. Additional files ================ {#Sec116} Additional file 1:Morphometric evaluation of the jejunum segment of the rabbits. Morphometric measurements of the villus height (Vh) and the crypt depth (Cd). H100 group, 2.5× haematoxylin-eosin stain. (TIF 7150 kb) Additional file 2Histopathological findings of the rabbits. (A) HI50 group. A normal liver is observed. 10× haematoxylin-eosin stain. (B) H100 group. Liver, periportal zone. Moderate and multifocal vacuolar degeneration of the hepatocytes (arrowheads), as well as mild and multifocal lymphoplasmacytic inflammation (arrows), are identified. 10× haematoxylin-eosin stain. (TIF 5050 kb) ADC : Apparent digestibility coefficient ADF : Acid detergent fibre ADFI : Daily feed intake ADG : Daily weight gain ADL : Acid detergent lignin ALP : Alkaline phosphatase ALT : Alanine amino-transferase aNDF : Neutral detergent fibre AST : Aspartate amino-transferase C : Control diet Cd : Crypt depth CP : Crude protein DE : Digestible energy DM : Dry matter DP : Digestible protein EE : Ether extract FA : Fatty acid FAME : Fatty acids methyl esters FCR : Feed conversion ratio GE : Gross energy GGT : Gamma-glutamyl transferase H : *Hermetia illucens* HE : Haematoxylin-eosin HR~i~ : Health risk index LDH : Lactic acid dehydrogenase LW : Live weight MUFA : Monounsaturated fatty acid PUFA : Polyunsaturated fatty acid S : Soybean oil SEM : Standard error of the means SFA : Saturated fatty acid T : *Tenebrio molitor* TP : Total proteins UFA : Unsaturated fatty acid Vh / Cd : Villus height to crypt depth ratio Vh : Villus height The authors gratefully acknowledge Dr. Andrea Dama, Dr. Enrico Bressan, Mr. Dario Sola and Mr. Mario Colombano for technical support, and Ynsect and Hermetia Deutschland GmbH & Co. KG for the provision of the insect fats. Funding {#FPar1} ======= Financial support for this work was provided by the University of Turin (ex 60%) grants (Es. fin. 2015--2016-2017). Availability of data and materials {#FPar2} ================================== The datasets analysed in the current study are available from the corresponding author on reasonable request. LG, SD, AT, GX, AS and FG conceived and designed the experiment. AT and GX formulated the experimental diets and carried out their analyses. LG, SD, MM, AS and FG prepare the diets and performed the trial. LG, SD, IB, MB, MM and AS collected the experiments data. MTC and IB performed the morphometric investigations. DD established the blood parameters. SD and IB performed the statistical analysis. All authors analysed and interpret the data. LG, SD, AT, IB and FG wrote the first draft of the manuscript. All authors critically reviewed the manuscript for intellectual content and gave final approval for the version to be published. Ethics approval and consent to participate {#FPar3} ========================================== The trial was designed according to the guidelines of the current European Directive (2010/63/EU) on the care and protection of animals used for scientific purposes. The experimental protocol was approved by the Ethical Committee of the University of Turin (Italy) (Ref. 386,638, 4/12/2017). Consent for publication {#FPar4} ======================= Not applicable. Competing interests {#FPar5} =================== The authors declare that they have no competing interests.

Introduction {#sec1} ============ Li-ion batteries (LIBs) have been widely used in many applications, for example, mobile phones, laptops, electric vehicles, and hybrid electric vehicles^[@ref1],[@ref2]^ because of their high energy densities (∼100--170 W h kg^--1^) and long cycle life.^[@ref3]−[@ref7]^ The present materials used in the cathode of LIBs consist of three main components: (i) ca. 80 wt % of active materials such as LiCoO~2~ (LCO), LiMn~2~O~4~ (LMO), LiNi~*x*~Co~*y*~Al~1--*x*--*y*~O~2~ (NCA), LiNi~*x*~Mn~*y*~Co~1--*x*--*y*~O~2~ (NMC), and LiFePO~4~ (LFP)^[@ref8],[@ref9]^ and composite materials,^[@ref10]−[@ref12]^ (ii) ca. 10 wt % conductive additives (i.e., spherical carbon black, Super P),^[@ref13],[@ref14]^ and (iii) ca. 10 wt % polymer binders \[i.e., polyvinylidene fluoride (PVDF) and poly(tetrafluoroethylene) (PTFE)\].^[@ref15],[@ref16]^ For example, LiMn~2~O~4~ having a spinel structure (space group *Fd*3*m*) with a poor electrical conductivity (∼10^--6^ S cm^--1^)^[@ref17]^ mixed with carbon black (e.g., Super P) and PVDF at a weight ratio of 8:1:1 can provide a discharge specific capacity of 82.6 mA h g^--1^ at 0.05 A g^--1^.^[@ref18]^ The LiMn~2~O~4~ rods were mixed with carbon black and PTFE (8:1:1 by weight), providing 96.8 mA h g^--1^ at 0.5 C.^[@ref19]^ The LiMn~2~O~4~ nanowires (NWs) mixed with acetylene black and PVDF at a weight ratio of 8:1:1 exhibit 94.7 mA h g^--1^ at 1 C.^[@ref20]^ Up to now, about 10--20 wt % spherical carbon black (Super P) is routinely used in the fabrication process of the LIB cathodes. However, those specific capacities are still far lower than a theoretical value of LiMn~2~O~4~. Besides, a simple question is why spherical carbon is extensively used as a conductive additive in LIBs. In this work, spherical carbon black nanoparticles (CNs) were turned to oxidized CNs (OCNs) and hollow carbon nanospheres (HCNs). They were then used as the conductive additive in fabricating the cathodes of LIBs. For comparison, four grades of the CN widely used, namely, EC300J (CN-1), Super P (CN-2), Denka (CN-3), and ENSACO (CN-4), were also employed as the conductive additives. The LIB cathodes include LiMn~2~O~4~, LiCoO~2~, LiNiMnCoO~2~, and LiFePO~4~, which are currently used in the commercial LIBs. Interestingly, we have found in this work that among carbon conductive additives, HCN can provide rather high specific capacities for all LIB cathodes because of its outstanding physicochemical property. The as-fabricated coin cell LiMn~2~O~4~ battery with a CR-2025 size using 10 wt % HCN as a conductive additive in the cathode exhibits 125 mA h g^--1^ at 0.1 C, which is 3.85-fold higher than that using the conventional conductive additive, CN (Super P), and higher than that in other previous reports. Results and Discussion {#sec2} ====================== Morphological and Structural Characterizations {#sec2-1} ---------------------------------------------- Transmission electron microscopy (TEM) images in [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}a--d show the spherical shapes of EC300J (namely, CN-1), Super P (CN-2), Denka (CN-3), and ENSACO (CN-4), with a diameter of around 30--50 nm in the primary spherical particles.^[@ref21],[@ref22]^ All of them are stable in an aggregated structure, forming a so-called high structure, leading to high electrical conductivity. Especially, CN-1 particles aggregated have a void fraction in their particles. Obviously, the spherical shape of the primary particles is not geometrically ideal for entirely coating the microscale particles such as LiMn~2~O~4~, LiCoO~2~, LiNiMnCoO~2~, and LiFePO~4~, which are used as the active materials of the LIB cathodes. {#fig1} After the oxidation process, the spherical shape of CN-2 was turned to OCN with graphene multilayers, which is obviously seen in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}a,b. After the reduction process of OCN with hydrazine, the structure of HCN ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}c,d) is not significantly different when compared with that of OCN. From high-resolution transmission electron microscopy (HR-TEM) images, the curvature graphene layers^[@ref22]^ are clearly observed for both OCN and HCN samples (see the inset images in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b,d). Notably, the electrical conductivity of carbon conductive additives is due to electron delocalization in the graphene sheets.^[@ref22]^ The resistance of LiMn~2~O~4~/HCN/PVDF (8:1:1 wt %) measured by a four-point probe (Jandel) is 1.07 Ω cm^--2^, which is lower than 1.80 Ω cm^--2^ of LiMn~2~O~4~/CN-2/PVDF (8:1:1 wt %) (see Table S1 and Figure S1 of [Supporting Information](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00763/suppl_file/ao7b00763_si_001.pdf)). This result indicates the high electrical conductivity of HCN over CN-2. {#fig2} Raman spectra in [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}a display two main peaks of D and G bands at ca. 1350 and 1587 cm^--1^, respectively.^[@ref23],[@ref24]^ Theoretically, the G band is attributed to the stretching mode of the C--C bond in a benzene ring. The D band is related to the vibrational mode of sp^3^ carbon atom from the disordered structure.^[@ref25],[@ref26]^ The amount of disordered carbon structures can be estimated by a peak intensity ratio between D and G bands (*I*~D~/*I*~G~). The *I*~D~/*I*~G~ values of CN-1, CN-2, CN-3, CN-4, OCN, and HCN are 1.07, 0.94, 1.00, 0.94, 1.27, and 1.10, respectively. The average lateral sizes (*L*~a~) of the layer structures in the samples calculated from Raman spectra^[@ref27]^ ranged from 3.42 to 4.65 nm, as listed in [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}. {#fig3} ###### Stacking Height, Lateral Size, *d*-Spacing, and BET Specific Surface Area of the Samples sample *L*~c~ (nm) *L*~a~ (nm) *d*~002~ (Å) *S*~BET~ (m^2^ g^--1^) -------- ------------- ------------- -------------- ------------------------ CN-1 1.39 4.06 3.62 723.47 CN-2 1.54 4.65 3.66 168.16 CN-3 2.00 4.33 3.58 279.18 CN-4 1.46 4.64 3.58 115.22 OCN 4.53 3.42 3.45 185.33 HCN 4.23 3.94 3.48 172.78 The functional groups of the samples characterized by Fourier transform infrared (FTIR) spectroscopy are shown in [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}b. A peak at 1724 cm^--1^ is due to the stretching vibrational mode of the carboxyl group (C=O).^[@ref28]^ The C--O stretching vibration is from the alcoholic and carboxylic groups at ca. 1232 cm^--1^. The peaks at 2660 and 1620 cm^--1^ indicate the C--H and C=C groups, respectively.^[@ref29],[@ref30]^ The FTIR results here confirm that the acid oxidation process can introduce the oxygen-containing functional groups, mainly the carboxylic group on the OCN surface. Consequently, the oxygen-functional peaks of HCN reduced and disappeared after the reduction process. Without oxygen-containing groups, HCN is an ideal conductive additive, not leading to the parasitic reactions when cycling the LIBs. [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}c shows the X-ray diffraction (XRD) patterns of the samples, exhibiting the diffraction peaks at 2θ of ca. 25° and 44° assigned to the (002) and (100) planes of the graphitic carbon, respectively.^[@ref31],[@ref32]^ The average stacking heights (*L*~c~) of CN-1--4, OCN, and HCN calculated by the Scherrer equation are 1.39--4.53 nm, indicating the expansion of the graphitic layers of the OCN after the acid oxidation process. The interlayer structures based on a (002) plane so-called *d*-spacing (*d*~002~) of the samples are 3.45--4.65 Å. These values were calculated by Bragg's law (see [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}). Overall, the crystalline structure of the HCN sample remains the graphitic carbon and is more or less the same structure as the CN samples. The result here indicates that the HCN retains high electronic conductivity because of its graphitic carbon structure. The N~2~ adsorption and desorption were characterized to investigate the specific surface area and porous characteristics of the samples ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}d). The gas adsorption isotherm of the HCN sample relates to the type IV isotherm, according to the IUPAC classification.^[@ref33]^ The carbon black samples exhibit the mesoporous characteristics, which are found in the range of 2--50 nm, according to the pore size distributions (the inset image of [Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}d). The Brunauer--Emmett--Teller (BET) equation was used to calculate the BET specific surface areas of all samples, which are 168.16--723.47 m^2^ g^--1^, as listed in [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}. The HCN shows higher specific surface area than that of CN-2 (Super P), the material precursor. [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}a shows a field-emission scanning electron microscopy (FE-SEM) image of the crystalline LiMn~2~O~4~ that has a hexahedron shape. [Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}b--f shows the morphology of the LiMn~2~O~4~ cathode mixed with conductive additive and PVDF (80:10:10 wt % ratio) before and after being charged/discharged for 1, 25, 50, and 100 cycles. The results clearly illustrate that the morphology of the tested LiMn~2~O~4~ remains almost the same as that tested before. This indicates the high stability of LiMn~2~O~4~. {#fig4} Electrochemical Property {#sec2-2} ------------------------ To further understand the electrochemical property of the HCN, the cyclic voltammograms (CVs) of HCN and its composite with 80 wt % active material LiMn~2~O~4~ and 10 wt % polymer binder PVDF versus a standard redox mediator (ferrocenemethanol, FcOH) with one electron-transfer reversible process were investigated. For comparison, the CVs of LiMn~2~O~4~ and CN as well as their composites coated on glassy carbon (GC) electrodes are also shown in [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}. [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}a shows a control experiment for which the CVs of bare GC in 1.6 mM FcOH in 0.1 M KCl at different scan rates have a reversible feature, indicating a reversible redox reaction of FcOH (Fe^2+/3+^). The CVs of bare LiMn~2~O~4~ coated on GC shown in [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}b with lower currents indicate that an insulating LiMn~2~O~4~ layer can block the redox reaction of FcOH at the GC electrode. This is because LiMn~2~O~4~ occupies an active surface area of GC. On the other hand, the CN layer clearly shows an adsorption effect, preconcentrating FcOH (Fe^2+^) mediator, leading to a significant enhancement in the anodic current (see [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}c). However, the CN layer on the GC surface has an irreversible feature with a lower cathodic current when compared to the anodic current. Interestingly, the HCN layer can provide both higher anodic and cathodic currents when compared with those of the bare GC. In addition, it has a reversible redox reaction characteristic and high capacitive current, which is good for the energy storage application. Also, the diffusion limit of FcOH through the HCN layer is not so high when compared with those of other systems. When CN and HCN were mixed with LiMn~2~O~4~ and PVDF at a ratio of 10:80:10 wt % and coated on the GC electrodes, the CVs in [Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}d,e clearly show that CN and HCN are great in terms of electrical conductivity, providing a reversible redox reaction of FcOH (Fe^2+/3+^) and significantly enhancing both anodic and cathodic currents when compared with those of the bare LiMn~2~O~4~-coated GC. In addition, the reversible behavior was quantitatively measured by the ratio of the anodic/cathodic peak current (*i*~pa~/*i*~pc~), as listed in [Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}. The GC shows a reversible behavior with *i*~pa~/*i*~pc~ equal to 1.00 at all scan rates. Also, LiMn~2~O~4~/GC has the reversible characteristics with an *i*~pa~/*i*~pc~ ratio of 0.98--1.0 for different scan rates. Surprisingly, CN-2, which is widely used as a conductive additive in commercial LIBs, and its composite with LiMn~2~O~4~ and PVDF provide rather high *i*~pa~/*i*~pc~ values (2.2--4.2), which are very much higher than 1.0, a theoretical value of an ideal reversible process (see [Table [2](#tbl2){ref-type="other"}](#tbl2){ref-type="other"}). By contrast, HCN provides desirable *i*~pa~/*i*~pc~ values that are closer to 1.0. Note that a reversible process is needed for an ideal energy storage material because it can provide high Coulombic efficiency. {#fig5} ###### Ratio of Anodic and Cathodic Current (*i*~pa~/*i*~pc~) Values of Bare GC Electrode and Coated Materials Using 1.6 mM FcOH as a Redox Mediator in 0.1 M KCl with Different Scan Rates scan rate (mV s^--1^) GC LiMn~2~O~4~ CN-2 HCN CN-2/LiMn~2~O~4~ HCN/LiMn~2~O~4~ ----------------------- ------ ------------- ------ ------ ------------------ ----------------- 10 1.00 0.99 4.16 0.96 0.89 1.02 25 1.00 1.00 3.00 0.82 0.73 0.99 50 1.00 0.99 2.56 0.88 0.67 1.03 75 1.00 0.98 2.28 0.86 0.73 0.96 100 1.00 1.00 2.21 0.87 0.66 0.99 [Figure [6](#fig6){ref-type="fig"}](#fig6){ref-type="fig"} shows the Nyquist plots of the samples with different rotation speed rates of GC rotating disc electrode (RDE) from 0 to 5000 rpm. The Nyquist plot of LiMn~2~O~4~ exhibits the highest resistance of charge transfer because of the kinetically sluggish of the insulating material. When the conductive additives CN and HCN were mixed with the active material, it can be clearly observed that HCN possesses lower charge-transfer resistance. The equivalent circuit (the inset image of [Figure [6](#fig6){ref-type="fig"}](#fig6){ref-type="fig"}a) was used to fit the experimental data, where *R*~s~ is the resistance in solution, *R*~ct~ is the charge-transfer resistance, *C*~dl~ is the double-layer capacitance, and *W* is the Warburg impedance, which represents the interfacial diffusive resistance.^[@ref34]^ The standard heterogeneous rate constant of electron transfer (*k*^0^, cm s^--1^) was calculated from [eq [1](#eq1){ref-type="disp-formula"}](#eq1){ref-type="disp-formula"}. The *k*^0^ values of all materials with different rotation rates as listed in [Table [3](#tbl3){ref-type="other"}](#tbl3){ref-type="other"} indicate that when HCN was used, the rate of charge transfer was increased, which is an ideal property for the conductive additive. {#fig6} ###### Standard Heterogeneous Rate Constant of FcOH (Fe^2+^/Fe^3+^) at GC RDE and CN, HCN, CN/LiMn~2~O~4~, and HCN/LiMn~2~O~4~ Coated on GC RDEs at Different Rotation Rates   10^3^*k*^0^ (cm s^--1^) ------ ------------------------- ------- ------- ------- ------- ------- 0 49.13 20.83 30.68 38.96 23.07 27.92 500 52.65 21.40 32.19 41.51 24.06 28.98 1000 56.03 22.41 33.86 43.10 24.82 30.02 2000 58.40 23.30 35.34 46.24 25.80 31.30 3000 60.66 24.01 37.18 48.72 26.62 32.19 4000 63.95 25.86 38.52 50.94 27.62 33.43 5000 70.88 26.50 40.37 53.00 28.18 35.02 Charge Storage Performance {#sec2-3} -------------------------- For the charge storage capacity, the as-fabricated LiMn~2~O~4~ electrode was electrochemically evaluated using a galvanostatic charge/discharge (GCD) technique. The specific capacity of the first charge/discharge profiles at 0.1 C from 3.0 to 4.3 V versus Li^+^/Li is shown in [Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}a. The results indicate that the battery using HCN as the conductive additive exhibits a specific capacity of ca. 125 mA h g^--1^, which is ca. 3.85-fold higher than those of the batteries using the pristine CN-2 (Super P) at 0.1 C. Note that [Table [4](#tbl4){ref-type="other"}](#tbl4){ref-type="other"} also compares the charge storage performances of the LiMn~2~O~4~-based batteries for which the as-fabricated battery here in this work provides significantly higher specific discharge capacity than that in other previous reports. {#fig7} ###### Charge Storage Performances of the LiMn~2~O~4~-Based Batteries cathode materials (wt %) specific discharge capacities (mA h g^--1^) refs ----------------------------------------------- --------------------------------------------- ------------ LiMn~2~O~4~/carbon black/PVDF (9:0.5:0.5) 92.76 ([@ref35]) LiMn~2~O~4~/carbon black/PVDF (9:0.5:0.5) 88 ([@ref36]) LiMn~2~O~4~ NWs/carbon black/PVDF (8:1:1) 94.7 ([@ref20]) LiMn~2~O~4~/carbon black/PVDF (8:1:1) 82.6 ([@ref18]) LiMn~2~O~4~ NWs/carbon black/PTFE (8:1:1) 96.8 ([@ref19]) LiMn~2~O~4~/carbon black/PVDF (8.5:0.75:0.75) 87.5 ([@ref37]) LiMn~2~O~4~ with graphite/PVDF (9.9:0.1) 96 ([@ref38]) LiMn~2~O~4~ thin film 85 ([@ref39]) LiMn~2~O~4~/carbon black/PVDF (7:1:2) 96.19 ([@ref40]) LiMn~2~O~4~/carbon black/PVDF (7.5:2:0.5) 87 ([@ref41]) LiMn~2~O~4~/carbon black/PVDF (7:2:1) 93.7 ([@ref42]) LiMn~2~O~4~/HCN/PVDF (8:1:1) 125 this work [Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}b also clearly shows that the LiMn~2~O~4~/HCN/PVDF (8:1:1 wt %) cathode has the highest discharge specific capacity for all applied C rates (0.1--4 C). The stability of the as-fabricated LiMn~2~O~4~/HCN/PVDF cathode was eventually evaluated over 800 cycles at 1 C. Its capacity retention remains over 95% with a Coulombic efficiency of 100% (see [Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}c). To further investigate the advantages of using HCN as the conductive additive in LIBs, other cathode materials of the present LIBs, such as LiCoO~2~, LiNiMnCoO~2~, and LiFePO~4~, were also employed, and their charge storage performances are shown in [Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}d--f. The LiCoO~2~ ([Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}d), LiNiMnCoO~2~ ([Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}e), and LiFePO~4~ ([Figure [7](#fig7){ref-type="fig"}](#fig7){ref-type="fig"}f) batteries using HCN exhibit significantly higher discharge specific capacities at 0.1 C when compared with those using CN (see their charge/discharge profiles in Figure S2 of [Supporting Information](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00763/suppl_file/ao7b00763_si_001.pdf)). The specific capacities of LiCoO~2~, LiNiMnCoO~2~, and LiFePO~4~ are ca. 106, 124, and 169 mA h g^--1^, respectively, which are also higher than those in other reports. As a result, it can be concluded here that including 10 wt % HCN to the cathodes of the present LIBs can significantly improve the overall charge storage performance of the cathodes of LIBs. The Mn K-edge X-ray absorption near-edge structure (XANES) spectra of the LiMn~2~O~4~ cathodes before and after being electrochemically tested by the GCD are shown in [Figure [8](#fig8){ref-type="fig"}](#fig8){ref-type="fig"}a. The average oxidation number of Mn in the LiMn~2~O~4~ cathode before being charged is +3.45. The average oxidation numbers of Mn after being tested for 25, 50, and 100 cycles are close to +3.45 (see [Figure [8](#fig8){ref-type="fig"}](#fig8){ref-type="fig"}b). This result indicates that the Li ions intercalate/deintercalate through the LiMn~2~O~4~ structure via a reversible reaction process without a parasitic reaction. {#fig8} Conclusions {#sec3} =========== The commercial spherical CNs such as EC300J (CN-1), Super P (CN-2), Denka (CN-3), and ENSACO (CN-4) as well as OCN and HCN were used as the conductive additives in the cathodes of the commercial LIBs, such as LiMn~2~O~4~, LiCoO~2~, LiNiMnCoO~2~, and LiFePO~4~. For example, the as-fabricated LiMn~2~O~4~ cathode using 10 wt % HCN as the conductive additive exhibits a specific capacity of 125 mA h g^--1^, which is 3.85-fold higher than that of the battery using CN at 0.1 C with a potential ranging from 3.0 to 4.3 V versus Li^+^/Li. The capacity retention is over 95% after 800 cycles with 100% Coulombic efficiency at 1 C. In addition, the HCN can significantly improve the charge storage performances of other commercially available LIB cathodes such as LiCoO~2~, LiNiMnCoO~2~, and LiFePO~4~ because of its high surface area and high ionic conductivity. This work indicates the influence of the superior conductive additive HCN to the charge storage performance of the LIB cathodes. The HCN in this work may be one of the best choices to increase the charge storage performance of LIBs for high-energy applications. Experimental Section {#sec4} ==================== Chemicals and Materials {#sec4-1} ----------------------- Spherical CNs including EC300J (Lion, Japan), Super P (Timcal), Denka (Japan), and ENSACO (Timcal), lithium manganese oxide, lithium cobalt oxide, lithium nickel manganese cobalt oxide, lithium iron phosphate (Gelon, Hong Kong), sulfuric acid (H~2~SO~4~, 98%, QRec), nitric acid (HNO~3~, 65%, QRec), PVDF (Sigma-Aldrich), ferrocenemethanol (97%, Sigma-Aldrich), hydrazine hydrate (99%, Loba Chemie), *N*-methylpyrrolidone (NMP, 99.5%, QRec), and 1 M lithium hexafluorophosphate (LiPF~6~, Sigma-Aldrich) in the mixture of ethylene carbonate (EC), ethyl methyl carbonate (EMC, Sigma-Aldrich), and dimethyl carbonate (DMC, Sigma-Aldrich) (1:1:1 v/v/v) (Gelon) were of analytical grade and used without further purification. Carbon fiber paper (CFP) (SGL Carbon SE, Germany) was used as a current collector. Deionized water was purified by using the Milli-Q system (\>18 MΩ·cm, Millipore). Teflon-mounted GC electrode with 3.0 mm in diameter was from Metrohm Siam Co., Ltd. Synthesis of OCNs {#sec4-2} ----------------- OCN was prepared via an oxidizing process. Briefly, CN-2 (5 g, Super P) and HNO~3~ (400 mL) were mixed together in a round-bottom flask and heated to 100 °C for 96 h by a reflux condenser process. The obtained suspension was washed several times with deionized water and then centrifuged at 6000 rpm for 30 min until neutral (pH 7). Afterward, OCN was collected by a vacuum filtration process and dried at 60 °C overnight. Synthesis of HCNs {#sec4-3} ----------------- HCN was synthesized by reducing OCN with hydrazine. First, OCN (100 mg) was mixed with deionized water (100 mL) and sonicated for 1 h. Then, 1 mL of 1 M hydrazine hydrate was added into the dispersion and heated by a reflux condenser process at 100 °C for 36 h. The suspension was centrifuged at 12 000 rpm for 30 min with deionized water (500 mL) and methanol (500 mL) until pH 7. The product was collected after being dried at 60 °C overnight. Characterizations {#sec4-4} ----------------- The morphology of the as-synthesized samples was characterized using FE-SEM and TEM. The structural properties were characterized using Raman spectra (Senterra dispersive Raman microscope, Bruker) and XRD (PANalytical with Cu Kα radiation, λ = 1.54056 Å) techniques. The functional groups on the surfaces of the as-synthesized materials were determined by an FTIR spectrometer (Frontier FT-IR, PerkinElmer). The surface area and porous structures of the samples were measured by an N~2~ adsorption/desorption technique (Autosorb 1 MP, Quantachrome). The BET model was used to calculate the specific surface area of the sample. Charge Storage Evaluation {#sec4-5} ------------------------- The cathode of the LIBs was fabricated by mixing 80 wt % active materials, such as LiMn~2~O~4~, LiCoO~2~, NMC, and LiFePO~4~, 10 wt % conductive additives, such as CN-1, CN-2, CN-3, CN-4, OCN, and HCN, and 10 wt % PVDF adhesive binder in an NMP solvent. The mixture was stirred for 24 h. The as-prepared slurry was casted on the CFP substrate with hydrophilic surface using a casting machine (GN-AFA-III, Gelon). The as-coated CFP was vacuum-dried at 50 °C overnight, cut into a circle (1.58 cm diameter), and used as the cathode. The polyethylene film separator (25 μm in thickness, Gelon) was soaked in 1 M LiPF~6~ in the mixture of EC, EMC, and DMC (1:1:1 v/v/v) electrolytes. Finally, the coin cell batteries (CR-2025 size) were assembled in an argon-filled glovebox (MBraun) (\<0.1 ppm H~2~O and O~2~) using a hydraulic crimping machine (GN-110, Gelon) at 2000 psi for 5 s. The electrochemical performance of the as-fabricated batteries was tested by a GCD technique using a Neware battery tester (GN-4008-SCT, Gelon). The Supporting Information is available free of charge on the [ACS Publications website](http://pubs.acs.org) at DOI: [10.1021/acsomega.7b00763](http://pubs.acs.org/doi/abs/10.1021/acsomega.7b00763).Resistivity of the cathodes using different conductive additives, digital photo of four-point probe equipment for measuring the resistivity of the as-prepared electrodes, and galvanostatic charge/discharge profiles of LiCoO~2~, LiNiMnCoO~2~, and LiFePO~4~ batteries ([PDF](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b00763/suppl_file/ao7b00763_si_001.pdf)) Supplementary Material ====================== ###### ao7b00763_si_001.pdf J.W. and N.P. synthesized and characterized the materials, performed the electrochemical experiment, and discussed the results. M.S. designed and directed the work, discussed the results, and wrote the manuscript. The authors declare no competing financial interest. This work was financially supported by the Thailand Research Fund and Vidyasirimedhi Institute of Science and Technology (RSA5880043). Synchrotron Light Research Institute (Public Organization), Thailand for XANES facilities, and the Frontier Research Centre at VISTEC are acknowledged.

INTRODUCTION ============ Deep transcriptome analyses have revealed that almost the entire genome of complex eukaryotes such as mammals is transcribed ([@B1; @B2; @B3]). Transcript length varies from as little as 16 nt (such as tiRNA) to \>100 kb (such as *Xist* RNA). Mammals contain a variety of small RNAs and include tiRNAs (16 nt), siRNAs/miRNAs/piRNAs (20--30 nt), tRNA halves (30--40 nt), PASR/TASR (22--200 nt) and snoRNAs (70--200 nt) that can modulate transcription, translation, replication and chromatin structure ([@B1],[@B3]). A growing number of ncRNAs have been described in *Saccharomyces cerevisiae*. Many of these are driven by RNA Pol II and include cryptic unstable transcripts (CUTs, ∼400 nt) ([@B4]) and stable unannotated transcripts (SUTs, ∼700 nt) ([@B5]). Most map to transcription start sites or the 3′-end of protein-coding genes and appear to be the result of bidirectional promoter activity. In general, CUTs are degraded rapidly by the Nrd1-exosome-Trf4-Air2-Mtr4p polyadenylation (TRAMP) complex ([@B4]). Several cases have shown where these ncRNAs interact with the ribosomal complex and are translated ([@B6],[@B7]). In the filamentous fungus *Neurospora crassa*, several new species of small RNAs have been recently described including miRNA-like small RNAs (milRNAs), Dicer-independent small interfering RNAs (disiRNAs) and qiRNAs ([@B8],[@B9]). qiRNAs arise in response to DNA damage and map to sense and antisense strands of the rDNA array. In this study, we undertook small RNA profiling in the ascomycete filamentous fungus, *Magnaporthe oryzae* (anamorph *Pyricularia oryzae* Cav), which causes blast, the most destructive disease of rice worldwide. The fungus not only destroys rice leaves, panicles and roots but also infects other cereals including wheat, barley, finger millet and grasses ([@B10; @B11; @B12]). Due to its agronomic significance and molecular genetic tractability, *M. oryzae* has emerged as a model to study fungal pathogenesis. In 2005, the genome (40 Mb) of *M. oryzae* was sequenced and ∼11 000 protein-coding genes identified ([@B13]). Studies using expressed sequence tags (EST), serial analysis of gene expression (SAGE), massively parallel signature sequencing (MPSS) and microarray expression profiling have revealed that the transcriptome is more complex than initially appreciated ([@B13; @B14; @B15]). Here, we conducted pyrosequencing of cDNA and describe a distinct class of small RNAs that are 5′- and 3′-modified, which we refer to as CPA-sRNAs (5′-methylguanosine-capped and 3′-polyAdenylated small RNAs) ([Figure 1](#F1){ref-type="fig"}A). CPA-sRNAs share no similarity to qiRNAs, milRNAs and disiRNAs discovered recently in *N. crassa*, which appear to possess no 5′- and 3′-modifications ([@B8],[@B9]). Figure 1.CPA-sRNA isolation and size distribution. (**A**) Strategy for CPA-sRNA preparation from mycelial total RNA. The protocol ensures capture of RNA species that possess both a 5′-cap and a 3′-polyadenylated tail. The first treatment with BAP prevents RNA containing a 5′-free phosphate from being able to ligate to the 5′-linker. The use of (dT)~20~VN oligo for single-strand cDNA priming allows cDNA to be synthesized exclusively from RNA containing polyA. Following amplification by PCR, small cDNAs (\<200 nt) were purified from a 3% agarose gel and subjected to 454 pyrosequencing. (**B**) Size distribution of CPA-sRNAs (≥16 nt) that matched to the *M. oryzae* genome (BLAST criteria: ≥80% coverage and ≥98% sequence identity). (**C**) CPA-sRNAs mapped to annotated protein-coding TUs. A vertical line represents the TSS and TTS for protein-coding genes. MATERIALS AND METHODS ===================== Fungal strain and growth ------------------------ *Magnaporthe oryzae* isolate 70--15 was used in this study because of the availability of genomic ([@B13]) and transcriptomic ([@B14],[@B15]) resources. Conidia were germinated and mycelia cultured in a liquid medium (0.2% yeast extract and 1% sucrose) by shaking at 200 rpm, 25°C for 3 days. The mycelia were filtered through cheesecloth and used for RNA isolation. RNA isolation, CPA-sRNA library construction and 454 sequencing --------------------------------------------------------------- Total RNA was isolated from 2 g of mycelia using the Trizol method ([@B15],[@B16]). PolyA^+^ RNA was purified using a PolyATtract mRNA Isolation System III (Promega) according to manufacturer's procedure. To construct the CPA-sRNA library, protocols used to generate full-length cDNA were followed, from which small molecules were size selected and sequenced ([@B16]). Briefly, the free phosphate at the 5′-ends of 1 µg polyA^+^ RNA from mycelia was removed by treating with bacterial alkaline phosphatase (BAP, Epicenter) followed by removal of the 5′-methylguanosine caps by treating with tobacco acid pyrophosphatase (Epicenter). PolyA^+^ RNA with an exposed 5′-phosphate was ligated to a 5′-RNA oligo linker (5′-AGCAUCGAGUCGGCCUUGUUGGCCUACUGG-3′) using T~4~ RNA ligase (Epicenter). The ligated polyA^+^ RNA was treated with DNase I (Invitrogen) to remove contaminating genomic DNA and re-purified using the PolyATtract mRNA Isolation System III. The 3′-oligo (dT)~20~VN linker (5′-GCGGCTGAAGACGGCCTATGTGGCC(T)~20~VN-3′) was used to synthesize cDNA using SuperScriptIII (Invitrogen) according to supplier's procedure. RNA was digested with RNase H (Invitrogen). Double-stranded cDNA was amplified with high fidelity Platinum Taq DNA polymerase (Invitrogen) using 5′-PCR primers specific for the 5′-RNA linker (5′-AGCATCGAGTCGGCCTTGTTG-3′) and 3′-PCR primers specific for the 3′-oligo(dT)~20~VN linker (5′-GCGGCTGAAGACGGCCTATGTG-3′). The conditions used for PCR amplification were 94°C for 2 min followed by 30 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 1 min and a final extension at 72°C for 10 min. PCR products were resolved on 3% agarose gels and cDNA between 60 and 200 nt were purified using a Gel and PCR Clean-Up System (Promega). Purified cDNA was ligated to 454 adapters and analyzed directly by 454 sequencing at the Joint Genome Institute, Walnut Creek, CA, USA. CPA-sRNA data analysis ---------------------- We obtained 127 330 raw reads in a FASTA format from a 454 sequencing run. 454 sequencing adaptemer and linkers at 5′- and 3′-ends were removed from raw reads and the remaining sequences were named CPA-sRNAs. Overall, we obtained a total of 80 111 CPA-sRNAs from mycelia with a size of ≥10 nts. We retained 25 389 reads with a size between 16 and 218 nts for matching to V6 *M. oryzae* genome assembly (GenBank ID; NZ_AACU00000000.2) ([@B13]). A detailed matching analysis was carried out using stringent BLASTN criteria of 80% coverage and 98% of sequence identity. We also utilized Magnaporthe transcriptome data ([@B14],[@B15]) including ESTs, MPSS tags and RL-SAGE tags to annotate CPA-sRNAs. All the genomic features (contigs, genes, tRNAs, rRNAs, snRNAs, repeats, mitochondrial genome) and transcriptomic data (ESTs, SAGE, MPSS) were visualized in a genome browser based on gbrowse ([@B17]). Defining the transcriptional unit --------------------------------- To define the transcriptional start and stop sites for protein-coding genes, we devised two approaches. First, we assigned a 5′-transcription start site (TSS) and 3′-transcription termination site (TTS) to gene models supported by ESTs. This provided a TSS and TTS for 2558 and 2551 genes, respectively. For the remaining annotated genes, we defined UTRs as 500 bp from start and stop codons. This is likely a slight overestimate of the average actual UTR length for protein-coding genes, but a value of 500 bp captured the vast majority of TUs. The average 5′-UTR for gene models supported by EST evidence was 327 nt. For other RNA species we defined the 5′-TSS and 3′-TTS as the first and last nucleotide of the mature RNA. For tRNAs, we used 150-nt upstream from 5′-mature tRNA for the 5′-leader and 150-nt downstream from 3′-mature tRNA for the 3′-terminator region. Alignments, read counts and prorating data ------------------------------------------ CPA-sRNAs may align to the genome one or more times. The genomic location of each alignment may correspond to features such as genes, tRNA, rRNA or transposable elements. Thus the alignments were used to map CPA-sRNAs to genomic features. We pursued three methods for describing CPA-sRNA mapped genomic data (alignment counts, read counts and prorating) that account for ambiguity in determining the genomic origin of each CPA-sRNA. Alignment counts are the simple summation of all CPA-sRNA alignments to a given genomic feature. Since CPA-sRNAs may align multiple times to the genome, use of alignment counts alone might result in over counting. This is most evident with CPA-sRNAs that map to transposable elements---there are 20 671 alignments to transposable elements that originate from only 325 CPA-sRNAs. We addressed this issue by defining read counts, such that each CPA-sRNA is counted only once for a given genomic feature to which it maps. As CPA-sRNAs may map to multiple features, the use of read counts does not directly reflect the CPA-sRNAs origin (multiple mappings arise from either multiple alignments to multiple features or a single alignment spanning adjacent features). We further refined our approach by taking into consideration multiple mappings. We prorated the counts for CPA-sRNAs by apportioning the counts across any multiple alignments and any features associated with that alignment (prorating). This was done as an iterative process: first, each CPA-sRNA was assigned a weight based on the number of copies found in sequencing data. Second, the weight from a given read was divided evenly between its genomic alignments. Third, each feature within a given alignment was given an equal portion of that alignments' weight. Last, each sub-feature divided the weight of the parent feature (sub-features exist as components of a feature---i.e. an exon is a sub-feature of gene). Summation of the apportioned CPA-sRNA weights for a given feature yields a balanced portrayal of CPA-sRNA coverage for that feature and summation of values for sub-features equals that of its feature. [Supplementary Figure S1](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1) provides a visualization of prorating using hypothetical examples. Purification of 5′-methylguanosine-capped RNA --------------------------------------------- 5′-methylguanosine-capped transcripts were purified using recombinant eIF4E~K119A,~ which binds 5′-m7GpppN RNA caps with a 10- to 15-fold higher affinity than wild-type eIF-4E ([@B18],[@B19]). GST-tagged eIF4E~K119A~ protein was bound to glutathione agarose beads (4E-beads) for 1 h at room temperature in PBS. The 4E-beads were washed in the binding buffer: 10 mM KHPO~4~, pH 8.0, 100 mM KCl, 2 mM EDTA, 5% glycerol, 0.005% Triton X-100, 1.3% poly(vinyl alcohol) 98--99% hydrolyzed (Aldrich), 1 mM DTT and 20 U/ml RNase inhibitor (Ambion). About 120 µg of total RNA was heat denaturated, diluted in the binding buffer, added to 200 µl (packed bead volume) of 4E-beads in a siliconized tube (Genemate, ISC BioExpress) and mixed for 1 h at room temperature. Samples were briefly centrifuged to pellet the beads with bound RNA and washed three times (5 min each) by mixing at room temperature in the binding buffer. The bound RNA on 4E-beads was phenol/chloroform extracted, precipitated and dissolved in RNase free water. The quantity of 5′-methylguanosine-capped RNA was measured by NanoDrop (Thermo Fisher) analysis and its integrity was determined with an Agilent 2100 Bioanalyzer. 3′-RACE analysis of CPA-sRNAs using 5′-capped RNA ------------------------------------------------- 5′-methylguanosine-capped RNA was treated with DNase I (NEB) to remove any contaminating genomic DNA. cDNA was synthesized in 20 µl reactions by adding the following reagents: 1 µg of 5′-methylguanosine-capped RNA, 50 picomole of 3′-oligo(dT) ~20~VN primer, 5 mM of dNTPs, 1 U of RNaseOut (Invitrogen) and 5 U of Superscript III (Invitrogen). [Supplementary Table S1](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1) lists all primer sequences used in this study. The reverse transcription reaction was incubated at 42°C for 2 h and heat inactivated. For evaluating CPA-sRNAs in 5′-methylguanosine-capped cDNA, PCR amplification was performed using a forward primer specific to the 5′-end of CPA-sRNAs of interest described in the 'Results' section and a reverse primer specific to 3′-oligo(dT)~20~VN linker. PCR was done with high fidelity Platinum Taq DNA polymerase (Invitrogen) and under the following conditions 94°C for 2 min followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 30 s and a final extension at 72°C for 5 min. PCR products were resolved on a 3% agarose gel, purified and cloned into the pGEM-T easy vector according to supplier's procedure (Promega). About 20 randomly selected white colonies were sequenced using the Sanger method. cDNA library characterization ----------------------------- Northern blot analyses were conducted using total RNA, RNA purified using eIF4E~K119A~ or oligo(dT) columns and separated on 15% denaturing polyacrylamide gels. Blots were hybridized with \[γ^32^P\]ATP-labeled oligo(dT)~20~ probes. To document that the cDNA contained long as well as short full-length cDNAs, we confirmed the presence of the full-length actin gene (MGG_03982.6) using specific PCR primers prior to size selection ([Supplementary Figure S2](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). Correlation analysis of CPA-sRNAs with mycelial gene expression and MPSS and SAGE tags -------------------------------------------------------------------------------------- Correlation analyses were conducted using normalized signal intensity values of microarray data for *M. oryzae* mycelia grown in complete media for 48 h and a further 12 h in minimal media (NCBI GEO Accession \#; GSE2716, Sample ID \#s; GSM 52525, GSM 52524, GSM 52520) with the number of assigned CPA-sRNAs using JMP (SAS Institute) software. Analyses were conducted for both CPA-sRNAs mapping in the sense and antisense orientation with expression values for individual genes. Genes were also grouped into 100 bins based on gene expression and the relationship between the mean gene expression and the mean number of CPA-sRNAs per bin compared. Likewise, the relationship between MPSS or SAGE tags, which were both derived from RNA extracted after 72 h growth on complete media, and CPA-sRNAs was determined by comparing the mean number of tags and the mean number of CPA-sRNA per bin. GO annotations for *M. oryzae* genes were obtained from the previously published work ([@B20]). RESULTS ======= CPA-sRNA discovery ------------------ Full-length cDNA was constructed from mycelial RNA, which was separated on an agarose gel and the fraction \<200 nt subjected to 454 sequencing. A total of 127 330 reads were obtained, from which 25 389 CPA-sRNAs (≥16 nt; excluding 3′-polyA sequences) were further analyzed ([Supplementary Table S2](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). Of the CPA-sRNAs, 57.4% (14 547) mapped to version 6 of the *M. oryzae* genome (BLASTN criteria of \>80% coverage and \>98% sequence identity). Interestingly, 84% (12 235) of CPA-sRNAs mapped to unique loci and 16% (2354) mapped to multiple locations in the genome. 10 265 (9780 prorated) CPA-sRNAs mapped to protein-coding TUs ([@B13]), and the remainder mapped to intergenic regions, transposable elements, rRNAs, tRNAs and snRNAs ([Table 1](#T1){ref-type="table"} and [Supplementary Table S3](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). 2778 (2498 prorated) CPA-sRNAs mapped to intergenic regions of *M. oryzae*. Of these, 1130 CPA-sRNAs overlapped EST or SAGE or MPSS sequences ([Table 2](#T2){ref-type="table"}). CPA-sRNAs ranged in length from 16 to 218 nt with a mean of 41 nt ([Figure 1](#F1){ref-type="fig"}B). Table 1.Distribution of CPA-sRNAs mapped to genomic and mitochondrial featuresRead Count[^a^](#TF1){ref-type="table-fn"}Prorated[^b^](#TF2){ref-type="table-fn"}Features[^c^](#TF3){ref-type="table-fn"}TotalSenseAntisenseTotalSenseAntisenseMappedTotalCoverage (%)Genes10 26588943507978075792201432711 04339    Introns68145631337823913946719 6512    Exons998586853386940173402062497730 70516      5′-UTR[^d^](#TF4){ref-type="table-fn"}2981252670523231967356132511 24112       EST Supp.12471201461105107431375255815       Unsupp.185313846601217893325950868311      CDS2227160377915901090500158111 05414       EST Supp.1676123351811668473201085519921       Unsupp.73142634042324318049658558      3′-UTR626056532276548942831206259711 07623       EST Supp.377236431482982288794801255131       Unsupp.3636209021312507139711111796852521tRNA[^e^](#TF5){ref-type="table-fn"}425394312891396628734184    5′-Leader26123724192191115134144    Mature2272261188188027434180    3′-Term1009374136518634155rRNA174117401164316421474898    5.8s828208282033100    8s66660464604141100    18s661660159359211250    28s9329320922922022100snRNA242401616051631Transp. Elements379325102320278422087344861Intergenic27782778--24982498--------Mitochondria4341343403143736    Genes118386381553      CDS33033021613    tRNA33022042020      Mature1101101205    rRNA313103030022100    Intergenic33--33--------[^1][^2][^3][^4][^5] Table 2.Association of CPA-sRNAs with other transcriptional evidenceGenome[^a^](#TF6){ref-type="table-fn"}IntergenicGenes[^b^](#TF7){ref-type="table-fn"}Read Count[^c^](#TF8){ref-type="table-fn"}Features[^d^](#TF9){ref-type="table-fn"}Read CountFeaturesRead CountFeaturesEST or ESS[^e^](#TF10){ref-type="table-fn"}10 2777898113074410 1494095ESS4747288860340410 1114035    MPSS229313323061581626712    SAGE2932155635224621501032EST8969501075634089132951[^6][^7][^8][^9][^10] CPA-sRNA validation ------------------- To validate CPA-sRNAs, 5′-methylguanosine-capped RNA was purified from total RNA using a high affinity variant of eIF-4E, which was previously used to prove that specific miRNA precursors have 5′-methylguanosine caps ([@B18],[@B19]). Gel blot analysis of 5′-methylguanosine-capped RNA using \[γ^32^P\]ATP labeled oligo(dT)~20~ revealed a smear from 20 to 200 nt confirming diversity of length and that CPA-sRNAs contain both a 5′-methylguanosine cap and polyA tract ([Supplementary Figure S2](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). The presence of a 3′-polyA tail was confirmed by 3′-RACE on individual CPA-sRNAs, which were subsequently cloned and sequenced. Sequencing of 3′-RACE products confirmed that CPA-sRNAs mapped to protein-coding genes, transposable elements, snRNAs, tRNAs, rRNA genes and to intergenic locations and is described in more detail below ([Figure 2](#F2){ref-type="fig"}B--F and [Supplementary Figure S2](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). Figure 2.CPA-sRNA validation using 3′-RACE. (**A**) Total RNA from *M. oryzae* was used to purify 5′ methylguanosine-capped RNAs using recombinant eIF4E~K119A~ bound to beads ([@B21]). 5′ methylguanosine-capped RNA was treated with DNase I and single-stranded cDNA synthesized using an oligo (dT)~20~VN primer. PCR amplification was performed using a forward primer to the 5′-end of specific CPA-sRNAs and reverse primer specific to the oligo (dT)~20~VN linker. PCR products were analyzed on 3% agarose gels, bands eluted, cloned into pGEM-T vectors and Sanger sequenced. PCR products were resolved on a 3% agarose gels for (**B**) protein-coding mRNA (MGG_0383.6, MGG_6594.6, MGG_0469.6, MGG_0592.6, MGG_02597.6, MGG_07928.6, MGG_10680.6, MGG_14279.6 and MGG_01210.6); (**C**) tRNAs (Ala: MGG_20297.6, Cys: MGG_20209.6, Gln: MGG_20266.6 and Leu: MGG_20218.6); (**D**) rRNAs (18S and 28S); **(E**) snRNAs (U6 and U2) and (**F**) retroelements (MAGGY-LTR). A DNA ladder is shown on the left of each panel. Arrows indicate PCR products that were sequenced. We also examined the genomic context of CPA-sRNAs to exclude the possibility that they may have arisen from loci corresponding to longer RNAs rich in adenosine. 83% (12 096 out of 14 547) of CPA-sRNAs aligned to genome regions lacking adenosine enrichment (≥5As) (data not shown), indicating that most CPA-sRNAs were not derived from internal poly-adenosine sequences of transcribed regions. We found that many CPA-sRNAs mapped to MPSS and SAGE tags derived from 3′-polyadenylated RNA located in intergenic regions and genes ([Table 2](#T2){ref-type="table"}). These tag associations were previously unexplained but in light of the present findings, they were likely derived from 3′-polyadenylated small RNAs. Taken together, these data from different approaches provide compelling evidence that CPA-sRNAs exist in fungal tissue and represent a distinct class of small RNAs. CPA-sRNAs associate with transcription termini of protein-coding genes ---------------------------------------------------------------------- A total of 10 265 (9780 prorated) CPA-sRNAs mapped to 4327 (39% of the total number) predicted protein-coding mRNAs (TUs), with more than a quarter (3507, 2201 prorated) mapping in the antisense orientation ([Table 1](#T1){ref-type="table"}). The majority of CPA-sRNAs mapped to UTRs (2981 (2323 prorated) to 5′-UTRs and 6260 (5489 prorated) to 3′-UTRs), whereas only 681 (378 prorated) mapped to introns. Examination of sense CPA-sRNAs mapping to 5′- or 3′-UTRs revealed that the vast majority associated with the transcript initiation (TSS) or termination (TTS) site, respectively ([Figure 1](#F1){ref-type="fig"}C). CPA-sRNAs were predominantly (4095 out of 4327) associated with genes supported by ESTs, MPSS and RL-SAGE tags ([Table 2](#T2){ref-type="table"}). A 3′-RACE was used to confirm the presence of CPA-sRNAs for nine randomly selected protein-encoding genes ([Figure 2](#F2){ref-type="fig"}B), which included *S*-adenosyl methionine synthetase (MGG_0383.6), chitinase 18--11 (MGG_06594.6), Sad1/UNC domain-containing protein (MGG_00469.6), cell wall glucanosyl transferase Mwg1 (MGG_00592.6), yjeF-related protein (MGG_02597.6), ubiquitin (MGG_07928.6), 40S ribosomal protein S24 (MGG_10680.6), glutamine synthetase (MGG_14279.6) and nuclear encoded mitochondrial hypoxia responsive domain containing protein (MGG_01210.6). The expected 3′-RACE product size of 80--200 nt were obtained for all nine genes. Sequencing of cloned 3′-RACE products confirmed they aligned with CPA-sRNAs obtained from pyrosequencing, including the splice junction for chitinase 18--11 (MGG_06594.6) ([Figure 3](#F3){ref-type="fig"}A). Figure 3.CPA-sRNA sequences validated for mRNA, tRNA and rRNA loci. (**A**) 454 and 3′ RACE PCR clone sequence location at the chitinase 18--11 gene (MGG_06594.6). A dashed line in the 3′-RACE sequence data represents the absence of intronic sequence (95 nt) for the chitinase gene. (**B**) CPA-sRNAs associated with Gln tRNA locus (MGG_20266.6). Underlined sequence data represent non-templated nucleotide. (**C**) CPA-sRNAs clustering at the 5′-end of 18S rRNA locus. To identify a possible role of CPA-sRNAs, we correlated their abundance with mycelia gene expression. Overall, we observed a positive correlation between CPA-sRNAs mapping in the sense orientation and mycelial gene expression, although not all individual genes followed this pattern ([Figure 4](#F4){ref-type="fig"}A and B and [Supplementary Table S4](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). Notably, the most highly expressed group of genes had highest numbers of mapped CPA-sRNAs. Inspection of 127 genes with ≥10 CPA-sRNAs mapped in the sense orientation showed that nearly all were functionally assigned with gene ontology (GO) terms involved in metabolism, with 67 (53%) being assigned to mycelial development and 43 (34%) to translation \[([@B20]) [Figure 4](#F4){ref-type="fig"}E and [Supplementary Table S5](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)\]. Of the latter, most ([@B42]) were assigned to structural components of the ribosome ([Figure 4](#F4){ref-type="fig"}F). Further analysis confirmed a strong positive correlation between CPA-sRNAs and gene expression for all (65) annotated structural ribosomal proteins (see asterisk in [Figure 4](#F4){ref-type="fig"}A and [Supplementary Table S4](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). In contrast, antisense CPA-sRNAs did not map primarily to TSS and TTS, nor was there evidence supporting a correlation with gene expression ([Figures 1](#F1){ref-type="fig"}C and [4](#F4){ref-type="fig"}C and D). In addition, we also observed a strong positive correlation between both sense-mapping MPSS or SAGE tags and sense-mapped CPA-sRNAs ([Supplementary Figure S3](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). Figure 4.Correlation of expression and GO annotation of genes with mapped CPA-sRNAs. Correlation analysis of gene expression and number of mapped CPA-sRNAs based on (**A**, sense mapping; **B**, antisense mapping) bins and (**C**, sense mapping; **D**, antisense mapping) individual genes. Genes were grouped into 100 bins based on mycelial gene expression; each bin contains 99 genes. Average gene expression per bin was plotted verses the average number of CPA-sRNAs for each bin. Asterisks indicates the average number of CPA-sRNAs and expression for 65 genes annotated as ribosomal structural proteins. (**E**) Biological process and (**F**) molecular function gene ontology annotation for 127 genes with ≥10 mapped CPA-sRNAs. Numbers on top of vertical bars indicate number of genes per category. CPA-sRNAs derived from RNA Pol I- and Pol III-transcribed genes --------------------------------------------------------------- We detected 425 (289 prorated) CPA-sRNAs that mapped to 287 tRNA loci, with 31 (6 prorated) that mapped in the antisense orientation ([Table 1](#T1){ref-type="table"}, [Supplementary Table S3](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). Of the 425 CPA-sRNAs, 114 mapped to pseudo-tRNAs, of which there are 141 essentially identical copies in the *M. oryzae* genome. Most CPA-sRNAs mapped around the beginning or end of the mature tRNA ([Figure 3](#F3){ref-type="fig"}B and [Supplementary Figure S4A](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). Several CPA-sRNAs corresponded to the entire tRNA, whereas others were shorter or longer than the corresponding tRNA. A number of CPA-sRNAs mapped to positions ∼--50 and ∼+50 nt from 5′- and 3′-ends, respectively, of the mature tRNA locus and likely correspond to the pre-tRNA transcript. A 3′-RACE confirmed CPA-sRNAs for five tRNAs ([Figure 2](#F2){ref-type="fig"}C and [Supplementary Figure S4B](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). The expected 3′-RACE PCR and sequence products were obtained for Ala tRNA (MGG_20297.6), Cys tRNA (MGG_20209.6), Gln tRNA (MGG_20266.6) and Leu tRNA (MGG_20218.6). The 3′-RACE and sequencing also revealed CPA-sRNAs corresponding to all three Pro tRNA paralogs (MGG_20065.6; MGG_20044.6 and MGG_20298.6). Analysis of CPA-sRNAs mapping to rRNA revealed 1675 (1597 prorated) that mapped to 18S-5.8S-28S rRNA repeat locus ([Table 1](#T1){ref-type="table"} and [Supplementary Figure S5A and B](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). We obtained diverse CPA-sRNAs for 5.8S rRNA, many of which were supported by SAGE tags ([Supplementary Figure S5C](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). We found 66 (46 prorated) CPA-sRNAs for the 8S rRNA locus, which has multiple copies dispersed throughout the *M. oryzae* genome ([Table 1](#T1){ref-type="table"} and [Supplementary Table S3](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). CPA-sRNAs associated with the 5′-end of 18S rRNA and 28S rRNA were validated by 3′-RACE analysis and sequencing ([Figures 2](#F2){ref-type="fig"}D and [3](#F3){ref-type="fig"}C). CPA-sRNAs derived from repetitive elements ------------------------------------------ More than 10% of the *M. oryzae* genome consists of repetitive elements ([@B13]). We found 379 (320 prorated) CPA-sRNAs mapping to transposable elements, including 102 (42 prorated) antisense mappings ([Table 1](#T1){ref-type="table"} and [Supplementary Table S3](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). Fifty (46 prorated) CPA-sRNAs mapped specifically to the LTR region of MAGGY, a gypsy-like element linked with pathogenicity ([@B21]), which were validated by 3′-RACE analysis ([Figure 2](#F2){ref-type="fig"}E and [Supplementary Figure S6](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). The LTR (250 nt) of MAGGY is structurally similar to retroviruses, which acts as a transcription initiator and terminator ([@B21]). We also identified a number of CPA-sRNAs that mapped to other retro-elements including MGR583 or SINE element (218; 155 prorated), PYRET (40; 29 prorated), OCCAN (11; 8 prorated) and MOLLY (5; 3 prorated), as well as to DNA transposon, POT2 (64; 20 prorated). Overall, we observed that CPA-sRNAs mapped primarily to the LTR (putative TSS and TTS) of retro-transposons while CPA-sRNAs were distributed across the entire transcript of DNA-transposons. Few CPA-sRNAs map to mitochondrial TUs -------------------------------------- Only 43 (43 prorated) CPA-sRNAs aligned to the mitochondria genome (∼35 kb) ([Table 1](#T1){ref-type="table"}; [Supplementary Table S3](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1) and [Figure S7](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). Of these, 31 (30 prorated) mapped to rRNA genes (rrnL, large subunit ribosomal RNA and rrnS, small subunit ribosomal RNA). In contrast to nuclear genes, only 11 (8 prorated) CPA-sRNAs were found to align to the coding sequence of two mitochondrial genes (MGG_21013.6, cytochrome b and MGG_21007.6, ATP synthase subunit 6). We did not detect CPA-sRNAs for mitochondrial tRNAs except for the SeC (MGG_21117.6) and Arg tRNA (MGG_21120.6) loci. Mitochondrial transcripts are typically transcribed by a simpler RNA polymerase, homologous to the bacteriophage T7/T3 RNA polymerase subunit as compared to the more complex nature of nuclear RNA Pol I, II and III ([@B22]). Although, we have limited knowledge of the mitochondrial RNA polymerase in filamentous fungi, our data suggest that CPA-sRNAs are biased toward nuclear RNA polymerase-derived transcripts. Analysis of CPA-sRNAs not matching the *M. oryzae* genome sequence ------------------------------------------------------------------ Deep sequencing projects of small RNA species typically reveal many sequences that do not align to the genome and are often disregarded as sequencing errors. However, evidence now suggests that these molecules result from post-transcriptional RNA modifications ([@B23; @B24; @B25]). Gowda *et al.* ([@B15]) reported previously that a large fraction of MPSS and RL-SAGE tags did not match the *M. oryzae* genome. Similarly, we found that ∼42% of CPA-sRNAs (10 824 out of 25 389) could not be aligned to the genome. Interestingly, we identified 388 CPA-sRNAs that matched only to *M. oryzae* ESTs but not to the genome (data not shown). In addition, 1585 CPA-sRNAs matched SAGE and/or MPSS tags but were unaligned to the genome. Further examination revealed that a small number (284) of unaligned CPA-sRNAs aligned to the genome sequence of strains P123 and Y34 (Y. Peng *et al.* unpublished data) suggesting that some CPA-sRNAs map to gaps in the 70--15 reference strain genome sequence. Manual inspection revealed evidence of 46 chimeric CPA-sRNAs ([Supplementary Table S6](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)), some of which were possibly derived from the fusion of RNAs from two or more non-contiguous genomic locations ([@B3]). We observed chimeric RNA fusions in head-to-head or head-to-tail orientations of the same exon, two exons of the same gene, exon--exon junctions of two genes, protein-coding region--intergenic or rRNA, intergenic--intergenic, rRNA--intergenic, rRNA--rRNA and rRNA--tRNA. Twenty-five percent of chimeric CPA-sRNAs were derived from exonic regions, where fusion points coincide with the canonical splicing sites ([Supplementary Table S6](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1)). We also found evidence for short homologous sequences (SHS) for 44% of chimeric CPA-sRNAs, similar to reports of animal ([@B26]) and plant ([@B27]) chimeric RNAs. Further examination of these chimeric CPA-sRNAs revealed a high rate (96%) of non-template nucleotide additions, 20% (9 out of 45) of which had 1--3 non-template nucleotides internally at the point of RNA fusion. We also observed non-templated nucleotides in many genome-matched CPA-sRNAs. For example, 54% of Gln tRNA-associated CPA-sRNAs (7 out of 13) had non-templated nucleotides (G, AAC, A, C, C, T) at the 3′-end ([Figure 3](#F3){ref-type="fig"}B). Similarly for 5.8S rRNA CPA-sRNAs, 81% (13 out of 16) and 88% (14 out of 16) had non-templated nucleotides at the 5′- and 3′-regions, respectively. Non-template sequence diversity at the 3′-end of 5.8S rRNA is supported by 26 RL-SAGE tags that matched the 3′-ends of CPA-sRNAs ([@B15]). Thus chimeric and non-templated nucleotide additions may explain why at least some CPA-sRNAs do not align to the genome. DISCUSSION ========== *Magnaporthe oryzae* has emerged as a model to study fungal pathogenesis due to its agronomic significance, genetic tractability, availability of genomic sequences and expression datasets including expressed sequence tags (EST), serial analysis of gene expression (SAGE) tags, massively parallel signature sequencing (MPSS) tags and microarray data ([@B13; @B14; @B15]). During the course of analyzing a full-length cDNA library, we identified CPA-sRNAs. These are a distinct class of small RNAs because these contain both 5′-methylguanosine-capped and 3′-polyadenylated and associate with TUs of RNA Pol I (rRNA), Pol II (mRNA/retrotransposons) and Pol III (snRNA/tRNA). RNA Pol II transcripts have been intensively studied with respect to 5′- and 3′-end modifications ([@B28]). Nuclear capping occurs co-transcriptionally by adding a 5′-*N*-methyl guanosine with an inverted 5′--5′-triphosphate bridge to the first gene-encoded nucleotide of RNA Pol II transcripts. A PolyA tail is added to 3′-ends of RNA Pol II transcripts by post-transcriptional events. These modifications protect RNAs from nucleases, and signal for RNA export and translation. Additional capping in the cytoplasm ([@B29]) and polyadenylation linked to nonsense-mediated mRNA decay ([@B6]) have been reported in eukaryotes. Recently, small ncRNAs such as CUTs and SUTs have been described in *S. cerevisiae* that appear to be the products of RNA Pol II and thus are likely capped and polyadenylated ([@B6]). This suggests that research on capping and polyadenylation is far from complete and further studies may shed further functions of capping and polyadenylation of small and long transcripts. Although the ends of RNA Pol I and III transcripts are less well defined as compared to RNA Pol II, it has been shown in yeast recently that some of these elements contain 3′-polyA tails ([@B30]). Furthermore, small RNAs (21--400 nt) in animals ([@B31],[@B32]) and plants ([@B33]) possess short stretches of adenosine (1--7 nt). The snRNA U6, which is transcribed by Pol III, carries a methylguanosine cap at the 5′-end ([@B34]). In our study, we obtained CPA-sRNAs for both U6 and U2 ([Figure 2](#F2){ref-type="fig"}F). Recently small RNAs from humans have been reported to possess cap structures at the 5′-ends ([@B35],[@B36]), many of which were associated with the TSS. Several reports have shown that some classes of small RNA (\<200 nt) associate with RNA Pol II TSS and TTS ([@B1],[@B37],[@B38]). Our study provides detailed evidence that CPA-sRNAs are not only associated with TSS and TTS of protein-coding genes but are also associated with snRNAs, tRNAs, rRNAs and retrotransposons. qiRNAs, milRNAs and disiRNAs reported recently in *N. crassa*, however, do not appear to possess 5′- and 3′-modified ends ([@B8],[@B9]). We have no knowledge of the mechanisms of CPA-sRNA biogenesis; however, it is likely they are either derived directly from the genome or are processed from longer transcripts. In the former case, they could be the product of an uncharacterized RNA polymerase, such as Pol IV, a homolog of DNA-dependent RNA polymerase II ([@B36],[@B39]), or Pol III ([@B40]), which are then processed, if necessary, by adding methylguanosine to the 5′-end and polyA to the 3′-ends, possibly by a cytosolic mechanism ([@B29]). Alternatively, CPA-sRNAs could be derived by the action of an endo-ribonuclease/splicing complex on long mRNAs releasing short RNA fragments, which might be spliced together, undergo addition of untemplated nucleotides before adding a 5′-methylguanosine cap and 3′-polyA tail. Currently, we have no direct evidence for a biological function for CPA-sRNAs. However, a growing body of research has shown that non-protein-coding small RNAs modulate many biological processes, including chromosome replication, chromatin remodeling, transcription regulation, RNA processing and stability as well as protein stability and translocation ([@B2],[@B3],[@B41]). Several studies have shown that ncRNA transcription is most predominant at the promoters (or TSSs), and also occurs at intergenic regions as well as within genes. Our finding of a strong positive correlation between CPA-sRNAs and gene expression suggests CPA-sRNAs may play a positive role in gene regulation. Small RNAs complementary to promoter regions have been shown to activate gene expression in several different cellular contexts ([@B42; @B43; @B44]). These RNAs are typically biased toward genes with higher levels of expression ([@B38]). Similarly CUTs are also typically positively correlated with gene expression ([@B4]). It is possible that CPA-sRNAs associate with transcriptionally active regions forming RNA--DNA hybrids, which create transcriptional bubbles (nucleosome-free single-stranded DNA) at the TSS and TTS ([@B45]). At TSS, CPA-sRNAs may facilitate transcription factor and RNA polymerase binding or act as primers for RNA synthesis, whereas CPA-sRNAs at TTS may block further transcription and facilitate the release of the RNA polymerase. On the other hand, as has been pointed out for CUTs, it is possible they simply reflect inefficient start site initiation and represent 'transcriptional noise' ([@B6]). Finally, we provide an explanation of why at least some small RNAs do not align with the genome sequence. While we cannot discount that many may be the result of sequencing error, our analyses reveal that some are the products of fragment fusion or contain non-templated additions at their termini or point of fusion such that they no longer align to the genome using strict criteria. We suggest that RNA editing and/or posttranscriptional modification may be involved in generating CPA-sRNA diversity, thus increasing the complexity of the small RNA transcriptome. However, their origin and significance remain to be determined. While there remains much to be learned about CPA-sRNA biogenesis and function, their discovery and characterization add another fascinating chapter in genome and RNA biology. SUPPLEMENTARY DATA ================== [Supplementary Data](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1) are available at NAR Online. FUNDING ======= Funding for open access charge: United States Department of Agriculture (Award \#2005-04936 to R.A.D.); National Institutes of Health grant (CA63640 to C.H.H.). *Conflict of interest statement*. None declared. Supplementary Material ====================== ###### Supplementary Data We appreciate all the members in the fungal genomics laboratory for their feedback on this project. We also thank Malathy Krishnamurthy for help editing this manuscript. [^1]: ^a^Read Count is the number of CPA-sRNAS that map on to particular genomic features. Note: values for genome features are typically less than the sum of component sub-features due to reads mapping to multiple locations and/or features. [^2]: ^b^Prorating divides the weight of any given read between alignments and features. This takes into account situations where reads map to multiple locations and/or are annotated with more than one feature (see 'Materials and Methods' section and [Supplementary Figure S1](http://nar.oxfordjournals.org/cgi/content/full/gkq583/DC1) for more detail). Both read counts and prorated are divided into sense/antisense with respect to the feature mapped. [^3]: ^c^For each feature, the number of members mapped by a CPA-sRNA is given, followed by the total number of possible features and the percentage mapped. [^4]: ^d^Values indicate number of CPA-sRNAs (read count or prorated) that map to features (5′-UTR, CDS or 3′-UTR) within gene models that are supported by ESTs (EST supp.) or have no supporting EST evidence (Unsupp.). [^5]: ^e^tRNA entries include alignments to pseudo-tRNA. [^6]: ^a^CPA-sRNAs that overlap the sequence of ESS and/or ESTs at the same genome (or intergenic) location. [^7]: ^b^CPA-sRNAs that map to transcriptionally (ESS and/or EST) supported gene loci. [^8]: ^c^Number of CPA-sRNAs that map to a given feature containing specified transcriptional evidence. [^9]: ^d^Number of features containing specified transcriptional evidence associated with CPA-sRNAs. [^10]: ^e^ESS (expressed short sequences) are either MPSS or SAGE tag annotations.

Sir, We read with interest the article on willingness of truckers for new HIV interventions published in the June 2013 issue[@ref1]. The study provides a useful insight into the willingness for newer interventions for HIV prevention among truckers. The study also included helpers in the sample, which is commendable since this group has so far been excluded from certain large scale surveys like the Integrated Behavioural and Biological Assessment survey[@ref2], even though they endure similar working conditions as drivers. However, we would like to raise certain issues which concerned us. The authors have used prevalence of HIV as 6 per cent for sample size calculation based on a previous study[@ref3]. However, on perusal of the quoted reference, the overall prevalence of HIV reported was 1.72 per cent and that among men in a transport related occupation was 4.79 per cent. Also, the said study was conducted among the general population of Guntur district of Andhra Pradesh. Thus, the use of these findings to calculate sample size in the context of truckers is questionable. The authors have mentioned that the number of truckers selected from each halt point was proportional to the number of trucks estimated to be present at each point, using a systematic random sampling method. If so, since the total number of truckers at each point would vary, the sampling interval at each point should have been chosen based on that number and the numbers of truckers required at that point; and not restricted to every 3^rd^ or 5^th^ truck as has been mentioned in the study. The method used to select one trucker from each truck is not mentioned. The rationale behind selecting drivers and truckers in a 2:1 ratio is also not explained. Similarly, the reason behind exclusion of trucks belonging to N3 (mass above 12 tonnes) category is not clear. Including ability to converse in one of three languages, *i.e*. English, Hindi and Telugu, as a criterion for selection into the study might have led to the exclusion of truckers belonging to States like Tamil Nadu and Karnataka (as is evident from the results section, which shows a very low proportion of truckers belonging to these States). This might be the reason for very low contact with HIV prevention services observed in the study. This is further supported by the fact that Targeted Interventions in Tamil Nadu were known to have a good coverage of bridge population like truckers at the time of the conduct of the study[@ref4]. These findings bring in the question of selection bias which has not been addressed as limitations and thus cast doubts on whether the results could be generalized to the whole of south India, as claimed by the authors in the discussion section. The low contact with HIV prevention programme reported might have also resulted from social desirability bias, as the information was gathered using direct interviews. Multiple logistic regression analysis was carried out to find various associations and to arrive at adjusted odds ratios. It would have been more relevant to report the *P* values arrived at through the multiple logistic regression analyses, instead of performing Chi square tests for each of the variables (Tables II and III in the study)[@ref1]. The categorization of some of the variables in Table III is not clear, *e.g* in Table III, working for 5 years and age 30 years falls in neither of the categories mentioned. It was also important to understand the characteristics of truckers willing for other newer interventions apart from circumcision (information not provided). One of the important variables which should have been reported is the economic background of the truckers, as this would have had a huge impact on the willingness to pay. In the discussion of results, two studies are quoted for having reported high acceptability of oral HIV testing in the high risk group of female sex workers (FSWs). However, neither of these pertains to FSWs: one of the studies was conducted among women admitted in a labour ward and the other is an editorial on applicability of oral HIV testing in developing countries[@ref5][@ref6].

1. Introduction =============== The occurrence of postoperative pulmonary complications (PPCs) is a common concern among surgical and critical care wards due to its association with morbidity, mortality, hospital and intensive care unit (ICU) readmission, prolonged hospital stay, and financial burden.^\[[@R1]--[@R5]\]^ The incidence of PPCs reported in the literature varies between 9% and 40% due to its association with different types of surgery, quantification of respiratory problems and other perioperative patient-related variables.^\[[@R3],[@R4],[@R6]\]^ Negative pressure pulmonary edema (NPPE) is a serious well-described pulmonary complication.^\[[@R7]--[@R11]\]^ The occurrence of NPPE is rare; the incidence ranges from 0.01% to 0.05% among all anesthetic procedures and 4% of all laryngospasm events.^\[[@R12],[@R13]\]^ It occurs after an intense inspiratory effort against an obstructed or closed upper airway and generates a large negative airway pressure, leading to severe pulmonary edema (transvascular fluid filtration and interstitial/alveolar edema) and hypoxemia.^\[[@R7]--[@R11],[@R14]\]^ The most common cause (50%) of upper airway obstruction documented in cases of NPPE is the laryngospasm; it usually starts with an isolated respiratory failure event that occurs during intubation or immediately after extubation or laryngeal mask removal.^\[[@R12]\]^ Two types of NPPE have been described in the literature: type I occurs when there is forced inspiration in the presence of acute obstruction of the airway,^\[[@R11],[@R15]\]^ while type II occurs after releasing a chronic airway obstruction (resection of laryngeal tumor and intrathoracic lesions).^\[[@R9],[@R10]\]^ There is not a strong consensus of the predictors for this complication, but the risk factors associated with NPPE include, but are not limited to obesity, short neck, acromegaly, obstructive sleep apnea, and upper airway surgery.^\[[@R12],[@R16]\]^ In addition, NPPE is prevalent in young, healthy and muscular patients with the ability and strength to create higher negative pressures.^\[[@R9],[@R17]\]^ The treatment of NPPE is supportive depending on the severity of the symptoms: it includes oxygen supplement to demand and positive end-pulmonary pressure in critical cases.^\[[@R8],[@R12],[@R18],[@R19]\]^ 2. Case description =================== A 39-year-old Hispanic male, 69 kg, 170 cm (body mass index: 23,), American Society of Anesthesiologist physical status (ASA) I, with a history of untreated peptic ulcer disease and appendectomy (10 years ago) was admitted to the hospital and scheduled to undergo median nerve neurorrhaphy with graft from lower left limb (sural nerve) due to sharp injury under general anesthesia. Preoperative vital signs, physical examination and laboratory assessments were unremarkable. Patient\'s verbal consent was obtained in order to publish the case report with de-identified data as per institutional standard procedures. Conventional intraoperative monitoring (electrocardiogram, noninvasive blood pressure, and peripheral oxygen saturation) and intravenous access for fluid/drug administration were performed per institutional routine. During anesthesia induction, the patient in supine position received intravenous propofol (140 mg), cisatracurium (6 mg), and remifentanil 0.5 mcg/kg/min. After loss of consciousness confirmation, a single endotracheal intubation attempt with an 8.0 tube was performed. Anesthesia maintenance was achieved with isoflurane 0.6 mean alveolar concentration (MAC) and intravenous remifentanil at 0.3 mcg/kg/min. Ventilator mode consisted on continuous mandatory ventilation (CMV) 500 mL, positive end expiratory pressure (PEEP) at 5 cmH~2~O, respiratory rate (RR) was 10/min, and ratio 1:2. The length of surgery was 150 minutes; the patient remained thermodynamically stable in supine position with 105-minutes tourniquet application on left arm. Intravenous dexamethasone (8 mg) and metoclopramide (10 mg) was administered as antiemetic prophylaxis. Analgesia was managed with intravenous morphine (6 mg). A total of 1500 mL of lactate ringer was infused during surgery. At the end of surgery, anesthetic agents were ceased and after patient responded to commands, maintained eye contact, and a TOF (Train-Of-Four) of 94% was documented, the endotracheal extubation was performed. However, immediately after extubation, anesthesia care providers observed marked respiratory distress, inspiratory effort, wheezing, cyanosis and rapid development of hypoxia with oxygen saturation level (SpO~2~) below 90% for approximately 30 seconds. Therefore, immediate positive pressure mask ventilation with 100% fraction of inspired oxygen (FiO~2~) was started and improved patient clinical status one minute after. Consequently, he was transferred to the postanesthesia care unit (PACU) with normal vital signs and no signs of respiratory distress. Nevertheless, 15 minutes after PACU admission, patient presented multiple episodes of hemoptysis, tachypnea (25 per minute), SpO~2~ of 82% and abundant bilateral pulmonary rales. Arterial blood gas revealed acute metabolic acidosis and included pH 7.33, partial pressure of oxygen (PaO~2~) 44.4 mm Hg, partial pressure of carbon dioxide (PaCO~2~) 40.0 mm Hg, bicarbonate (HCO3) 20.5 mmol/L, base excess −5.5 mmoL and oxygen saturation of 79.3%, lactate 2.4 mmol/L. A baseline chest x-ray revealed symmetric parenchymal opacities with ground-glass attenuation and bilateral multilobar consolidations patterns (Fig. [1](#F1){ref-type="fig"}). {#F1} Based on the aforementioned information, a diagnosis of NPPE was established and supportive treatment was immediately initiated with noninvasive mechanical ventilation with a PEEP at 10cmH2O, intravenous furosemide (20 mg) every 12 hours, and fluids restriction. Patient remained in PACU for continuing monitoring and laboratory/imaging follow-up testing until next morning. On postoperative day 1, patient responded satisfactory to supportive treatment; his arterial blood gas revealed a pH 7.43, PaO~2~ 58.6 mm Hg, PaCO~2~ 39.0 mm Hg, HCO~3~ 25.3 mmol/L, base excess 1.0 mmoL and oxygen saturation of 92.8%; therefore, patient was transferred to the general care floor requiring no oxygen supplementation 12 hours after extubation time. Subsequently, on postoperative day 3, patient was discharged to home in stable conditions after the evaluation of a chest x-ray that revealed right basal parenchymal opacity related to atelectasis with non-clinical significance (Fig. [2](#F2){ref-type="fig"}). {#F2} 3. Discussion ============= We described a case of pulmonary edema due to negative pressure after endotracheal extubation. The classic clinical evolution of NPPE begins immediately after the emergence phase of general anesthesia; however a late onset (up to 24 hours) has been reported in the literature, so patients presenting with laryngospasm should be monitored for up to 24 hours after the emergence from general anesthesia.^\[[@R20]\]^ A rapid diagnosis and intervention is required for NPPE as it could eventually lead to acute severe hypoxemia.^\[[@R21],[@R22]\]^ For this particular patient, Type 1 NPEE was promptly determined which is usually the result of forced inspiration against a partially obstructed or closed glottis, creating an excessive decrease in intrathoracic pressure of approximately −100 to −140 cmH~2~O (normal value: −4 cmH~2~O).^\[[@R23],[@R24]\]^ This causes an increase in venous return to the right side of the heart (preload), producing an increase in the hydrostatic pressure of the pulmonary venous circulation, which generates a trans-pulmonary hydrostatic gradient with movement from a space with high pressure (pulmonary capillaries) to one with low pressure (pulmonary and alveolar interstitium) and disruption of alveolar capillary membrane.^\[[@R25]\]^ Consequently, gas exchange decreases producing hypoxemia, catecholamine release, and systemic and pulmonary hypertension.^\[[@R26]\]^ The degree of "alveolar flooding" depends on the extent of interstitial edema, the presence or absence of alveolar epithelial lesion or the ability of the alveolar epithelium to actively remove the fluid.^\[[@R27]\]^ Identification of abundant rales in pulmonary fields, tachypnea, and gradual oxygen desaturation were all symptoms and signs that are consistent with NPPE diagnosis.^\[[@R21]\]^ Patient\'s risk factors are his relatively young age and his muscular appearance.^\[[@R23]\]^ Noninvasive respiratory support performed in this case represents the common treatment for NPPE patients and it is used as a valid approach by other recent studies.^\[[@R28]--[@R30]\]^ Focusing on the prevention and early identification of postoperative laryngospasm could further lower the incidence of NPPE. Prevention usually involves lidocaine topical administration or laryngotracheal anesthesia, use of throat packs and cautious oropharyngeal suction in order to reduce stimulation/manipulation and irritation of the larynx.^\[[@R16]\]^ 4. Conclusion ============= In conclusion, despite NPPE being reported in the literature as a self-limited event with favorable prognosis and simple management, its prevention and/or early identification diminishes the financial burden and the severity of clinical outcomes with the avoidance of prolonged hospitalizations or intensive care unit admissions. The occurrence of NPPE in the operating room could be managed successfully with supportive regimens, effective clinical team coordination, and awareness of the importance of its rapid diagnosis. Potential risk factors and accessible preventive methods for patients undergoing surgery should be considered, in order to maximize safety in surgical and intensive care settings. Author contributions ==================== **Conceptualization:** Alberto Uribe, Byron Rosero-Britton. **Data curation:** Alberto Uribe, Byron Rosero-Britton. **Formal analysis:** Alberto Uribe, Byron Rosero-Britton. **Investigation:** Alberto Uribe, Byron Rosero-Britton. **Methodology:** Alberto Uribe, Byron Rosero-Britton, Nicoleta Stoicea. **Project administration:** Alberto Uribe, Byron Rosero-Britton, Nicoleta Stoicea. **Resources:** Byron Rosero-Britton. **Supervision:** Alberto Uribe, Byron Rosero-Britton, Nicoleta Stoicea, Sergio D. Bergese. **Validation:** Alberto Uribe, Byron Rosero-Britton, Nicoleta Stoicea, Sergio D. Bergese. **Visualization:** Alberto Uribe, Byron Rosero-Britton. **Writing -- original draft:** Alberto Uribe, Byron Rosero-Britton, Nicoleta Stoicea, Luis Periel, Sergio D. Bergese. **Writing -- review & editing:** Alberto Uribe, Byron Rosero-Britton, Nicoleta Stoicea, Luis Periel, Sergio D. Bergese. Alberto Uribe orcid: 0000-0001-7897-8322. Abbreviations: ASA = American Society of Anesthesiologist physical status, CMV = continuous mandatory ventilation, FiO~2~ = fraction of inspired oxygen, HCO~3~ = bicarbonate, MAC = mean alveolar concentration, NPEE = negative pressure pulmonary edema, PaCO~2~ = pressure of carbon dioxide, PACU = postanesthesia care unit; PaO~2~ = pressure of oxygen, PEEP = positive end expiratory pressure, PPCs = postoperative pulmonary complications, SpO~2~ = oxygen saturation, TOF = train-of-four. The authors have no conflicts of interest to disclose.

Background {#Sec1} ========== Obesity (body mass index (BMI) ≥ 30) is defined as a condition of abnormal or excessive fat accumulation that may impair health \[[@CR1]\]. An elevated BMI is a major risk factor for cardiovascular diseases (mainly heart disease and stroke), diabetes, and some cancers. Although once considered problems only affecting high-income countries, the prevalence of overweight and obesity is now dramatically increasing in low- and middle-income countries as well, particularly in urban areas. In 2014, more than 1.9 billion adults (18 years and older) were overweight, and of these, over 600 million were obese \[[@CR1]\]. Overall, approximately 13 % of the world's adult population (11 % of men and 15 % of women) was obese in 2014 \[[@CR1]\]. Female obesity often begins during pregnancy or soon after childbirth \[[@CR2]\]. Promoting health management tailored to the stage of life may ensure that changes in body weight and physique caused by pregnancy and childbirth are not overlooked. With many women choosing to marry and have children later in life, a trend towards older women becoming pregnant is increasing in Japan. Women who become pregnant at an older age often find it difficult to return to their pre-pregnancy body weight \[[@CR3]\]. Since weight gain during pregnancy is associated with a variety of perinatal risks \[[@CR4]--[@CR6]\], the United States Institute of Medicine (IOM) has published guidelines that have been revised by the Japanese Ministry of Health, Labor and Welfare on how much weight a woman should gain during pregnancy, highlighting the importance of intervention during pregnancy to prevent postpartum weight retention \[[@CR7], [@CR8]\]. Women with normal-weight obesity (NWO; normal BMI with an elevated percentage fat mass (%FM) ≥ 30 %) are said to be at increased risk of metabolic disorders and cardiovascular disease \[[@CR9]\], underscoring the importance of weight management using %FM as a useful marker in addition to BMI. However, few investigations have evaluated the changes in the %FM of postpartum women. The MONA LISA hypothesis \[[@CR10]\] proposed in 1991 purports that autonomic nervous system (ANS) activity and the energy metabolism regulatory functions of the sympathetic nervous system have a major influence on obesity and fat metabolism, highlighting the possibility of ANS activity being a cause of weight gain and obesity. ANS activity is also thought to be involved in the course of postpartum body weight and %FM, but details regarding these relationships remain unclear. The aim of this study is to clarify the change of ANS activity, body weight, percentage fat mass (%FM), and body mass index (BMI) and the factors regulated to the return to the pre-pregnancy weight in the first year postpartum. Methods {#Sec2} ======= This study was conducted from 2012 through 2016. Subjects were recruited from among mothers who gave birth at the University Hospital, Kyoto Prefectural University of Medicine (Kyoto, Japan), and a nearby obstetrics and gynecology department clinic. As shown in Table [1](#Tab1){ref-type="table"}, significant differences in socioeconomic and demographic characteristics were not found between the two health care center subgroups. Further, all subjects neither smoked nor drank from before pregnancy, and all subjects were married. We displayed a written summary (including the date and time, location, and the methods) of the study at the facilities. We studied 51 mothers with no underlying diseases who agreed to participate in three surveys from 1 to 12 months after delivery (first survey: 30--150 days, second survey: 150--270 days, third survey: 270--360 days). The nature and purpose of the study were explained to all participants, who gave written informed consent to participate. The study protocol was approved by the Ethics Review Board of Kyoto Prefectural University of Medicine.Table 1Socioeconomic status and demographic characteristics of the participantsALL (*n* = 51)University Hospital, Kyoto Prefectural University of Medicine (*n* = 17)Obstetrics and gynecology department clinic (*n* = 34)*P* valuesAge33.7 ± 0.634.1 ± 1.033.5 ± 0.70.554Employment status^ab^ Employed31(60.8)8(15.7)23(45.1)0.156 Unemployed20(39.2)9(17.6)11(21.6)Family structure^ab^ Nuclear family50(98.0)17(33.3)33(64.7)1.000 Extended family1(2.0)01(2.0)Period of pregnancy (week)39.2 ± 0.239.4 ± 0.339.1 ± 0.20.633Pre-pregnancy BMI (kg/m2)20.1 ± 0.320.0 ± 0.620.2 ± 0.40.849Pre-pregnancy BMI group^ab^ BMI of \<18.514(27.5)6(11.8)8(15.7)0.555 BMI of 18.5-25.035(68.5)10(19.6)25(48.9) BMI of \> 25.02(4.0)1(2.0)1(2.0)Mean ± standard error or ^a^N (%); Mann-Whitney-U test or ^b^χ^2^ test Questionnaire survey {#Sec3} -------------------- Subjects completed a standardized questionnaire which included age, occupation, family structure, marital status, pre-pregnancy and postnatal exercise habits, medical history (mother and baby), parity, day of delivery, expected delivery date, method of childbirth, breast feeding, height, pre-body weight, and gestational weight gain. Measurement of body weight and fat mass {#Sec4} --------------------------------------- Body weight and %FM were determined using a bioelectrical impedance analyzer (BIA) (DC-320, Tanita Corp., Tokyo, Japan) \[[@CR11]\]. Subjects wore indoor clothing with no shoes. Half a kilogram (0.5 kg) was deducted from the weight of each participant to account for the weight of clothing. The %FM was calculated as fat mass/weight × 100, and BMI was calculated as the weight (kg)/height (m)^2^. The study defined +1 kg difference from the pre-pregnancy weight as a return to the pre-pregnancy weight. Subjects who returned to the pre-pregnancy weight within a year of delivery were categorized as the weight loss group, and those who did not were categorized as the no weight loss group. NWO was defined as a BMI of 18.5--25 kg/m^2^ and a %FM of ≥30 %. We defined "pre-NWO" as a BMI of 18.5--25 kg/m^2^ and a %FM of 25--30 %. Measurement of ANS activity {#Sec5} --------------------------- All examinations were performed in the Kyoto Prefectural University of Medicine or the nearby obstetrics and gynecology department clinic. All data acquisition was performed in our investigation room, which was kept quiet and comfortable, at a temperature of approximately 25--28 °C. For the assessment of ANS activity, after at least 20 min of rest, and confirmation that participants did not have tachycardia or hypertension, the investigation was started \[[@CR12]\]. Subjects were fitted with a photoplethysmography (PPG) monitor (Biocom Technologies, Inc., Washington DC, USA) on the earlobe, which acquired electrocardiographic (ECG) data for 5 min. The ECG was assessed for heart rate variability (HRV) with an analyzer (Heart Rhythm Scanner; HRV Analysis System; Biocom Technologies, Inc.) equipped with software that performs short-term HRV analysis algorithms. This system was based on the recommendations of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology \[[@CR13]\]. The reliability of these instruments has been confirmed by comparing the results of measurements using PPG with those of 12-lead ECG \[[@CR14], [@CR15]\]; this technique has also been used by Suetake et al. \[[@CR16]\]. Subjects were asked to listen to a metronome set at 15 clicks/min and synchronize their breathing rate accordingly \[[@CR12]\]. R--R spectral analysis {#Sec6} ---------------------- We assessed resting ANS activity by noninvasively analyzing HRV using power spectral analysis of the ECG. Cardiac rhythm is modulated by the sympathetic and parasympathetic components of the ANS, which exert antagonistic effects. The R--R interval on ECG, the inter-beat interval of heart rate, is determined by the net effect of inputs from the sympathetic and parasympathetic systems \[[@CR13]\]. This interval constantly fluctuates, and the extent of fluctuation constitutes HRV, which is a surrogate for ANS activity \[[@CR13], [@CR17]\]. Spectral transformation of this R--R interval variability generates a high-frequency band (HF; approximately 0.15--0.4 Hz), a low-frequency band (LF; approximately 0.04--0.15 Hz), and a very-low-frequency band (VLF; approximately 0.003--0.04 Hz). In addition, the sympathetic nervous system (SNS) and parasympathetic nervous system (PNS) activities were also calculated as ratio of (VLF + LF)/HF and HF/TOTAL, respectively, according to the previous studies \[[@CR18]\]. Statistical analyses {#Sec7} -------------------- All statistical analyses were performed using a commercial software package (SPSS 24.0 J for Windows; SPSS Inc., Tokyo, Japan). Since variation was large, the TP at the first survey was considered to be 100, and TP, LF, HF, VLF, LF/HF, SNS, and PNS activities were normalized by this TP at first survey value. Repeated measure analysis of variance was used for comparisons between measurement times. A multivariable Cox proportional hazards model was conducted to determine factors associated with the return to pre-pregnancy weight in the first year postpartum. Return or not to the pre-pregnant weight was used as the objective variable, while SNS activity, PNS activity, pre-pregnancy exercise habits, parity, pre-pregnancy BMI, and age were used as explanatory variables. The category division of SNS activity, PNS activity, BMI of pre-pregnancy, and age were carried out based on average value. All *P* values less than 0.05 were considered statistically significant. Data are expressed as mean ± standard error. Results {#Sec8} ======= Table [2](#Tab2){ref-type="table"} shows the demographic characteristics of the participants, in total and by parity. Significant differences were observed only for age (*P* \< 0.05). All subjects had single pregnancies and vaginal delivery, and all their children were normal.Table 2Demographic characteristics of the participantsALL (*n* = 51)Primipara (*n* = 35)Multipara (*n* = 16)*P value*Age33.7 ± 0.632.8 ± 0.735.6 ± 0.935.6 ± 0.90.029Height (cm)159.0 ± 0.7159.2 ± 0.9158.7 ± 1.20.831Pre-pregnancy body weight (kg)50.9 ± 1.049.9 ± 1.153.3 ± 2.00.102Pre-pregnancy BMI (kg/m^2^)20.1 ± 0.319.6 ± 0.321.2 ± 0.80.090Pre-pregnancy BMI group^ab^ BMI of \<18.514(27.5)11(21.6)3(5.9)0.081 BMI of 18.5-25.035(68.6)24(47.0)11(21.6) BMI of \> 25.02(3.9)02(3.9)Gestational weight gain (kg)9.5 ± 0.59.5 ± 0.69.7 ± 1.10.959Period of pregnancy (week)39.2 ± 0.239.5 ± 0.238.5 ± 0.50.069Exercise habits in pre-pregnancy^ab^ have9(17.6)8(15.7)1(2.0)0.242 no42(82.4)27(52.9)15(29.4)Breast feeding have43(84.3)30(58.8)13(25.5)0.694 no8(15.7)5(9.8)3(5.9)Mean ± standard error or ^a^N (%); Mann-Whitney-U test or ^b^χ^2^ test weight loss group, no weight loss group Body weight, %FM, and BMI {#Sec9} ------------------------- Body weight, %FM, and BMI at each measurement time are also shown in Table [3](#Tab3){ref-type="table"}. Body weight, %FM, and BMI decreased significantly over time after delivery (*P* \< 0.001, *P* \< 0.001, *P* \< 0.001). However, 13 (25.5 %) subjects had not returned to their pre-pregnancy body weight by the second measurement, and 10 (19.6 %) had still not returned to their pre-pregnancy body weight by the third measurement. Of the 35 subjects who had average pre-pregnancy BMI, 8 (22.9 %) had not returned to their pre-pregnancy body weight by the second measurement and 7 (20.0 %) had not returned by the third measurement.Table 3Body weight, percentage fat mass, body mass index, and normalized autonomic nervous system activity at three time points (*n* = 51)30--150 days150--270 days270--360 days*P values*Body weight (kg)51.5 ± 1.050.4 ± 1.049.9 ± 1.0\<.001Percentage fat mass (%)27.4 ± 0.826.1 ± 0.825.3 ± 0.8\<.001Body mass index20.3 ± 0.319.9 ± 0.319.7 ± 0.3\<.001Body weight reduction (kg)8.8 ± 4.29.9 ± 4.410.4 ± 5.0\<.001TP100.0122.5 ± 14.9133.9 ± 18.60.216LF27.6 ± 1.639.9 ± 5.947.4 ± 8.30.081HF39.5 ± 2.545.4 ± 6.048.5 ± 7.10.473VLF32.8 ± 2.437.2 ± 4.537.9 ± 4.60.541SNS activity2.8 ± 0.62.1 ± 0.22.0 ± 0.20.662PNS activity0.4 ± 0.020.4 ± 0.020.4 ± 0.020.662Exercise habits^a^ have4(7.8)4(7.8)4(7.8)- no47(92.2)47(92.2)47(92.2)Mean ± standard error or ^a^N (%); repeated measures analysis of variance*TP* total power, *LF* low frequency, *HF* high frequency, *VLF* very low frequency, *SNS* sympathetic nervous system, *PNS* parasympathetic nervous systemTP at 30-150 days was considered to be 100, and TP, LF, HF, VLF, SNS activity, and PNS activity were normalized by this value Of the 51 subjects, NWO was observed in 12 subjects (23.5 %) at the first measurement, in 9 (17.6 %) at the second, and in 9 (17.6 %) at the third, and pre-NWO was observed in 20 (39.2 %) at the first measurement, in 16 (31.4 %) at the second, and in 15 (29.4 %) at the third. In addition, overweight (BMI ≥ 25 kg/m^2^) was observed in 3 (5.9 %) each at the first and second measurements, and in 1 (2.0 %) at the third measurement. ANS activity {#Sec10} ------------ Table [3](#Tab3){ref-type="table"} shows TP, LF, HF, VLF, LF/HF, SNS, and PNS activities normalized by the TP value at the first measurement point. No significant differences in activity were noted among the three time points. Comparison between the weight loss group and no weight loss groups {#Sec11} ------------------------------------------------------------------ Average age was 33.9 (0.7) years in the weight loss group (*n* = 41) and 32.8 (0.8) years in the no weight loss group (*n* = 10). Among other variables, pre-pregnancy BMI was 20.3 (0.4) kg/m^2^ and 19.3 (0.5) kg/m^2^; the period of pregnancy was 39.3 (0.2) weeks and 39.0 (0.5) weeks; the number of primiparas was 28 (54.9 %) and 7 (13.7 %); the number of multiparas was 13 (25.5 %) and 3 (5.9 %); and the number of those who had exercise habits before pregnancy was 8 (15.7 %) and 1 (2.0 %), respectively. The results of ANS activity are indicated to Table [4](#Tab4){ref-type="table"}. No significant differences were seen among the two groups for any item.Table 4Comparison between weight loss group and no weight loss group30--150 days*P* values150--270 days*P* values270--360 days*P* valuesweight loss groupno weight loss groupweight loss groupno weight loss groupweight loss groupno weight loss groupSNS activity2.9 ± 0.82.1 ± 0.40.4062.1 ± 0.31.9 ± 0.20.6182.0 ± 0.22.0 ± 0.50.776PNS activity0.4 ± 0.030.4 ± 0.040.3930.4 ± 0.020.4 ± 0.030.6180.4 ± 0.020.4 ± 0.050.776Mean ± standard error; Mann-Whitney-U test*SNS* sympathetic nervous system, *PNS* parasympathetic nervous systemTP at 30-150 days was considered to be 100, and SNS activity, PNS activity were normalized by this value Factors related to the return to pre-pregnancy weight {#Sec12} ----------------------------------------------------- To identify factors that affected the return to pre-pregnancy weight within a year of delivery, an analysis using a multivariable Cox proportional hazards model (forced entry method) was performed. All confidence intervals straddled 1, and the results showed no significant differences regardless of variable (Table [5](#Tab5){ref-type="table"}).Table 5Analysis of factors affecting the return to pre-pregnancy weight using a multivariable Cox proportional hazards modelFactorCategory advantage/disadvantagePartial regression coefficient*P* valueHazard ratio95 % CISNS activity^a^First survey: 30--150 days after delivery≦2.80/\>2.81−1.0450.0640.3520.116 to 1.061Second survey: 150--270 days after delivery≦2.10/\>2.110.1030.8071.1080.487 to 2.524Third survey: 270--360 days after delivery≦2.00/\>2.01−0.9870.0690.3730.129 to 1.079PNS activity^a^First survey: 30--150 days after delivery≦0.4/\>0.41−0.9030.0710.4050.152 to 1.080Second survey: 150--270 days after delivery≦0.4/\>0.41−0.6750.1680.5090.195 to 1.329Third survey: 270--360 days after delivery≦0.4/\>0.41−0.4640.3740.6290.226 to 1.750Exercise habits in pre-pregnancy±0.2190.6231.2440.520 to 2.975ParityPrimipara/multipara0.4370.2891.5480.691 to 3.467BMI in pre-pregnancy≦24/≧25−1.2460.1640.2880.050 to 1.666Age≦34/≧35−0.3810.3020.6830.332 to 1.408*95 % CI* 95 % confidence interval^a^TP at 30--150 days was considered to be 100, and SNS and PNS activities were normalized by this value Discussion {#Sec13} ========== The weight of mothers gradually approached the pre-pregnancy weight over 6 months after giving birth \[[@CR19]\]. However, approximately 25 % of participants had not returned to their pre-pregnancy body weight by 150--270 days after delivery, and approximately 20 % had still not returned to their pre-pregnancy body weight by 270--360 days after delivery. In addition, sizeable percentages of subjects, depending on the measurement time, were found to have NWO or pre-NWO. High body weight---or more specifically, a high BMI---is associated with metabolic syndrome and elevated rates of morbidity and mortality \[[@CR1], [@CR20]\]. NWO is also associated with metabolic syndrome and cardiovascular diseases \[[@CR21]\]. The prevalence of NWO is higher in women than in men \[[@CR21]\], and BMI and %FM tend to increase with age \[[@CR22]\]. In Japanese women in particular, the percentage of women thought to be at risk for metabolic syndrome increases with age, with frequencies of 1.4 % in the third decade, 6.1 % in the fourth decade, 8.0 % in the fifth decade, and 15.1 % in the sixth decade \[[@CR23]\]. The average delivery age of 31.5 years among Japanese women \[[@CR24]\] may explain the sharp increase between the third and fourth decades, as pregnancy and childbirth have been hypothesized to trigger metabolic syndrome. The age of menopause among Japanese woman is an average of 50 years \[[@CR25]\], and the increase in the sixth decade may be attributable to menopause-related weight gain \[[@CR26]\]. Thus, addressing %FM is seen as increasingly important, in addition to managing body weight postpartum and later in life. ANS activity is involved in energy expenditure and appetite as well as in adjustment of body weight. Reductions in ANS activity, particularly decreases in sympathetic nervous activity, can lead to overweight \[[@CR27]\]. It is therefore considered that body weight is affected by age, ANS activity, and exercise habits. Because the multiparas were older than the primiparas, parity is also considered to affect body weight. However, these factors had no effect on the return to pre-pregnancy weight for the following reasons: (1) there were no significant differences in age or ANS activity between those who returned to their pre-pregnancy weight within a year of delivery and those who did not; (2) only a small number of subjects were obese; (3) many subjects were likely to have had normal ANS activity; and (4) only a small number of subjects who had had exercise habits before pregnancy maintained their exercise habits after delivery. Conclusions {#Sec14} =========== No marked postpartum changes were observed in ANS activity as body weight, %FM, and BMI decreased over a period of months following delivery. However, approximately 20 % had still not returned to their pre-pregnancy body weight by 270--360 days after delivery. In addition, NWO and pre-NWO were observed in some subjects at different measurement points, suggesting that managing weight and fat mass are both important for preventing postpartum obesity. Also, ANS activity was not correlated with the return to pre-pregnancy weight. These findings warrant confirmation in a larger population. Limitations and future directions {#Sec15} --------------------------------- The low number of subjects and low percentage of obese subjects may limit the generalization of these results. In the future, we intend to examine a larger population of subjects to clarify postpartum weight changes in obese subjects and to examine the relationship or otherwise between changes in weight and ANS activity. In this study, ANS activity at rest was used for analysis. We will further investigate ANS activity using the Scholling test. Funding {#FPar1} ======= This work was supported by a Grant-in-Aid for Scientific Research ((C) 23593312) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. Availability of data and materials {#FPar2} ================================== The datasets supporting the conclusions of this article are included within the article. All of the data is included in the text and tables in the manuscript. Authors' contributions {#FPar3} ====================== MI, SU, and AW contributed to the conception and design of this study; MI performed the statistical analysis and drafted the manuscript; and EM and TM critically reviewed the manuscript and supervised the entire study process. All authors read and approved the final manuscript. Competing interests {#FPar4} =================== The authors declare that they have no competing interests. Ethics approval and consent to participate {#FPar5} ========================================== The nature and purpose of the study were explained to all participants, who gave written informed consent to participate. The study protocol was approved by the Ethics Review Board of Kyoto Prefectural University of Medicine.

Introduction ============ Thyroid follicular cells are found in the thyroid gland, specifically in the epithelial monolayer. In total, \>95% of thyroid tumors are derived from these follicular cells ([@b1-ol-0-0-7146]). In 2016, the incidence of thyroid tumors rose globally, largely due to technological and diagnostic advances ([@b2-ol-0-0-7146]). However, it remains difficult to distinguish whether a thyroid nodule is benign or malignant. Follicular thyroid tumors may be divided into malignant follicular thyroid carcinoma (FTC) and benign follicular thyroid adenoma (FA). Only 5--10% of thyroid nodules are malignant ([@b3-ol-0-0-7146]). Patients with follicular tumors usually must undergo thyroid lobectomy for diagnosis, which is often an unnecessary surgery, as the disease is usually benign. Fine-needle aspiration cytology is considered the most accurate method for the diagnosis of FTC and FA ([@b4-ol-0-0-7146]). Previously, microRNAs (miRNAs/miRs) have been demonstrated to be involved in the pathogenesis of various diseases, like cancer, diabetes and osteoarthritis ([@b5-ol-0-0-7146]--[@b7-ol-0-0-7146]). miRNAs are small (18--25 nucleotides) non-coding, single-stranded RNA molecules that bind to targets in a base pair-mediated manner, resulting in the degradation or inhibition of the expression and function of protein-coding mRNAs. miRNAs often bind to the 3′-untranslated region (3′UTR) of target genes ([@b8-ol-0-0-7146]), although they are usually only partially complementary to the target ([@b9-ol-0-0-7146]). miRNAs regulate \~30% of the human genes associated with proliferation, apoptosis, metastasis, cell immunity and differentiation ([@b10-ol-0-0-7146]). Each miRNA is able to regulate several hundred mRNAs, and each mRNA may be the target of several miRNAs. Therefore, a regulatory control network exists between miRNAs and mRNAs ([@b11-ol-0-0-7146]). Furthermore, miRNAs have been associated with several types of tumors, including non-small cell lung cancer, colon and esophageal cancer, and FTC ([@b12-ol-0-0-7146]--[@b14-ol-0-0-7146]). However, there are few studies of specific miRNA and mRNA analyses of follicular thyroid tumors. Several microarray studies have already described the differentially expressed genes (DEGs) between malignant and benign thyroid nodules. However, these studies have several restrictions, including the fact that the samples are limited, they contain significant false-negatives, and they require external analysis at an offsite company laboratory ([@b15-ol-0-0-7146]--[@b17-ol-0-0-7146]). Certain studies have aimed to reveal the potential miRNAs associated with follicular thyroid tumors ([@b18-ol-0-0-7146]). In the present study, an integrated analysis of differentially expressed miRNAs (DEMs) and DEGs between FTC and FA was performed. A Gene Ontology (GO) analysis of the DEGs was performed. A total of 36 miRNA-gene pairs were identified between the DEGs and the target genes of the DEMs. A miRNA-mRNA network analysis was then performed to additionally investigate the pathogenesis of FTC. Materials and methods ===================== ### Analysis of mRNA and miRNA profiling datasets Expression profile datasets containing mRNA and miRNA were acquired from the Gene Expression Omnibus (GEO; <http://www.ncbi.nlm.nih.gov/geo/>). The expression profiling data of GSE29315 (Tomas *et al*, unpublished) are mRNA profiling data, originally obtained from a cohort of 9 FTC and 17 FA samples. The GSE62054 dataset contains miRNA profiling data, which was originally obtained from 17 FTC and 8 FA samples ([@b19-ol-0-0-7146]). Additionally, GSE29315 was hybridized on the Affymetrix U95 GeneChip platform (Affymetrix; ThermoFisher Scientific, Inc., Waltham, MA, USA) and GSE62054 was performed on the Illumina Human v2 miRNA expression BeadChip (Illumina, Inc., San Diego, CA, USA). ### Preprocessing of profiling data GSE29315 and GSE62054 data were first preprocessed by the Affy package in R language version 3.4.0 and then were processed by log~2~ transformation, background correction and data normalization using the Robust Multi-array Average algorithm ([@b20-ol-0-0-7146]). ### Identification of DEMs, DEGs and GO enrichment analysis Identification of DEMs and DEGs were conducted by the Limma package version 3.32.5 in R software ([@b21-ol-0-0-7146]). The threshold values for different expression were log~2~ (fold-change)\>0.5 or log~2~ (fold-change)\<-0.5 with P\<0.05 ([@b22-ol-0-0-7146]). GO enrichment analysis for DEGs was performed with the Database for Annotation, Visualization and Integrated Discovery (DAVID) ([@b23-ol-0-0-7146]). ### Overlapping genes of DEGs and the predicted target genes of the DEMs The predicted target mRNAs of the DEMs were generated using the miRWalk ([@b24-ol-0-0-7146]), miRecords ([@b25-ol-0-0-7146]) and TarMir databases ([@b26-ol-0-0-7146]). The overlapping DEGs and the predicted target mRNAs of the DEMs were identified for additional network analysis. ### Construction and analysis of miRNA-mRNA regulatory network To obtain an improved understanding of the biological function of the miRNA-mRNA regulatory network, node-degree analysis was performed, based on the overlapping genes and their upstream miRNAs. The network was visualized using the Cytoscape platform software version 3.0.1 ([@b27-ol-0-0-7146]). Results ======= ### Identifying DEMs and DEGs between FTC and FA GSE29315 and GSE62054 were downloaded from GEO and then normalized, and corrected by the quantile normalization method and hierarchical clustering analysis using R software. DEMs and DEGs were identified between the FTC and FA. A total of 86 DEGs and 32 DEMs were obtained when the threshold values were set at P\<0.05 and log~2~(fold-change)\>0.5 or log~2~ (fold-change)\<-0.5. The top 5 downregulated DEMs were miR-7, miR-1179, miR-7-2, miR-486-5p and miR-130b. The top 5 upregulated DEMs were miR-663b, miR-137, miR-30c-1, miR-767-5p and miR-603 ([Table I](#tI-ol-0-0-7146){ref-type="table"}). As for the DEGs, the top 5 downregulated genes were fatty acid binding protein 4 (FABP4), cytidine monophospho-N-acetylneruaminic acid (CMAHP), integral membrane protein 2A (ITM2A), carbonic anhydrase 4 (CA4) and family with sequence similarity 189 member A2 (FAM189A2), and the top 5 upregulated genes were erythrocyte membrane protein band 4.1 like 3 (EPB41L3), secretogranin V (SCG5), paired box 1 (PAX1), methylenetetrahydrofolate dehydrogenase (NADP + dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) and cadherin 2 (CDH2) ([Table II](#tII-ol-0-0-7146){ref-type="table"}). A volcano plot was constructed to identify the DEGs ([Fig. 1](#f1-ol-0-0-7146){ref-type="fig"}). ### GO enrichment analysis of DEGs GO analysis of all the DEGs ([Table III](#tIII-ol-0-0-7146){ref-type="table"}) identified 8 associated biological processes: Positive regulation of macromolecule metabolic process, regulation of cell motion, cell proliferation, tube development, regulation of response to external stimulus, regulation of locomotion, response to drug and T cell activation ([Fig. 2](#f2-ol-0-0-7146){ref-type="fig"}). ### Integrated network analysis of miRNA-mRNA interaction From the miRWalk, miRecords and TarMir databases, target genes of the DEMs were identified. A total of 24 overlapping genes were identified between the targets genes and DEGs ([Table IV](#tIV-ol-0-0-7146){ref-type="table"}). Furthermore, 36 miRNA-gene pairs were obtained among the 24 overlapping genes and 9 DEMs ([Table V](#tV-ol-0-0-7146){ref-type="table"}). Node-degree analysis is summarized in [Table VI](#tVI-ol-0-0-7146){ref-type="table"}. The regulation network between those overlapping genes and their upstream miRNAs is presented in [Fig. 3](#f3-ol-0-0-7146){ref-type="fig"}. Discussion ========== The important roles of miRNAs in the pathogenesis of FTC have been identified previously ([@b28-ol-0-0-7146]). miRNAs exhibit different expression patterns within different tumor types, and are closely associated with the diagnosis, treatment and prognosis of tumors ([@b29-ol-0-0-7146]--[@b31-ol-0-0-7146]). Ak *et al* ([@b21-ol-0-0-7146]) observed that DEMs and differentially expressed mRNAs vary between benign and malignant tumors, which may suggest the different roles of these miRNAs and mRNAs. miR-197 and miR-346 have been indicated to be overexpressed in FTC, resulting in the dysregulation of their target genes ([@b32-ol-0-0-7146]). However, studies regarding DEMs and DEGs in FTC are rare. In the present study, the difference between miRNA-mRNA regulatory networks from FTC and FA samples were compared in order to investigate the mechanism of FTC. It was identified that miR-7, miR-1179, miR-7-2, miR-486-5p and miR-130b were the top downregulated miRNAs, and that miR-663b, miR-137, miR-30c-1, miR-767-5p and miR-603 were the top upregulated miRNAs. For the DEGs, the top downregulated genes were FABP4, CMAHP, ITM2A, CA4 and FAM189A2, and the top upregulated genes were EPB41L3, SCG5, PAX1, MTHFD2 and CDH2. In addition, miR-7, miR-296-5p, miR-10a, miR-144, miR-139-5p, miR-452 and miR-145 were downregulated, and miR-137 and miR-493 were upregulated in the FTC miRNA-mRNA regulatory network compared with those in FA. The gene arrays identified DEGs, in which leucine rich repeat neuronal 3, chromodomain helicase DNA binding protein 9, PKIA, zinc finger protein 148 (ZNF148), TGFB induced factor homeobox 1, transforming growth factor β receptor 2, gap junction protein α1 and CDH2 were observed to be target genes inversely correlated with miR-7, miR-144, miR-139-5p, miR-145 and miR-137. In other studies, FTC or FA have been compared with normal tissue, and differences in miRNA expression were observed to occur in the range between 1.2- and 2-fold, which was similar to the data of the present study ([@b33-ol-0-0-7146]--[@b35-ol-0-0-7146]). In the present study, it was identified that miR-7, miR-296-5p, miR-10a, miR-144, miR-139-5p, miR-452, miR-145, miR-137 and miR-493 are important miRNAs that are differentially expressed between carcinoma and adenoma samples. Certain studies have suggested that miR-7 is not only a tumor promoter, but also a tumor suppressor. As a tumor suppressor, miR-7 is downregulated in tumors, such as thyroid cancer, breast cancer and castration-resistant prostate cancer, leading to a derepression of the oncogenes epidermal growth factor receptor, insulin receptor substrate 1, Raf-1 proto-oncogene, serine/threonine kinase, tyrosine kinase non-receptor 2, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit δ, mechanistic target of rapamycin kinase, Ribosomal protein S6 kinase β-1 and phosphatidylinositol-4,5-bisphosphate 3-kinase ([@b36-ol-0-0-7146]--[@b38-ol-0-0-7146]). miR-296-5p has been revealed to be significantly inversely correlated with post-contrast T1 values for diffuse myocardial fibrosis in patients with hypertrophic cardiomyopathy, and is a downstream effector under conditions that promote glioblastoma stem cell stemness, and inhibit glioblastoma cell stemness and their capacity to self-renew as spheres and propagate glioma xenografts *in vivo* ([@b39-ol-0-0-7146],[@b40-ol-0-0-7146]). miR-10a has been identified as a downregulated miRNA associated with human metastatic medullary thyroid carcinoma, and it may be important for tumor development and/or reflect C-cell lineage ([@b41-ol-0-0-7146],[@b42-ol-0-0-7146]). miR-144 may suppress the invasion and migration capability of thyroid cancer and suppress the expression of zinc finger E-box-binding homeobox (ZEB)1 and ZEB2, the two E-cadherin suppressors, by directly binding their 3′UTRs ([@b43-ol-0-0-7146]). miR-137 was indicated to participate in hematopoiesis, particularly in the efficacy of warfarin, wherein miR-137 may cause aberrant vitamin K epoxide reductase complex subunit 1 expression ([@b44-ol-0-0-7146]). miR-139-5p is an oncogenic molecule in the process of tumorigenesis, and has been demonstrated to be a sensitive and specific biomarker for the diagnosis of thyroid tumors and others tumor types ([@b45-ol-0-0-7146]). Furthermore, it may be of use as a tractable therapeutic target to decrease the mortality rate and increase the survival rate ([@b46-ol-0-0-7146]). miR-145 has primarily been indicated as being downregulated in colorectal tumors. Previously, certain studies have identified that miR-145 is highly expressed in mesenchymal cells such as fibroblasts and smooth muscle cells ([@b47-ol-0-0-7146]). The miRNA was demonstrated to directly regulate the expression of thyroid hormone receptor TRβ1 in renal cancer cells and to correlate with intracellular triiodothyronine concentrations in renal tumors ([@b48-ol-0-0-7146]). miR-493 also promoted the invasion and chemoresistance of gastric cancer cells. However, dickkopf related-protein 1 overexpression reversed its effects on proliferation, invasion and chemo-sensitivity ([@b49-ol-0-0-7146]). Based on these data, we hypothesize that these miRNAs serve important roles in FTC with different pathways. In the present study, several genes that were overlapping were identified between the DEGs and the target genes of the DEMs. These may be upregulated or downregulated. However, they all contributed to the development of FTC. Certain functions of these genes in cancer have been studied. For example, ZNF148 is a member of the human zinc finger Krüppel family and it maps to regions implicated in recurrent chromosomal rearrangements in hematological malignancies ([@b50-ol-0-0-7146]). The present study identified spalt-like transcription factor 1 (SALL1), which is one of the four human family members of the Spalt family. Members of the Spalt family are highly conserved zinc-finger transcription factors that are conserved from *Caenorhabditis elegans* to vertebrates, with regulatory functions in organogenesis, limb formation and cell fate assignment during neural development. SALL1 expression has been identified to correlate with the expression of CDH1, which is consistent with its tumor suppressive function and suggests its potential involvement in epithelial-to-mesenchymal transition ([@b51-ol-0-0-7146],[@b52-ol-0-0-7146]). Cell division cycle 27 (CDC27) is a core component of the anaphase-promoting complex and is involved in the regulation of mitotic checkpoints to ensure chromosomal integrity ([@b53-ol-0-0-7146]). CDC27 may significantly affect the function of the polymeric protein complex and is also a target of certain anticancer drugs ([@b54-ol-0-0-7146],[@b55-ol-0-0-7146]). Nuclear receptor subfamily 2 group F member 2 (NR2F2), also known as chicken ovalbumin upstream promoter transcription factor, is highly prioritized as a candidate gene associated with hypertension ([@b56-ol-0-0-7146]). Certain studies have demonstrated that NR2F2 is nuclear receptor transcription factor vital for angiogenesis and heart development ([@b57-ol-0-0-7146]). These data suggest that several genes have functions in numerous pathways involved in tumorigenesis and progression. Each miRNA is able to regulate several hundred mRNAs. In addition, each mRNA may be targeted by several miRNAs and each mRNA participates in several biological functions in the human body. Therefore, each miRNA may affect different biological processes and pathways through a miRNA-mRNA network ([@b58-ol-0-0-7146],[@b59-ol-0-0-7146]). It is important to understand the pathogenesis and treatment of tumors by investigating the specific miRNA-mRNA co-regulation effects. In the present study, mRNAs and their functions were described with GO enrichment analysis. There were 86 mRNAs and 8 biological functions involved. In total, \~80% of follicular carcinomas contain Ras gene mutations or a paired box gene 8/peroxisome proliferator-activated receptor γ gene rearrangement, which leads to uncontrolled proliferation. Mutations in the phosphatase and tensin homologue suppressor gene and the phosphatidylinositol 3-kinase pathway may be an important factor in the development of more aggressive thyroid cancer types and may be more common in follicular cancer, which is responsible for cell motility, locomotion and response to external stimulus ([@b60-ol-0-0-7146]--[@b62-ol-0-0-7146]). Other factors that have been implicated in the pathogenesis of FTC include gene mutations in p53, c-myc, c-fos and the thyrotropin receptor ([@b63-ol-0-0-7146]--[@b66-ol-0-0-7146]). These molecules serve functions in cell proliferation, apoptosis, cytoskeleton rearrangement and responses to drugs. Additionally, FTC, but not adenoma, recruits tumor-associated macrophages by releasing Chemokine (C-C motif) ligand 5; therefore, an abnormal immune response, including T cell activation, may be involved in follicular cancer. Other GO terms may be validated in future studies ([@b3-ol-0-0-7146],[@b67-ol-0-0-7146]). In conclusion, the present study identified 86 DEGs and 32 DEMs between FTC and FA. A total of 24 overlapping genes were identified between the DEGs and the target genes of the DEMs. Network analysis indicated a co-regulatory association between miR-296-5p, miR-10a, miR-139-5p, miR-452, miR-493, miR-7, miR-137, miR-144, miR-145 and corresponding targeted mRNAs in FTC. However, the present study has limitations, such as the small sample size, although attention was paid to ensure the use of two genetically homogenous populations to avoid population stratification. The mechanism of the miRNA-mRNA network and the roles of these genes in FTC require additional study and validation *in vitro* and *in vivo*. {#f1-ol-0-0-7146} {#f2-ol-0-0-7146} {#f3-ol-0-0-7146} ###### Top 5 differentially expressed miRNAs of malignant follicular thyroid carcinoma compared with benign follicular thyroid adenoma. miRNA P-value log~2~(fold-change) --------------- ----------- --------------------- Downregulated   miR-7 0.0041392 −1.7320437   miR-1179 0.0081728 −1.3950195   miR-7--2 0.0006626 −1.2525509   miR-486-5p 0.0412501 −1.0502825   miR-130b 0.0028172 −0.9176468 Upregulated   miR-663b 0.0009353 0.9881272   miR-137 0.0088044 0.9341108   miR-30c-1 0.0059237 0.8695624   miR-767-5p 0.0036048 0.7353497   miR-603 0.0392875 0.6646499 miR/miRNA, microRNA. ###### Top 5 differentially expressed mRNAs of malignant follicular thyroid carcinoma compared with benign follicular thyroid adenoma. mRNA P-value log~2~(fold-change) --------------- ------------- --------------------- Downregulated   FABP4 0.001621719 −2.100023748   CMAHP 0.024414059 −1.066127774   ITM2A 0.016922069 −1.060957028   CA4 0.003105875 −1.030691864   FAM189A2 0.001993414 −1.000814956 Upregulated   EPB41L3 0.000527208 1.020917517   SCG5 0.044745635 0.990761798   PAX1 0.040281329 0.936795356   MTHFD2 0.002917107 0.870767506   CDH2 0.038829964 0.862357097 ###### Differentially expressed mRNAs of malignant follicular thyroid carcinoma compared with benign follicular thyroid adenoma. Gene P-value log~2~ (fold-change) ----------- ------------- ---------------------- FABP4 0.001621719 −2.100023748 CMAHP 0.024414059 −1.066127776 ITM2A 0.016922069 −1.060957028 CA4 0.003105875 −1.030691864 FAM189A2 0.001993414 −1.000814956 MPPED2 0.007206792 −0.981309728 HGD 0.023615869 −0.852452356 TNFRSF11B 0.036234193 −0.836887107 SLC16A4 0.020876545 −0.826437495 BZRAP1 0.006323889 −0.784389089 PDGFRL 0.019724555 −0.769696713 TFF3 0.004160978 −0.758375163 LAMB1 0.019590536 −0.754903323 UBR2 0.001897906 −0.746515077 TGFBR2 0.016942031 −0.736954014 CPQ 0.040123761 −0.722559518 RDX 0.014625862 −0.700582583 PTPRN2 0.008418827 −0.692563257 SALL1 0.039302461 −0.687132893 LRRN3 0.006382367 −0.685656353 TRAM2 0.016638751 −0.664343953 SLITRK5 0.040721395 −0.652918137 RGS16 0.027819411 −0.648828034 STARD13 0.013643606 −0.642603503 THBD 0.010984131 −0.635676793 GJA1 0.035287842 −0.633044868 TNFSF10 0.009577025 −0.620052341 PKIA 0.022943634 −0.617442203 CLDN8 0.014099121 −0.606720582 IL11RA 0.016027339 −0.589253369 CDC27 0.012718038 −0.584208837 SLC35D2 0.010299633 −0.579905832 IFI44L 0.015727697 −0.576791484 NR2F2 0.013187137 −0.575425266 CHD9 0.048840737 −0.573993055 FCGRT 0.011662768 −0.566086321 AGTR1 0.013773676 −0.538397573 CXCL12 0.003888448 −0.538189987 HSD17B8 0.019222754 −0.533271237 LOC728093 0.035060908 −0.524392385 SELE 0.017742851 −0.507241011 AKAP12 0.010628133 −0.506141506 PVALB 0.045712444 0.509393237 NNT 0.027397598 0.513526356 ADORA1 0.036372247 0.513841883 GPI 0.018981531 0.514219746 AREG 0.042790083 0.515091119 ELF4 0.001468499 0.516939945 NAB2 0.011359365 0.527142113 GPC1 0.030827537 0.529427807 SLC25A5 0.029858761 0.534298394 RYR1 0.014981235 0.538436907 PEG10 0.018255692 0.549699307 SLIT2 0.005701378 0.551929669 ESYT1 0.003009396 0.561757654 CRLF1 0.029356956 0.562850973 DPP4 0.018431425 0.567301902 CES2 0.003842477 0.571035541 CYCS 0.030715945 0.585863898 BASP1 0.026758782 0.594217929 KLHL21 0.022505203 0.599053201 LCN2 0.017669691 0.601124995 EEF1A2 0.018392179 0.602560272 TGIF1 0.030147532 0.605396529 TSPYL2 0.046736047 0.617648983 CKS2 0.026759687 0.623716633 SPAG5 0.027923235 0.624868361 CKMT2 0.043578063 0.645621487 ALDH1A3 0.010848438 0.653685247 ZNF148 0.031227695 0.661077991 UCHL1 0.003566593 0.663217388 ASNS 0.006539605 0.667304137 NPTXR 0.009448284 0.674187256 FKBP5 0.027541727 0.694255151 GOT1 0.001282557 0.730143285 IGFBP3 0.009116606 0.771327366 SERPINE2 0.020069598 0.785032654 SOX4 0.006616428 0.808876177 BSG 0.001981905 0.809699733 SCNN1A 0.027983691 0.825462627 NPC2 0.005654992 0.837641446 CDH2 0.038829964 0.862357097 MTHFD2 0.002917107 0.870767506 PAX1 0.040281329 0.936795356 SCG5 0.044745635 0.990761798 EPB41L3 0.000527208 1.020917517 ###### Significant regulation of mRNAs in the specific miRNA-mRNA interacting regulatory network. Gene P-value log~2~ (fold-change) ----------- ---------- ---------------------- TNFRSF11B 0.036234 −0.836892 BZRAP1 0.006324 −0.784391 TGFBR2 0.016942 −0.736951 SALL1 0.039302 −0.687134 LRRN3 0.006382 −0.685662 TRAM2 0.016639 −0.664343 RGS16 0.027819 −0.648836 STARD13 0.013644 −0.642635 THBD 0.010984 −0.635682 GJA1 0.035288 −0.633043 PKIA 0.022944 −0.617443 CDC27 0.012718 −0.584214 NR2F2 0.013187 −0.575433 CHD9 0.048841 −0.573992 CXCL12 0.003888 −0.538192 NAB2 0.011359 0.527142 SLC25A5 0.029859 0.534298 PEG10 0.018256 0.549699 SLIT2 0.005701 0.551934 BASP1 0.026759 0.594218 TGIF1 0.030148 0.605397 ALDH1A3 0.010848 0.653685 ZNF148 0.031228 0.661078 CDH2 0.038832 0.862357 miRNA, microRNA. ###### Significant regulation of miRNAs in the specific miRNA-mRNA interacting regulatory network. miRNA P-value log~2~ (fold-change) ------------ ----------- ---------------------- miR-7 0.0041392 −1.7320437 miR-137 0.0088043 0.9341108 miR-144 0.0059723 −0.8403724 miR-139-5p 0.0147834 −0.5824534 miR-145 0.0087553 −0.5871226 miR-296-5p 0.0124989 −0.5545413 miR-10a 0.0061396 −0.7961598 miR-452 0.0325921 −0.5399825 miR-493 0.0186862 0.6062014 miR/miRNA, microRNA. ###### Node-degree analysis of miRNA-mRNA interactions. Node Degree ------------ -------- miR-144 10 miR-137 9 miR-145 5 miR-7 5 ZNF148 4 miR-139-5p 3 PKIA 3 TGFBR2 3 TRAM2 3 TGIF1 2 miR/miRNA, microRNA.

{#sp1 .285} {#sp2 .286} {#sp3 .287} {#sp4 .288} {#sp5 .289} {#sp6 .290} {#sp7 .291} {#sp8 .292} {#sp9 .293} {#sp10 .294} {#sp11 .295}

Background ========== Pulmonary arterial hypertension (PAH) results from proliferative vasculopathy caused by pulmonary vascular remodeling which involves all layers of the blood vessel. Pathologic findings include intimal hyperplasia and fibrosis, medial hypertrophy, and thrombi of the small pulmonary arteries and arterioles with occasional plexiform lesion formation \[[@b1-amjcaserep-18-941],[@b2-amjcaserep-18-941]\]. Similar pathologic changes may be present in pulmonary hypertension (PH) associated with microscopic tumor embolism, which is thought to be related to the phenomenon of pulmonary tumor thrombotic microangiopathy (PTTM). Pulmonary tumor embolism is described as direct and entire occlusion of the pulmonary vessel by tumor emboli. In PTTM, tumor emboli do not occlude affected vessels; however, the microscopic tumor cell emboli induce the activation of the coagulation system at the surface of the tumor emboli, with fibrocellular intimal proliferation, resulting in stenosis and occlusion of the vessel \[[@b3-amjcaserep-18-941]\]. PTTM manifests clinically as subacute respiratory failure with pulmonary hypertension and progressive right ventricular failure due to increased right ventricular afterload, and sudden death \[[@b4-amjcaserep-18-941],[@b5-amjcaserep-18-941]\]. We present a case of PTTM developing in a patient with gastro-esophageal (GE) junction adenocarcinoma who was also treated with trastuzumab and a new experimental medication that is a human monoclonal antibody blocking the activity of ErbB3 receptor tyrosine kinase (RTK). This case was challenging in view of the rapid progression of right heart failure and a trans-bronchial biopsy suggesting an alternative diagnosis. We highlight the limitations of trans-bronchial biopsies in evaluating PTTM. The final diagnosis of PTTM was not made until the autopsy was done. Case Report =========== A 49-year-old man with stage IV human epidermal growth factor receptor (HER2) positive gastro-esophageal (GE) junction adenocarcinoma presented to the hospital with complaints of cough, shortness of breath, lightheadedness, and increasing fatigue. Two years earlier, when his malignancy was diagnosed, he was initially started on FOLFIRINOX (fluorouracil, leucovorin, oxaliplatin, and irinotecan) with later transition to the single agent trastuzumab (anti-HER2 monoclonal antibody). FOLFOX was added to trastuzumab due to the signs of disease progression on the PET scan. However, restaging scans demonstrated progression of his malignancy; therefore, he was included in a phase 1 clinical trial of a new experimental medication with receptor tyrosine kinase (RTK) blocking activity. He had completed 2 cycles of this medication by the time of his admission to the hospital. Upon admission, he was found to have hypoxemic respiratory failure. He had mildly elevated troponin T and troponin I at 0.03 ng/ml (normal range: \<0.01 ng/mL) and 0.25 ng/ml (normal range: 0.00--0.08 ng/mL), respectively. His EKG showed new T wave inversions in precordial leads, suggestive of anterolateral ischemia. There was also a change of axis from the left axis deviation to the normal axis. A trans-thoracic echocardiogram (TTE) demonstrated severely increased right ventricle (RV) cavity size with severely decreased RV systolic function. McConnell's sign (RV dysfunction with characteristic sparing of the apex) was noted. RV systolic pressure was estimated at 70 mmHg. Based on these results, a computed tomography angiography was obtained, which was negative for pulmonary embolism but demonstrated multiple nodules in both lungs, with surrounding ground-glass opacities and evidence of tree-in-bud pattern ([Figure 1](#f1-amjcaserep-18-941){ref-type="fig"}). Due to concern for possible fungal or atypical bacterial infection, the patient underwent bronchoscopy with bronchoalveolar lavage (BAL). BAL stains and cultures ruled out an infectious etiology. After prolong discussion with the family, who opted to proceed with the trans-bronchial biopsies due to concerns for possible drug-related lung injury rather than further advancement of malignancy, the procedure was performed with enormous caution and vigilance due to the newly diagnosed pulmonary hypertension, which in general is considered a contradiction for the procedure. The patient was also empirically started on treatment with prednisone 1 mg/kg daily due to concern for possible drug-related lung injury. Trans-bronchial biopsies showed arteriole obliteration by smooth muscle proliferation, consistent with pulmonary vasculopathy ([Figure 2A](#f2-amjcaserep-18-941){ref-type="fig"}) and suggestive of pulmonary arterial hypertension (PAH) \[[@b1-amjcaserep-18-941],[@b2-amjcaserep-18-941]\]. PAH was previously reported in patients receiving dasatinib, which can be reversed following discontinuation of the therapy \[[@b6-amjcaserep-18-941]\]. Due to the RTK-blocking activity of the experimental medication, it was argued at the time that it may have played a role in the pathogenesis of this patient's condition, in a similar way that dasatinib causes pulmonary vasculopathy. Given the elevated estimated (by echo) RV pressure with hemodynamic stability at the time, the patient was planned for consideration of initiation of infused prostanoid analogues therapy and thus required right heart catheterization (RHC). The RHC confirmed severe pulmonary hypertension with mean pulmonary artery pressure of 70 mmHg and a pulmonary vascular resistance of 20 Wood units. A vasodilatory challenge test was not done because of his low cardiac index (1.25 liters/minute/m^2^). Cytologic examination of blood aspirated from the pulmonary artery catheter (not in a wedged inflated balloon position) showed presence of circulating tumor cells. Unfortunately, shortly after the RHC, the patient developed pulseless electrical activity cardiac arrest and died after resuscitation efforts were unsuccessful. Autopsy results confirmed dilated cardiomyopathy of the right ventricle, and postmortem lung pathologies showed arteriolar smooth muscle proliferation and fibrosis with recanalization of thromboemboli ([Figure 2B](#f2-amjcaserep-18-941){ref-type="fig"}), and the precise thickness of the RV was measured at 8 mm. These findings are largely similar to those of the trans-bronchial biopsies, except for diffuse dissemination of tumor cells in the lymphatic vessels ([Figure 2C](#f2-amjcaserep-18-941){ref-type="fig"}) and small pulmonary vessels ([Figure 2D](#f2-amjcaserep-18-941){ref-type="fig"}), consistent with PTTM \[[@b3-amjcaserep-18-941]--[@b5-amjcaserep-18-941]\]. Discussion ========== PTTM is a very rare complication of metastatic cancer. Unfortunately, it is difficult to identify clinically and is predominantly diagnosed postmortem \[[@b7-amjcaserep-18-941]\]. Evidence of tree-in-bud pattern on the chest CT was very evocative of PTTM in the setting of PH \[[@b8-amjcaserep-18-941]\], but infectious processes were also high on the differential in this immunocompromised patient undergoing chemotherapy. In this case, trans-bronchial biopsies were suggestive of PAH without any evidence of lymphangitic tumor spread. Moreover, the patient had a history of being treated with RTK-blocking medications. Tyrosine kinase inhibitors (TKI) are known for their inhibition of platelet-derived growth factor (PDGF), which is known to be implicated in the development of PAH \[[@b3-amjcaserep-18-941],[@b4-amjcaserep-18-941]\]. The trans-bronchial biopsies could not rule out this possibility (drug-associated pulmonary vascular disease), which turned out (postmortem) to not be the case. Taking into consideration all the differentials at the time, we performed cytologic examination of blood aspirated from the pulmonary artery catheter, which confirmed the presence of circulating tumor cells. Unfortunately, due to the rapidly progressive nature of the disease, the patient died right after the RHC. Even if he had been properly diagnosed, this is almost uniformly fatal with a background of advanced malignancy that does not respond to first-line therapies. Recent reports give us hope that with the appropriate clinical suspicion, the outcomes may be less dismal in a select group of patients \[[@b9-amjcaserep-18-941]\]. The data on the effective treatment of pulmonary tumor emboli is insufficient but it is generally directed at the primary tumor. Different proposed therapies, such as combination chemotherapy with dexamethasone, warfarin, and aspirin, and pulmonary vasodilators, do not improve the prognosis under such circumstances, but may improve patient symptoms \[[@b4-amjcaserep-18-941],[@b5-amjcaserep-18-941],[@b7-amjcaserep-18-941]\]. In this perplexing and unfortunate case, PTTM was missed on the trans-bronchial biopsy but confirmed on autopsy. Diagnosing PTTM on trans-bronchial biopsies would not have changed the inevitable outcome of this case, as the patient rapidly deteriorated and there are no known effective therapies, especially in the setting of such an advanced aggressive malignancy. Conclusions =========== It is important to consider PTTM as a differential diagnosis for rapidly progressive respiratory failure in patients with metastatic tumors. Antemortem diagnosis of pulmonary tumor embolism may be difficult to establish, and we show that even trans-bronchial biopsies may be misleading. {#f1-amjcaserep-18-941} {#f2-amjcaserep-18-941} [^1]: Authors' Contribution: [^2]: Study Design [^3]: Data Collection [^4]: Statistical Analysis [^5]: Data Interpretation [^6]: Manuscript Preparation [^7]: Literature Search [^8]: Funds Collection [^9]: **Conflict of interest:** None declared

Introduction {#sec1-1} ============ Health sector reform under the National Rural Health Mission (NRHM), aims at increasing functional, administrative and financial autonomy of various field units.([@ref1]) Under NRHM, up-gradation of health service delivery facilities for better community participation and accountability is strategized as a vehicle of flexible funding by the government to help raise the quality of care, by making the health service delivery system more responsive to meet people\'s needs with equitable access. This required the development and functioning of proper management structure, which was called Rogi Kalyan Samitis (RKS). RKS is state\'s attempt to make "health everyone\'s business" by de-mystifying health-care delivery at district and sub-district levels especially with reference to facility based health- care delivery systems and encouraging citizen\'s participation in the management bodies.([@ref2][@ref3]) RKS is now constituted as a registered society under the Societies Registration Act of government of each state, which lays down detail guidelines for registering the Samiti.([@ref4]) In Maharashtra, RKS comprises of two bodies-governing body (GB), which is responsible for policy formulation and decision making while executive body (EB) for implementing these decisions. There exists a charter/guideline of RKS which includes a set of well-defined roles and responsibilities of each committee in order to make RKS fully operational in the state.([@ref5]) The District Program Managing unit (DPMU) functions as a support system under the NRHM to assist GB and EB on functioning of RKS in the district.([@ref6]) We aimed to analyze operational aspects and challenges of RKS under health systems strengthening. Materials and Methods {#sec1-2} ===================== Study design {#sec2-1} ------------ Mixed method, multi-site, collective case study. The cases {#sec2-2} --------- Three RKS, i.e., one RKS at district level and two at block levels. Study locale {#sec2-3} ------------ The district of Pune was selected for study after interactions with District Program Manager (DPM) revealed that, RKS were set up in all the health service delivery facilities. Study subjects {#sec2-4} -------------- DPM, Block Program Manager (BPM)/Taluka Health Officer (THO), RKS Members belonging to EB at District Hospital and selected Community Health Center/Rural Hospital (CHC/RH) at block levels, beneficiaries of the out-patient department (OPD) and in-patient department (IPD). Sampling frame-work {#sec2-5} ------------------- All the study subjects were selected for in depth interviews. The number of members of EB to be interviewed in depth was restricted to 03 from each facility under study as it was observed that, the level of data saturation was achieved after this. Two CHCs/RH were selected as either high performing or low performing according to their reported performance at DPMU. High performing CHC/RH means adhering to RKS Charter and closely following the well-defined set of roles and responsibilities, whereas low performing CHC/RH lacked in these areas. The names of these CHCs/RH are anonymized, and they would be mentioned as high performing and low performing CHC/RH. A total of 10% of patients from OPD, and 20% of patients from IPD were selected randomly at each CHC/RH. The inclusion criteria for respondents were patients 18 years and above. Informed consent was taken from every official selected for the study beforehand; with the agreement of maintaining his confidentiality. This study was conducted over a period of 7 months (from August 2011 to February 2012). Data collection methods {#sec2-6} ----------------------- Primary data were collected through semi-structured interviews, researcher\'s observations, field notes, and quantitative data. Secondary data were collected from the documented records and reports available at the facilities under study. Data analysis {#sec2-7} ------------- Qualitative data were analyzed thematically. The interviews were read word by word; data were extracted and then coded inductively by the authors. Data were triangulated by combining responses from interviews with the researcher\'s observations and obtained documentary data. Results and Findings {#sec1-3} ==================== Even after repeated visits, researchers were denied facilitation of interaction with authorities/stakeholders at District Hospital. Medical Superintendent (Clinical) who is also the secretary of GB of RKS at DH prohibited the researchers to interact with members of EB though he failed to justify his act. Researchers were sent to the clerk from where they managed to obtain information related to previous years expenditure conducted under RKS grant. Hence, researchers were unable to obtain complete data related to functioning of RKS at this health facility. Organization of RKS {#sec2-8} ------------------- Organization and activities conducted under RKS is determined on the basis of data taken from the respective health facility. Criteria used are whether the health facility under study abides by guidelines provided by Government.([@ref7]) [Table 1](#T1){ref-type="table"} represents the performance of health facilities. Events such as the formation of GB and EB, their composition, frequency of meetings, outreach activities, biomedical waste management, etc., has been taken into organizational consideration in high performing CHC/RH. However, there is no provision of all the above services that aim to achieve patient welfare at low performing CHC/RH and district hospital. RKS was yet to develop and display charter of citizen\'s health rights at low performing CHC/RH and DH. ###### Details of organization and activities conducted under RKS at health facilities under study  Compliance with citizen\'s charter is to be ensured through setting up of grievance redressal mechanism, which seems to be the missing link in operationalization of RKS. RKS guidelines {#sec2-9} -------------- Guidelines are available with DPMU and at high performing CHC/RH, which are not the case for low performing CHC/RH and DH. From conversation, it was clear that the Medical Superintendent at low performing CHC/RH was unaware of the guidelines. Also, secretary of GB at DH denied working of RKS as per guidelines, though he refused to reveal reasons for the same. Medical Superintendent at DH mentioned "why should we follow the guidelines right from the district hospital to the Primary Health Centers (PHC), when nobody follows RKS guidelines?" At high performing CHC/RH, the Medical Superintendent mentioned that guidelines were briefed by him to the committee members. However, no photo-copies of any reading material/guidelines related to RKS were provided to members at any of these health facilities. Functioning of RKS {#sec2-10} ------------------ ### Meetings {#sec3-1} In high performing CHC/RH frequency of meetings was regular and according to guidelines i.e., the frequency of meetings of GB was once in 3 months whereas, meetings of EB were conducted on a monthly basis. In low performing CHC/RH and DH, it was difficult for the stakeholders to elaborate on documentation details pertaining to the attendance of members attending the meeting and their outcome decision. The THO of low performing CHC/RH admitted that the meetings were not conducted on a regular basis. At high performing CHC/RH, attendance of members attending the meeting was recorded in an updated meeting register and signature of RKS members were obtained across their respective names. ### Meeting agenda for the financial year 2010-11 {#sec3-2} Finding at low performing CHC/RH was that Medical Superintendent decides agenda in advance, and the committee was held only for formal approval of pre-decided issues whereas in high performing CHC/RH the agenda to be executed was unanimously decided by the RKS members in the meetings. As quoted by the Medical Superintendent at low performing CHC/RH "all the powers to carry out expenditure from RKS fund should be given to the Medical Superintendent. What is the need to conduct meetings and increase the load of unnecessary documentation?" Interactions with members of RKS revealed that; meetings and agenda were organized in democratic style in high performing CHC/RH whereas; the same were conducted in autocratic style at low performing CHC/RH. [Table 2](#T2){ref-type="table"} infers that, the committees are more concerned with purchasing basic material requirements, repairing and beautification at low performing CHC/RH. High performing CHC/RH reveals setting up of an agenda, which addresses community health needs by the provision of medicine, drinking water facilities, and organization of outreach services. Though the documented agenda set of the DH reflects expenses to be made towards patient welfare, i.e., IEC material and appliances; it is not clear whether the expenditure was carried out under the decided heads as the researchers could not observe any development made in this health facility. ###### List of the agenda of meetings held in the financial year 2010-11  Capacity building and training {#sec2-11} ------------------------------ None of the stakeholders could elaborate on the importance of capacity building. Formal training for health systems and support systems functionaries is yet to be strategized. The DPM said "training sessions are organized periodically from the state level, which includes a component on RKS." However, the DPM could not furnish any such documented records related to previously organized training sessions. Similarly, on being interviewed THOs, Medical Superintendent and members of both the blocks declined that any training sessions have been conducted. All of them felt the need of training sessions to be organized as they felt that these sessions would help them to perform effectively. As quoted by the Taluka Health Officer/Block Program Manager of low performing block, "we do not get satisfactory answers for our difficulties from our superiors, for executing the RKS scheme in our block. Hence, periodic training sessions will certainly empower us!" Finance management {#sec2-12} ------------------ [Table 3](#T3){ref-type="table"} depicts that, regardless of consistency in fund allocation, high performing CHC/RH, low performing CHC/RH, and DH have failed to utilize funds according to the RKS guidelines for budget expenditure.([@ref8]) In low performing CHC, fund has been under-utilized under one head of repair and maintenance (Rs. 58,695); whereas, the maximum limit is Rs. 100,000. Rest of the funds is left unutilized. In high performing CHC/RH and DH, funds are left unutilized in areas like organization of outreach services and innovative schemes for welfare of the community. Fund utilization has exceeded under heads such as purchase of stationary (Rs. 140,000), purchase of appliances (Rs. 150,000), and drugs for below the poverty line beneficiaries (Rs. 200,000) whereas, the maximum limit is up to Rs. 100,000 only for each head under discussion. ###### Fund utilization pattern at health facilities  Monitoring system {#sec2-13} ----------------- As quoted by DPM, "appropriate monitoring system is yet to be operationalized in the District. Though the periodic progress is monitored by the GB at some health facilities in the District; much of it needs to be documented." A fully operational monitoring system is essential to initiate mid-course correctiveness for improvement and monitoring progress of RKS. Strengths and concerns about RKS as perceived by key stakeholders {#sec2-14} ----------------------------------------------------------------- The major strengths of RKS as commonly perceived by stakeholders were community participation and availability of funds at the level of devolution. Whereas, the concerns were lack of capacity building strategies, involvement of people from a political background leading to lack of consensuses among members and excess documentation. Community perceptions {#sec2-15} --------------------- Out of total 28 respondents (8 IPD and 20 OPD) none were aware about the existence of the RKS in low performing CHC/RH. Yet except for two respondents none complained about the quality of services being provided through this health-care facility. One patient said "we are poor people, yet we are receiving these services. We have to be satisfied as we do not have any better option due to our inability to bear the costs of health- care services." 42 out of 50 (20 OPD and 30 IPD) i.e., 84% respondents were aware of the existence of RKS in high performing CHC. Nearly 72% agreed upon availability of drinking water and clean toilet facility. Nearly 76% knew about availability of free referral services. The patients availing indoor services reported satisfactory diet facility and also agreed that medicines were always made available to them. A total of 94% respondents seemed to be extremely satisfied with the facilities provided. Discussion {#sec1-4} ========== In the context of decentralized decision-making, RKS is a strategy to improve the quality of management responses and thereby facilitate strengthening of health systems as well as health outcomes. This was reflected through developments made in infrastructure and improvements experienced by beneficiaries who approved provision of quality health-care through high performing CHC/RH. Study revealed that, inadequate support systems for capacity building and training are constraints, which weaken the public-health system.([@ref9][@ref10][@ref11]) Failure to build managerial capacities will result in inadequacies in building of technical expertise at level of devolution where the money has been decentralized. Financial management capacity of health facilities, related to management of flexible funds needs to be strengthened; as utilization of funds is not being undertaken as per mandates indicated in guidelines.([@ref9][@ref12]) This reflects managerial failure of the support systems resulting in operational challenges of RKS model at district level. Regular meetings are yet to be conducted in accordance with guidelines resulting in decisions to be taken autocratically at the level of devolution; thus, deviating from the purpose of establishing such Samitis. Though the GB of each RKS is mandated to act as a monitoring body also; a fully operating monitoring system is yet to be functional. An appropriate monitoring system engaging support systems and health systems functioning can contribute to functionality by providing supportive supervision and better updating of mandatory registers observe mid-course corrective measures at all levels of health facilities delaying, which the mandates of the RKS will remain a distant reality.([@ref9][@ref13]) Compliance of Citizen\'s charter is ensured through grievance redressal mechanism, as per the guidelines. However, this mechanism is yet to be fully functional; which reveals structural and functional defect in managerial mechanisms, which gives a picture that these health facilities are still functioning in isolation of the need of local communities.([@ref3][@ref9][@ref11][@ref14]) Health system strengthening requires a holistic view and approach in its management. [Figure 1](#F1){ref-type="fig"} represents that RKS strategy is based on the following building blocks, which contribute to strengthen health systems: {#F1} Support systems capacity needs to be coordinated with functional health system by adequate supervision, monitoring and motivation of RKS members.Financial management and adequate accountability of reporting and documentation.Effective community participation through social mobilization and partnerships.Capacity building for systems strengthening. Conclusion {#sec1-5} ========== A review of RKS functioning has revealed that this can become a viable and operationally feasible model by building capacities of RKS members and support systems for effectiveness in RKS functioning, contributing to reporting and documentation of challenges and scopes, bringing out transparency, accountability and partnerships can contribute to transform a community led sustainable RKS instead of bureaucratic led non-participatory approach. This will ensure community led partnerships for scaling up of local specific innovations and sustainable RKS strategies on benefiting health services meaningfully to the local community. **Source of Support:** Nil **Conflict of Interest:** None declared.

Attachment is seen in both a child's protest and proximity-seeking behavior if he or she is distressed or involuntarily separated from a primary caregiver as well as in children's confident exploration of novelty when they feel safe in the presence of their caregiver ([@bibr24-0963721420904967]). Theoretically, secure attachment relationships develop when caregivers are sensitively responsive to the signals and needs of their child, whereas insecure attachment relationships may develop when caregivers ignore or respond only intermittently to signals. Accordingly, research on attachment has examined predictors and outcomes of both secure and insecure attachment relationships. To synthesize the empirical evidence on these associations, attachment researchers were early adopters of meta-analytic methodology (e.g., [@bibr6-0963721420904967]; [@bibr27-0963721420904967]). In 1985, Main, Kaplan, and Cassidy proposed that caregivers' own mental representations regarding attachment, identified as autonomous (secure), dismissing, preoccupied, or unresolved on the basis of their responses to the Adult Attachment Interview, predict the quality of children's attachment relationships via the sensitivity of caregivers' responses to children. The importance of intergenerational transmission for developmental and clinical psychology, as well as for developmental psychopathology, lies in what it can tell us about caregivers' contributions to their children's social functioning and mental health and about factors that interrupt this contribution. In [@bibr25-0963721420904967], van IJzendoorn published a meta-analysis of 18 studies examining the intergenerational transmission of attachment and found an effect size of a strength rarely observed in psychological science (*r* = .47, *d* = 1.06; *N* = 854), which increased confidence among the research community in Main et al.'s Adult Attachment Interview as a strong predictor of individual differences in infant--caregiver attachment. However, van IJzendoorn's meta-analysis also showed that Main et al.'s model could only partly account for how transmission came about. This finding became known as the "transmission gap" and led to numerous theoretical and empirical efforts to further understand and bridge it ([@bibr26-0963721420904967]). Yet over the years, there were also studies, including ones with relatively large sample sizes, for which the authors reported null findings for intergenerational transmission (e.g., [@bibr2-0963721420904967]; [@bibr3-0963721420904967]). These findings cast doubt on the replicability of the intergenerational transmission, prompting a new meta-analysis more than two decades later. With more than four times as many data available (i.e., 83 samples), [@bibr31-0963721420904967] reported a considerably lower, moderately large effect size (*r* = .31, *d* = 0.65; *N* = 4,102). However, studies showed significant and unexplained heterogeneity in effect sizes, prompting an effort to conduct individual-participant-data (IPD) meta-analysis ([@bibr15-0963721420904967]) for testing more complex models. The purpose of the current article is to discuss how attachment researchers have turned to IPD to overcome the limitations of single studies with small sample sizes and traditional aggregate-data meta-analysis. Some hurdles on the road to creating IPD data sets, and their potential solutions, will also be discussed. The Promise of Data Pooling {#section1-0963721420904967} =========================== IPD meta-analysis has been a gold-standard method of meta-analysis for some time in the biomedical sciences ([@bibr23-0963721420904967]), but it has only recently found its way to psychology ([@bibr17-0963721420904967]). IPD meta-analysis involves obtaining, harmonizing, and synthesizing the raw data for the individual participants in studies pertaining to common research questions ([@bibr15-0963721420904967]). Compared with meta-analysis based on study-level aggregate data, IPD meta-analysis thus adds the data on the level of the participants to the analyses (see [Fig. 1](#fig1-0963721420904967){ref-type="fig"}). {#fig1-0963721420904967} The method of IPD meta-analysis is precisely what was needed in attachment research, as the field had hit the saturation stage foreseen by van IJzendoorn and Tavecchio in [@bibr28-0963721420904967]. In this stage, many of the major questions seemed to have been settled and, despite countless but fragmented efforts, progress was slow in resolving the remaining gaps. Combining data from primary studies, whether large or small, capitalized on the benefits of this saturation stage and offered exciting prospects for the renewal of the attachment-research paradigm ([@bibr4-0963721420904967]). We started the Collaboration on Attachment Transmission Synthesis (CATS) to both overcome stagnation in understanding intergenerational transmission of attachment and to test the feasibility of IPD meta-analysis for our field. On the basis of discussions, the participating investigators drafted a protocol (see <https://osf.io/9p3n4/>) with the aim of advancing our insight into the mechanisms underlying intergenerational transmission of attachment. All authors of the studies identified in the [@bibr31-0963721420904967] meta-analysis were invited to participate in this project. This led to a data set of 59 samples with 4,498 parent--child dyads, but new samples continue to be added. Advantages of and Approaches to IPD Meta-Analyses for Attachment Research {#section2-0963721420904967} ========================================================================= The main advantage of a pooled set of raw data over a meta-analysis of aggregate data is the increase in power and degrees of freedom ([@bibr15-0963721420904967]) so that increasingly complex models and auxiliary hypotheses may be tested. In attachment research, data collection through labor-intensive methods constrains sample sizes, resulting in few adequately powered studies ([@bibr20-0963721420904967]). In the 2016 meta-analysis, only 18% (15/83) of the studies reached the .80 power threshold of 82 parent--child dyads required to assess secure--insecure attachment transmission ([@bibr31-0963721420904967]). Testing more complex models, such as the ones to answer pertinent questions on moderating or mediating factors of attachment transmission, require a much larger sample for drawing replicable conclusions. The CATS data set makes this venture possible. Our first study on ecological factors that might affect intergenerational transmission of attachment is an example of moderator testing that would not have been possible without IPD ([@bibr29-0963721420904967]). In this study, we examined, for example, whether attachment transmission differed by age of the child. The preceding meta-analysis of aggregate data had looked into this issue as well, but given that attachment was measured with different instruments in studies with younger children versus older children, there was no way of separating the effects of age from the effects of using a different instrument ([@bibr31-0963721420904967]). In the IPD meta-analysis, we controlled for the type of instrument and found that the transmission effect was stronger for older children than for younger children ([@bibr29-0963721420904967]). This finding provides support for the theoretical notion that cumulative experiences with parents lead to more stable, ingrained attachment patterns and helps to recalibrate expected intergenerational transmission effect sizes. Several other manuscripts are currently under way. These report, for example, on patterns of nontransmission (e.g., from secure to insecure classifications or between different types of insecure classifications) using pooled data, a procedure that is necessary because of low base rates for these transmission patterns ([@bibr10-0963721420904967]), and on a moderated mediation model of attachment transmission explaining why the transmission gap could not be solved with additional mediators ([@bibr30-0963721420904967]). Empirical findings may also be made more robust as a guide for theory development by controlling "researcher degrees of freedom," which represent the diversity of choices a researcher makes during the research process ([@bibr19-0963721420904967], p. 1359). All choices made during study design, data collection, data analysis, and reporting may affect study outcomes and hence theory development on a given topic. In the field of attachment research, there are historical reasons that allow for a variety of ways in which attachment variables may be parsed. Originally, only three categories of parent--child attachments were identified (secure, avoidant, and resistant; [@bibr1-0963721420904967]), but later, [@bibr12-0963721420904967] discovered insecure disorganized attachment. From that moment, researchers could parse their attachment variables as a secure/insecure dichotomy, an organized/disorganized dichotomy, three- or four-way categorical variables, or combinations of categories and a rating scale, a set of options which was also mirrored in the variables for adult attachment representations as assessed in the Adult Attachment Interview ([@bibr11-0963721420904967]). In all, 38 different ways of examining the intergenerational transmission of attachment have been described in the literature ([@bibr18-0963721420904967]). The absence of substantive or statistical reasons for choosing one variant over the other may indicate underspecification in the theoretical model, making it harder to design tests that could expose the theory's flaws and thus undermining the credibility of the theory. The meta-analytic finding that the effect size for unpublished data was lower than the effect size for published data, even within the same studies, also hints at selective reporting of significant findings ([@bibr31-0963721420904967]). Secondary analyses are just as vulnerable to *p* hacking as primary studies ([@bibr32-0963721420904967]), which, given their suggestive high power (uncorrected for multiple exploratory analyses of data sets), may be even more misleading. Paradoxically, controlling access to a data source that may be used to answer a plethora of questions may increase transparency, compared with not controlling access to open data. Registration of hypotheses and analysis plans for IPD meta-analysis of pooled data helps to control researcher degrees of freedom and false-discovery rates. Transparency of the analytic process is vital to ensure replicability of the results, and this is facilitated in a consortium of researchers monitoring the workflow from research question, theorizing, and hypothesizing to data collection and analysis planning. In CATS, study hypotheses and exploratory questions are included in the protocol that accompanies the invitation to the study authors and are posted on our OSF page (see <https://osf.io/9p3n4/>). Furthermore, manuscript proposals are circulated across the entire CATS group as an internal registration when new studies are started, thus preventing cherry-picking results for publication. Finally, for each manuscript, we conduct sensitivity analyses ([@bibr29-0963721420904967]) or multiverse analyses ([@bibr21-0963721420904967]) to account for alternative ways to examine the data. The recently published registration format for secondary analysis (see <https://osf.io/x4gzt/>) will facilitate public registration for future IPD meta-analyses. In attachment research, a categorical model of attachment was taken up early on as the most likely representation of reality, perhaps under the influence of emerging diagnostic classification systems ([@bibr4-0963721420904967]). The categorical assumption started to be put to the test much later ([@bibr5-0963721420904967]; [@bibr16-0963721420904967]). Latent structure analyses and taxometric analyses, however, require large data sets, so that smaller studies supporting dimensional measurement models have thus far insufficiently impacted research practices. With the pooled data on parental attachment representations in the CATS data set, we were able to show that individual differences in adult attachment representations may also be consistent with a latent dimensional rather than categorical model ([@bibr14-0963721420904967]), but incremental validity is still an outstanding question for which the IPD approach might be perfectly suited. Researchers could go one step further and pool raw materials such as interview transcripts or video-recorded observations, which could also be beneficial for methodological refinement. This was shown very early on by [@bibr12-0963721420904967], who made a case for the existence of disorganized attachment on the basis of videotapes that were impossible to code with the regular rating system shared with them by researchers working with high-risk samples. This project had an enormous impact on attachment research and the use of attachment constructs in clinical practice. Thirty years later, we aim to refine attachment measurements again by sharing the raw materials. A first project regarding the structure of the scale for unresolved loss or trauma in the Adult Attachment Interview has been registered (see <https://osf.io/bu5cx>). The Challenges of IPD Meta-Analysis {#section3-0963721420904967} =================================== Like any method, IPD meta-analysis comes with challenges and limitations. In this section, we describe three broad challenges for this type of research; for more practical challenges and concrete tips, see [Table 1](#table1-0963721420904967){ref-type="table"}. ###### Practical Challenges to Data Pooling and How the Collaboration on Attachment Transmission Synthesis (CATS) Dealt With Them  ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Challenge and recommendation Tip ----------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Obtaining the data Invest time and effort to validate and share (archived) data. • Plan enough time for this stage. For CATS, it took 18 months.\ • Establish the meta-analysis as a collaborative group effort by researchers who contribute data. Define roles within the collaboration for which members may volunteer (group coauthor, named coauthor, lead author) and that have academic value. Establish a transparent procedure for assigning roles and for involving members in study plans and ideas (e.g., collaborator meetings, circulating paper proposals).\ • Make contributing as easy as possible: Inform potential collaborators of exactly which variables are needed, provide a template for the data, be flexible in data format, and offer assistance.\ • Provide a secure shared folder or server to share the data; data sharing through e-mail is not compliant with privacy laws. Determine whether data sharing is ethically or legally allowed. • Include institutional privacy officers from the outset in making a data-protection impact assessment and in determining the infrastructure needs and the minimal set of joint agreements for the collaboration members.\ • Provide information on where data are located, who controls access, and who has access and under which conditions.\ • Provide clear directions to collaboration members for checking the local or study-specific ethical and legal conditions under which they are allowed to share their data and for anonymizing their data. Creating the overall data set Get insight into the quality of the received data. • Perform checks for inconsistencies with article, anomalies (e.g., out-of-range scores on questionnaires), and missing data. Try to resolve issues that arise with study authors.\ • Ask for data quality indicators, such as interrater reliability and internal consistency.\ • Exclude data that are not up to a priori standards. Securing access and analysis of the data Set up a secure and accessible storage facility. • Determine whether the data need to be accessible to researchers (data analysts) outside the organization. If so, consider building a data commons with secure remote access. If not, store the data with the university secure storage facility.\ • Place only the final files that need to be accessed by researchers outside the organization on the remotely accessible server. Anonymize the origin of the data sets. Store data files received from primary study authors or files used for data cleaning on a separate secure server.\ • Partition the remotely accessible server so that researchers have access only to the parts they need to access.\ • Add the necessary analysis software and other processing software to the remotely accessible server so that the researchers do not have to copy data to their own computers.\ • Have researchers with access to the data sign a data-sharing agreement. Defining authorship • Clearly define contributor roles for the project and provide transparent information about who fulfills these roles and how (e.g., using the Contributor Roles Taxonomy; [www.casrai.org/credit.html](https://www.casrai.org/credit.html)).\ • For manuscript preparation, consider the use of tools that allow for simultaneous working on a manuscript so that authors can see and respond to each other's feedback, comments are given in the same document, and a record of writing contributions is maintained.\ • Give reasonable deadlines for responding and keep them.\ • Map contributor roles on authorship roles. In CATS, we have three layers of involvement with articles: Participation in drafting the manuscript leads to named authorship, sharing data and reading and approving the draft before submission leads to group authorship, and sharing data without manuscript involvement leads to a mention as a nonauthor collaborator. These types of involvement were based on the guidelines by the [@bibr8-0963721420904967]. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- First, pooling data is useful only when the same underlying constructs are measured in enough studies, whether or not they are measured with different instruments. Before requesting data from study authors, it is necessary to review these instruments of the constructs of interest and assess the feasibility of harmonizing different ways of operationalizing a construct. The attachment field proved eminently suited for IPD meta-analysis because it has honed a limited and well-calibrated set of standard instruments such as the Strange Situation procedure and the Adult Attachment Interview. Harmonizing the measures for parental sensitivity already required making multiple assumptions, however. Second, sharing participant data is increasingly regulated under privacy-protection laws, which vary across countries. Consulting with institutional privacy officers is key when setting up a data-pooling project to discuss the ethical and legal basis for data sharing and making data-sharing agreements. Furthermore, to ensure the privacy of participants, it is important to establish secure ways to transfer, store, and analyze anonymized data. In CATS, we have built a data commons for storage and analysis, which is a secured, remote-access information-technology infrastructure holding the pooled data set, syntax codes, and various analysis software packages ([@bibr7-0963721420904967]). A final challenge is that likely not all data from eligible studies can be acquired. This can occur for several reasons, such as authors who cannot be traced or data that have been destroyed, but also because of priority claims of authors who want to publish their research on their arduously collected data sets before sharing the data. Fortunately, in CATS, authors of 67% of the original studies contributed their raw data, but sharing rates are often lower ([@bibr9-0963721420904967]). It is therefore important to decide in advance what percentage of data would be enough to proceed and to compare aggregate data of the missing studies with the IPD data of the included studies and, whenever possible, to include them if they differ ([@bibr22-0963721420904967]). Conclusion {#section4-0963721420904967} ========== Wide-scale collaboration among attachment researchers in CATS has brought rigorous testing of complex attachment-theoretical propositions within reach while enabling the exploration of the boundaries of these propositions. Capitalizing on the advantages of the saturation stage of attachment research offers new and exciting horizons that pull attachment research back into the stage of construction of attachment theory. Data pooling holds these same promises for other fields in psychology: addressing theoretical challenges, increasing methodological rigor and transparency, and strengthening the capacity to inform applied research. Together with other efforts to make psychological science more robust ([@bibr13-0963721420904967]), IPD meta-analyses show both the value and the viability of moving to new levels of collaboration. Recommended Reading {#section5-0963721420904967} =================== [@bibr15-0963721420904967]. (See References). An accessible description of what individualparticipant-data (IPD) meta-analysis is, how it is different from meta-analysis of aggregate data, when it is the preferred method of meta-analysis, and how to conduct an IPD meta-analysis. [@bibr22-0963721420904967]. (See References). Contains the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for reporting on IPD meta-analyses, which are originally from the medical field but can be applied to psychological research. [@bibr26-0963721420904967]. (See References). Reviews previous research on the intergenerational transmission of attachment and the difficulty in explaining the "transmission gap"; also provides a novel theoretical framework including contextual factors and differential susceptibility to fill in the gap. [@bibr29-0963721420904967]. (See References). Describes the first IPD metaanalysis on the intergenerational transmission of attachment by the Collaboration on Attachment Transmission Synthesis. [@bibr32-0963721420904967]. (See References). Explains the various uses of secondary data analysis as a tool for the generation of hypotheses, confirmatory work, methodological innovations, and analytical methods, with caveats for using secondary data. For a full list of collaborators in the Collaboration on Attachment Transmission Synthesis (CATS) and their affiliations, see the CATS Open Science Framework project at <https://osf.io/56ugw/>. **ORCID iDs:** Carlo Schuengel  <https://orcid.org/0000-0001-5501-3341> Marinus H. van IJzendoorn  <https://orcid.org/0000-0003-1144-454X> Transparency {#section6-0963721420904967} ============ *Action Editor*: Randall W. Engle *Editor*: Randall W. Engle **Declaration of Conflicting Interests:** The author(s) declared that there were no conflicts of interest with respect to the authorship or the publication of this article. **Funding:** This work was supported by a grant from Stichting tot Steun Nederland to M. Oosterman and C. Schuengel, a grant from the Social Sciences and Humanities Research Council Canada (No. 430-2015-00989) to S. Madigan, a grant from the Wellcome Trust (WT103343MA) to R. Duschinsky, and a Veni grant by the Dutch Research Council (No. 451-17-010) to M. L. Verhage.

Background {#Sec1} ========== Health related patient reported outcome measures (PROMs) is considered essential to determine the impact of disease on the life of individuals, taking account not only of the clinical diagnosis of a disease but also of its impact \[[@CR1]\]. Shoulder pain is one of the most common causes of musculoskeletal pain, with a prevalence of 20--33 % in the general population \[[@CR2]\]. Shoulder disorders are responsible for major medical, social, and economic costs \[[@CR3]\]. They are often accompanied by pain and restricted movement, hampering certain activities \[[@CR4]\] and compromising psychological and social wellbeing \[[@CR5]\]. Various questionnaires are available in English to evaluate the impact of shoulder disorders on function. With regard to other languages, it is recommended to translate, culturally adapt, and validate existing instruments in order to avoid the further proliferation of different methods \[[@CR6]--[@CR8]\]. The original version of the Shoulder Pain Disability Index (SPADI) is a quality of life questionnaire developed to evaluate the pain and disability associated with shoulder dysfunction \[[@CR9]\]. The SPADI is a 13-item shoulder function index on the ability of responders to carry out basic activities of daily living. Each item is scored by a numeric rating scale that ranges from 0 (no pain/no difficulty) to 10 (worst pain imaginable/so difficult it required help). SPADI provides a pain scale (5 items; scale score range 0--50 points, expressed as percentage) and a disability scale (8 items; scale score range 0 -- 80 points, expressed as percentage). The two scale scores are averaged to derive a total Spanish Version of the SPADI score (0--100 points). A higher score indicates greater pain-related disability \[[@CR9]\]. The original version of the SPADI was initially proposed as a visual analog scale (VAS) and subsequently validated as a numerical scale to enable administration by telephone, obtaining reliable and valid results \[[@CR10]\]. Previous systematic reviews have found no single questionnaire to superior to others in terms of administrative burden or measurements properties \[[@CR10]\]. There are other validated questionnaires already available as Disability of Arm, Shoulder, and Hand (DASH) or Simple Shoulder Test (SST) but several studies have recommended SPADI also, as specific scale for the shoulder due to its easy administration and rapid completion (3--10 min), which is facilitated by the brevity of the questions and the numerical response scale used (from 0 to 100) \[[@CR11]--[@CR13]\]. Systematic reviews have described SPADI as one of the highest-quality questionnaires related to the upper extremity and have endorsed its utilization \[[@CR10], [@CR13]\]. Although the SPADI questionnaire has been validated in German \[[@CR14]\], Slovenian \[[@CR15]\], Turkish \[[@CR16]\], Italian \[[@CR17]\], Portuguese \[[@CR18]\], Persian \[[@CR19]\], and Danish \[[@CR20]\], it has not yet been validated in Spanish. The objective of this study was to report the procedure followed for the cross-cultural adaptation and subsequent validation of a Spanish version of SPADI, including an examination of its psychometric properties. Methods {#Sec2} ======= Design {#Sec3} ------ A two-stage observational study was conducted. The first stage comprised the translation and cross-cultural adaptation of SPADI, while the second stage consisted of a prospective evaluation of the internal consistency, reliability, construct validity and measurement error of Spanish Version of the SPADI. ### Stage 1- Translation and cross cultural adaptation {#Sec4} Two physicians, with adequate expertise in shoulder disorders management and both lenguages, and an independent native professional interpreter translated the English version of SPADI into Spanish and organized a meeting to take account of possible cultural issues. A back-translation process was carried out by a specialist translator to guarantee the conceptual equivalence of the terms used, as recommended in the literature \[[@CR21], [@CR22]\]. People involved in translation worked independently. In a second meeting, we compared the two versions and found no appreciable differences between them. A final version of the Spanish Version of the SPADI was agreed and tested in a pilot study with 40 patients (24 females, age = 45,6 ± 13.0 years) with shoulder problems (fractures and tendinopaties) recruited from among rehabilitation outpatients at hospital setting. This pilot study included cognitive debriefing standardised interviews carried out for one member of the research staff to assess its comprehensibility and ensure that the items retained the meaning of the original version. ### Stage 2- Evaluation of psychometric properties {#Sec5} #### Participants and procedure {#FPar1} This questionnaire validation study included 136 volunteers with different shoulder disorders recruited from among rehabilitation outpatients in a hospital setting. Inclusion criteria were the presence of a shoulder disorder and the availability of a diagnosis by a specialist rehabilitation physician; diagnoses were classified into six subcategories (Table [1](#Tab1){ref-type="table"}). Exclusion criteria were age under 18 years and inadequate command of Spanish to complete the questionnaires. All patients signed their informed consent to participation in the study, which was approved by the Research Ethics Committee of our hospital.Table 1Demographic characteristics of the study population and the distribution of diagnosesCharacteristicCases (%)Age (years) Mean (sd)Study Population13649.8 ± 15.0 Male61 (44.9 %)46.8 ± 15.3 Female75 (55.1 %)52.2 ± 14.4 Diagnosis  Humerus Fractures29 (21.3 %)  Calcific Tendinopathy16 (11.8 %)  Rotator cuff tear24 (17.6 %)  Osteoarthritis3 (2.2 %)  Hemiplegic Shoulder Pain2 (1.5 %)  Biceps Tendinopathy30 (22.1 %)  Frozen shoulder10 (7.4 %)  Complex Regional Pain Syndrome4 (2.9 %)  Avascular Necrosis3 (2.2 %)  Glenohumeral Instability9 (6.6 %)  Others^a^6 (4.4 %)Questionnaires Scores Mean (SD) Shoulder Pain Disability Index58.5 (22.2) Mental Health SF-1238.2 (9.0) Physical Health SF-1247.5 (11.0) Simple Shoulder Test34.5 (24.14) Disability of Arm, Shoulder and Hand48.1 (20.4) Visual Analogue Scale4.0 (2.6)^a^Minor-Heterogeneous shoulder disorders: Polymyalgia reumathic (2), Acromioclavicular Luxation (2), Unspecific Shoulder Pain (2) Participants who met the selection criteria completed a Spanish version of the SF-12 \[[@CR23]\], SST \[[@CR24]\], DASH \[[@CR25]\] questionnaires and VAS in the hospital with the assistance of rehabilitation service staff. SF-12 (Version 1) is a self-administered instrument with 12 items on physical and mental health status; responses are scored (for intensity or frequency) on a Likert-type scale (3--6 points according to the item). These items are used to calculate the physical and mental summary measures. This instrument has shown adequate reliability (ICC = 0.73-0.86) \[[@CR23]\]. SF12 was used to check discriminant construct validity. The SST is a 12-item shoulder function scale on the ability (yes/no) of respondents to perform 12 activities of daily living (ADLs). The total SST score (0 to 100) expresses the percentage of items with a positive response. The Spanish version of the SST was recently validated and showed adequate reliability (ICC = 0.69--0.94) \[[@CR24]\]. The 30-item DASH measures the function and symptoms of patients with upper extremity musculoskeletal disorders. The total score ranges from 0 (best state) to 100 (worst). The Spanish version of DASH has been validated and showed adequate reliability (Cronbach alpha = 0.96) \[[@CR25]\]. SST and DASH were usded to check convergent validity. Pain intensity was tested using a VAS (0 to 10). A randomly selected subgroup (*n* = 56) of the total sample repeated the questionnaires after an interval of 24--48 h to study the reliability of the Spanish Version of the SPADI using a test-retest methodology. This sample was selected using a random numeric sequence generated by a computer. A brief interval of 24--48 h was selected to avoid fluctuations in the severity of the symptoms. The questionnaires were administrated in same conditions used in the all sample. Statistical analysis {#Sec6} -------------------- Sample size was selected in accordance with recommendations to include 4--10 subjects per variable, with a minimum sample size of 100 subjects to ensure stability of the variance--covariance matrix in the confirmative factor analysis \[[@CR26]\]. Means and standard deviations of the demographic variables were calculated. Construct validity and factor structure were then determined from maximum likelihood extraction (MLE) with varimax rotation, establishing the satisfaction of the following three criteria as a priori extraction requirement: scree-plot inflection, eigenvalue \>1.0, and variance \>10 %. The fit of confirmatory factor analysis was considered to be acceptable if the comparative fit index (CFI) and the normalized fit index (NFI) were greater than 0.90, with root mean square error of approximation (RMSEA) values equal to or less than 0.08 \[[@CR27], [@CR28]\] The internal consistency of measures was evaluated by determining the Cronbach alpha coefficient in an expected range of 0.70 to 0.90 \[[@CR29]\]. The test-retest reliability was analyzed by using the type 2.1 interclass correlation coefficient, and the error sensitivity was calculated with the MDC95 analysis of Stratford. Where MDC = 1.96 × SEM × square root of 2 \[[@CR30]\]. The construct validity was determined by comparing Spanish Version of the SPADI with SF-12, SST, DASH and VAS scores. Correlations were calculated using Pearson approach. A correlation value below 0.25 indicates a weak relationship, a value between 0.25 and 0.50 a fair relationship, a value between 0.50 and 0.75 a moderate to strong relationship, and a correlation above 0.75 a strong relationship \[[@CR31]\]. Moderate to strong positive (DASH, VAS) and negative (SST) correlations with Spanish Version of the SPADI were expected, with a similar pattern of expected correlations for both dimensions of the index. A lower correlation was expected between Spanish Version of the SPADI and SF-12, which is a generic functionality scale. SPSS version 21.0 for IOS (IBM, Chicago, IL) and LISREL v.8.8 (SSI Inc., Lincolnwood, IL) were used for the statistical analysis. Results {#Sec7} ======= Potentially eligibles participants in database during the period of the study were 234 patients wich were invited for eligibilty 172 patients. 36 patients refused to participate and finally 136 participants were included. 40 participants completed the second questionnaire of the 56 invited. All participants comprehend the questionnaire and found it easy to complete. No conceptual ambiguities or language difficulties were encountered in the translation of the SPADI (Additional file [1](#MOESM1){ref-type="media"}). Minor changes in the Spanish version included the replacement of imperial with metric measures, e.g., "10 lb" becomes "4.5 Kg" in item 12. After the cognitive debriefing interviews ensured that the items retained the meaning of the original version. Table [1](#Tab1){ref-type="table"} exhibits the demographic characteristics of the participants and the distribution of diagnoses. In the factorial analysis, the correlation matrix for Spanish Version of the SPADI was adequate according to results of the Kaiser-Meyer-Oklin (0.92) and Bartlett's sphericity test (*p* \< 0.001). A 62.8 % of the variance was explained by two factors. The item loading is shown in Table [2](#Tab2){ref-type="table"}: factor 2 comprised items 1, 9, and 10, while factor 1 included the remaining items with the exception of item 13, which showed cross-loading. The confirmatory factor analysis showed a Comparative Fit Index of 0.82 and a Normed Fit Index of 0.80. The Root Mean Square Error of Aproximation was 0.12. The *x*2 test for the 2-factor model was significant (*x*^2^ = 185.41, df = 62, *p* \< 0.01 (Fig. [1](#Fig1){ref-type="fig"}).Table 2Factor loading items for the two-factor solutionComponent12At its worst?,22**,55**When lying on the involved side?**,63**,36Reaching for something on a high shelf?**,81**,32Touching the back of your neck?**,70**,44Pushing with the involves arm?**,68**,44Washing your hair?**,83**,25Washing your back?**,82**,17Putting on an undershirt or pullover sweater?**,67**,52Putting on a shirt that buttons down the front?,28**,85**Putting on your pants?,24**,88**Placing an object on a high shelf?**,87**,22Carrying a heavy object of 10 lb?**,57**,40Removing something from your back pocket?^a^**,61,56**^a^Cross loadingThe bold numbers represent the main factor load in each componentFig. 1The *x*2 test for the 2-factor model was significant For the Spanish Version of the SPADI, there were no significant missing responses (\<7 missing response in 136 patients), similar level of missing response was found in the rest of questionnaires used in this study. After check the origen of this missing data a missing at random was assumed. A high degree of internal consistency was obtained for each factor α = 0.92 (CI95 % 0.91 to 0.95) and 0.82 (CI95 % 0.76 to 0.86), The test-retest reliability was also evaluated with ICC, with an item ranging from 0.89 to 0.93. The ICC for the total score was 0.91 (95 % CI 0.88 to 0.94). Measurement error by MDC~95~ was 12.2 %. The construct validity was confirmed by strong positive correlations between the Spanish Version of the SPADI and DASH (pain: *r* = 0.80; *p* \< 0.01; disability: *r* = 0.76; *p* \< 0.01), moderate positive correlations between SPADI and VAS (pain: *r* = 0.67; *p* \< 0.01; disability: *r* = 0.65; *p* \< 0.01), moderate negative correlations between SPADI and SST (pain: *r* = −0.71; *p* \< 0.01; disability: *r* = −0.75; *p* \< 0.01) and weak positive correlation with physical and mental components of SF-12 (both *r* = 0.40; *p* \< 0.01). Discussion {#Sec8} ========== SPADI demonstrated good internal consistency and convergent validity and reliability in a sample of 136 patients with different shoulder disorders. The confirmatory factor analysis showed an acceptable fit with a CFI of 0.82 and NFI of 0.80, but the error was higher (RMSEA = 0.12) than the recommended value of 0.08 \[[@CR28]\]. The internal consistency value calculated for this version was similar to that obtained for versions validated in other languages \[[@CR14]--[@CR20]\] and within an acceptable range, in common with the original questionnaire and all other published versions \[[@CR26]\]. The Spanish version showed a clear difference in the loading of the two factors, with items that evidenced a high correlation with one of the factors and a low correlation with the other. Our finding contrasts with the similar loading of the two factors reported for the original questionnaire \[[@CR9]\] and subsequent versions \[[@CR32]\], which hindered a clear separation between pain and disability dimensions. The fit indices associated with the confirmatory factor analysis model were satisfactory, although the error of approximation was an exception and did not indicate an optimal fit, which may possibly be due to an effect of our specific study population on the factor structure. It should be borne in mind that a slightly increased error does not necessarily imply that the structure of the scale is poor. The good construct validity obtained with this Spanish version supports its usefulness for evaluating patients' perception of the impact of shoulder lesions. The test-retest reliability of Spanish Version of the SPADI (0.89--0.93) was superior to that reported for the original questionnaire (0.64--0.66), slightly higher than that of the Persian \[[@CR19]\] and Danish \[[@CR20]\] versions, and similar to that of the Brazilian \[[@CR18]\], German \[[@CR14]\], Turkish \[[@CR16]\], and Slovenian \[[@CR15]\] versions. Perhaps, the reason could be a Spanish sample is more homogenous. The Measurement error by MDC~95~ was 12.2 %, lower than the original version, was of 18 % \[[@CR11]\]. As expected, the lowest correlation (divergent construct validity) was observed between SPADI and SF-12, reflecting the discriminative validity of the instrument, given that SF-12 is a generic functionality scale. Moderate-strong correlations were found with the specific shoulder-related instruments (SST and DASH), indicating adequate convergent validity. Low-moderate correlation was found between SPADI and VAS, as observed in previous cross-cultural validation studies \[[@CR14], [@CR20]\]. The present study population was limited to hospital outpatients in a Spanish urban setting, and different results may be obtained in other types of population. The greater reliability observed for the Spanish version than for the original questionnaire may be attributable to the shorter interval between tests (48 h) which could induce an artificial inflation of correlation coefficients due to recall bias. Studies of this version of the questionnaire are warranted to test its validity in other Spanish-speaking countries (e.g., Latin America, Philippines, etc.). A study strength is that the sample size was larger (*n* = 136) than in previous validation studies of this instrument, reducing standard errors and supporting its application in individual and group studies \[[@CR33]\]. Further research is needed to establish longitudinal validity, responsiveness and thresholds for minimal important change in the Spanish version of SPADI. Conclusions {#Sec9} =========== In conclusion, the psychometric properties of the Spanish version of SPADI are similar to those of the original questionnaire and subsequent adaptations in different languages, supporting its utilization as a reliable clinimetric instrument in the setting of shoulder disorders. Additional file {#Sec10} =============== Additional file 1:**Shoulder and Pain Disability Index (SPADI-Sp).** (PDF 60 kb) **Competing interests** The authors declare that they have no competing interests. **Authors' contributions** AIC-V and MAM has made a contribution to the conception and design of this study and to analyze the data. RPC, VTF, LMM and MDMM recruit the patient, collect data and made substantial contributions to the manuscript. All the authors have given final approval of the version to be published. This study received a grant (8.06/5.42.3064-3) from the Research Office of the University of Malaga.

All relevant data are within the paper. Introduction {#sec001} ============ Consider the two-dimensional elliptic partial differential equation $$\begin{array}{r} {\frac{\partial^{2}u}{\partial x^{2}} + \frac{\partial^{2}u}{\partial y^{2}} = D\left( x \right)\frac{\partial u}{\partial x} + g\left( x,y \right),\left( x,y \right) \in \Omega} \\ \end{array}$$ which is defined in the solution domain Ω = {(*x*, *y*):0 \< *x*, *y* \< 1}, where functions *D*(*x*) and *g*(*x*, *y*) ∈ *C* ^2^(Ω). The corresponding Dirichlet boundary conditions are given by $$\begin{array}{r} {u\left( x,y \right) = \psi\left( x,y \right),\left( x,y \right) \in \partial\Omega} \\ \end{array}$$ where ∂Ω is its boundary. These types of problems arise very frequently in different areas of applied mathematics and physics such as convection-diffusion equation which describes the transport phenomena, and the Poisson's equation which is broadly used in electrostatics, mechanical engineering and theoretical physics. Thus, solving elliptic differential equation have been of interest to many authors \[[@pone.0132782.ref001]--[@pone.0132782.ref003]\]. In 1968, Bickley \[[@pone.0132782.ref004]\] suggested the use of cubic splines for solving a linear ordinary differential. Following this, Albasiny and Hoskins \[[@pone.0132782.ref005]\] approximated the solutions by applying the cubic spline interpolation introduced by Ahlberg *et al.* \[[@pone.0132782.ref006]\], which leads to a matrix system of tri-diagonal instead of upper Hessenberg form which was obtained by Bickley \[[@pone.0132782.ref004]\]. The cubic spline method suggested by Bickley \[[@pone.0132782.ref004]\] was then examined by Fyfe \[[@pone.0132782.ref007]\]. Fyfe concluded that spline method is better than the usual finite difference method in terms of its accuracy and also its flexibility to get the approximation at any point in the domain. Due to its simplicity, many researchers started to work on spline methods for solving boundary value problems \[[@pone.0132782.ref008]--[@pone.0132782.ref011]\]. To mention a few, Bialecki *et al.* \[[@pone.0132782.ref012]\] formulated a new fourth order one step nodal bicubic spline collocation methods for the solution of various elliptic boundary value problems. Mohanty and Gopal \[[@pone.0132782.ref013]\] proposed a high accuracy cubic spline finite difference approximation of *O*(*k* ^2^ + *h* ^4^) accuracy for the solution of non-linear wave equation. Goh *et al.* \[[@pone.0132782.ref014]\] discussed the solution for one-dimensional heat and advection-diffusion by using a combination of finite difference approach and cubic B-spline method. Over the last few decades, we have seen the formulation of group iterative methods for solving the two dimensional elliptic partial difference Eqs \[[@pone.0132782.ref015]--[@pone.0132782.ref018]\]. In 1991, a half-sweep iterative method had been introduced by Abdullah \[[@pone.0132782.ref019]\] via the explicit decoupled group (EDG) iterative method which was shown to be faster and computationally economical than the existing explicit group (EG) method due to Yousif and Evans \[[@pone.0132782.ref018]\] for solving elliptic partial differential equation. Inspired by Abdullah \[[@pone.0132782.ref019]\], Othman and Abdullah \[[@pone.0132782.ref020]\] proposed a quarter-sweep iteration through the modified explicit group (MEG) method. Following this, Ali and Ng \[[@pone.0132782.ref021]\] extended the idea and formulated the modified explicit decoupled group (MEDG) method for solving two-dimensional Poisson equation. The MEDG method exhibits a better convergence rather than the existing group schemes of the same family, namely EG, EDG and MEG methods. In 1986, Yousif and Evans \[[@pone.0132782.ref018]\] developed the explicit group (EG) iterative method where a small group of 2, 4, 9, 16 and 25 points were constructed in the iterative processes for solving Laplace's equation. The numerical results show that the EG method is simpler to program compared to the block (line) iterative methods and it requires less storage. However, this method was solely formulated using the usual standard finite difference discretization which restricts the solutions at only certain points of the solution domain. This, thus, motivate us to adopt the idea in using splines in the formulation of the group methods. In this paper, a new method, namely spline explicit group (SEG) iterative method, which incorporates cubic spline with group iterative scheme, is developed for solving the elliptic problems. Using a cubic spline approximation in the *x*-direction and central difference in the *y*-direction, we obtain a new three level implicit nine-point compact finite difference formulation. Then, a four point explicit group iterative scheme is applied to the obtained system. The performance of the method will be investigated via two benchmark problems, that is the convection-diffusion equation and Poisson's equation. The Cubic Spline Approximation and Numerical Scheme {#sec002} =================================================== Here, the solution domain Ω = \[0, 1\] × \[0, 1\] is divided by *h* \> 0 in *x*-direction and *k* \> 0 in *y*-direction. Therefore, the grid points (*x* ~*l*~, *y* ~*m*~) are represented as *x* ~*l*~ = *lh* and *y* ~*m*~ = *mk*, *l* = 0, 1, ..., *N* ~*x*~, *m* = 0, 1, ..., *N* ~*y*~, where *N* ~*x*~ and *N* ~*y*~ are positive integers. Let *U* ~*l*,\ *m*~ be the approximation solution of *u* ~*l*,\ *m*~ at the grid point (*x* ~*l*~, *y* ~*m*~). Suppose that *S* ~*m*~(*x*) is the *m*-th mesh row cubic spline polynomial which interpolates the value *U* ~*l*,\ *m*~ at (*x* ~*l*~, *y* ~*m*~), is given by \[[@pone.0132782.ref006]\] $$\begin{array}{r} \begin{array}{cl} {S_{m}\left( x \right) =} & {\frac{\left( x_{l} - x \right)^{3}}{6h}M_{l - 1,m} + \frac{\left( x - x_{l - 1} \right)^{3}}{6h}M_{l,m} + \left( \frac{x_{l} - x}{h} \right)\left( U_{l - 1,m} - \frac{h^{2}}{6}M_{l - 1,m} \right)} \\ & {+ \left( \frac{x - x_{l - 1}}{h} \right)\left( U_{l,m} - \frac{h^{2}}{6}M_{l,m} \right)} \\ \end{array} \\ \end{array}$$ for *x* ~*l*−1~ ≤ *x* ≤ *x* ~*l*~, where *l* = 1, 2, ..., *N* ~*x*~ and *m* = 0, 1, 2, ..., *N* ~*y*~. For each *m*-th mesh row, the cubic spline *S* ~*m*~(*x*) satisfies the following properties *S* ~*m*~(*x*) coincides with a polynomial of degree three on each \[*x* ~*l*−1~, *x* ~*l*~\], *l* = 1, 2, ..., *N* ~*x*~, *m* = 0, 1, 2, ..., *N* ~*y*~*S* ~*m*~(*x*) ∈ *C* ^2^\[0, 1\], and*S* ~*m*~(*x* ~*l*~) = *U* ~*l*,\ *m*~, *l* = 0, 1, 2, ..., *N* ~*x*~, *m* = 0, 1, 2, ..., *N* ~*y*~ The derivatives of cubic spline *S* ~*m*~(*x*) can be obtained as below $$\begin{array}{r} {S_{m}^{\prime}\left( x \right) = \frac{- \left( x_{l} - x \right)^{2}}{2h}M_{l - 1,m} + \frac{\left( x - x_{l - 1} \right)^{2}}{2h}M_{l,m} + \frac{U_{l,m} - U_{l - 1,m}}{h} - \frac{h}{6}\left\lbrack M_{l,m} - M_{l - 1,m} \right\rbrack} \\ \end{array}$$ $$\begin{array}{r} {S_{m}^{\prime\prime}\left( x \right) = \frac{\left( x_{l} - x \right)}{h}M_{l - 1,m} + \frac{\left( x - x_{l - 1} \right)}{h}M_{l,m}} \\ \end{array}$$ And, from [Eq (1)](#pone.0132782.e001){ref-type="disp-formula"}, it gives $$\begin{array}{r} {M_{l,m} = S_{m}^{\prime\prime}\left( x_{l} \right) = U_{xxl,m} = - U_{yyl,m} + D_{l}U_{xl,m} + g_{l,m}} \\ \end{array}$$ When *x* = *x* ~*l*~, [Eq (4)](#pone.0132782.e004){ref-type="disp-formula"} becomes $$\begin{array}{r} {S_{m}^{\prime}\left( x_{l} \right) = U_{xl,m} = \frac{U_{l,m} - U_{l - 1,m}}{h} + \frac{h}{6}\left\lbrack M_{l - 1,m} + 2M_{l,m} \right\rbrack} \\ \end{array}$$ Similarly, for *x* ∈ \[*x* ~*l*~, *x* ~*l*+1~\], it gives $$\begin{array}{r} {S_{m}^{\prime}\left( x_{l} \right) = U_{xl,m} = \frac{U_{l + 1,m} - U_{l,m}}{h} - \frac{h}{6}\left\lbrack M_{l + 1,m} + 2M_{l,m} \right\rbrack} \\ \end{array}$$ Combining both Eqs ([7](#pone.0132782.e007){ref-type="disp-formula"}) and ([8](#pone.0132782.e008){ref-type="disp-formula"}), the following approximation can be obtained $$\begin{array}{r} {S_{m}^{\prime}\left( x_{l} \right) = U_{xl,m} = \frac{U_{l + 1,m} - U_{l - 1,m}}{2h} - \frac{h}{12}\left\lbrack M_{l + 1,m} - M_{l - 1,m} \right\rbrack} \\ \end{array}$$ Further, we have $$S_{m}^{\prime}\left( x_{l + 1} \right) = U_{xl + 1,m} = \frac{U_{l + 1,m} - U_{l,m}}{h} + \frac{h}{6}\left\lbrack {M_{l,m} + 2M_{l + 1,m}} \right\rbrack$$ $$S_{m}^{\prime}\left( x_{l - 1} \right) = U_{xl - 1,m} = \frac{U_{l,m} - U_{l - 1,m}}{h} - \frac{h}{6}\left\lbrack {M_{l,m} + 2M_{l - 1,m}} \right\rbrack$$ By using the continuity of first derivative at (*x* ~*l*~, *y* ~*m*~), which is, $S_{{}^{{}_{m}}}^{\prime}(x_{l}^{+}) = S_{{}^{{}_{m}}}^{\prime}(x_{l}^{-})$, the following relation can be obtained $$\begin{array}{r} {U_{l + 1,m} - 2U_{l,m} + U_{l - 1,m} = \frac{h^{2}}{6}\left( M_{l + 1,m} + 4M_{l,m} + M_{l - 1,m} \right)} \\ \end{array}$$ The following approximations are considered $$\begin{array}{l} {{\overline{U}}_{yyl,m} = \left( {U_{l,m + 1} - 2U_{l,m} + U_{l,m - 1}} \right)/k^{2}} \\ \end{array}$$ $$\begin{array}{l} {{\overline{U}}_{yyl + 1,m} = \left( {U_{l + 1,m + 1} - 2U_{l + 1,m} + U_{l + 1,m - 1}} \right)/k^{2}} \\ \end{array}$$ $$\begin{array}{l} {{\overline{U}}_{yyl - 1,m} = \left( {U_{l - 1,m + 1} - 2U_{l - 1,m} + U_{l - 1,m - 1}} \right)/k^{2}} \\ \end{array}$$ $$\begin{array}{l} {{\overline{U}}_{xl,m} = \left( {U_{l + 1,m} - U_{l - 1,m}} \right)/\left( {2h} \right)} \\ \end{array}$$ $$\begin{array}{l} {{\overline{U}}_{xl + 1,m} = \left( {3U_{l + 1,m} - 4U_{l,m} + U_{l - 1,m}} \right)/\left( {2h} \right)} \\ \end{array}$$ $$\begin{array}{l} {{\overline{U}}_{xl - 1,m} = \left( {- 3U_{l - 1,m} + 4U_{l,m} - U_{l + 1,m}} \right)/\left( {2h} \right)} \\ \end{array}$$ Eqs ([14b](#pone.0132782.e018){ref-type="disp-formula"}) and ([14c](#pone.0132782.e019){ref-type="disp-formula"}) are obtained from the second-order one-sided finite difference scheme. For the derivatives of *S* ~*m*~(*x*), we consider $$\begin{array}{l} {{\overline{M}}_{l,m} = - {\overline{U}}_{yyl,m} + D_{l}{\overline{U}}_{xl,m} + g_{l,m}} \\ \end{array}$$ $$\begin{array}{l} {{\overline{M}}_{l + 1,m} = - {\overline{U}}_{yyl + 1,m} + D_{l + 1}{\overline{U}}_{xl + 1,m} + g_{l + 1,m}} \\ \end{array}$$ $$\begin{array}{l} {{\overline{M}}_{l - 1,m} = - {\overline{U}}_{yyl - 1,m} + D_{l - 1}{\overline{U}}_{xl - 1,m} + g_{l - 1,m}} \\ \end{array}$$ $$\begin{array}{l} {{\overline{\overline{U}}}_{xl + 1,m} = \frac{U_{l + 1,m} - U_{l,m}}{h} + \frac{h}{6}\left\lbrack {{\overline{M}}_{l,m} + 2{\overline{M}}_{l + 1,m}} \right\rbrack} \\ \end{array}$$ $$\begin{array}{l} {{\overline{\overline{U}}}_{xl - 1,m} = \frac{U_{l,m} - U_{l - 1,m}}{h} - \frac{h}{6}\left\lbrack {{\overline{M}}_{l,m} + 2{\overline{M}}_{l - 1,m}} \right\rbrack} \\ \end{array}$$ $$\begin{array}{l} {{\hat{U}}_{xl,m} = \frac{U_{l + 1,m} - U_{l - 1,m}}{2h} - \frac{h}{12}\left\lbrack {{\overline{M}}_{l + 1,m} - {\overline{M}}_{l - 1,m}} \right\rbrack} \\ \end{array}$$ By using Taylor series expansion about the grid point (*x* ~*l*~, *y* ~*m*~), [Eq (1)](#pone.0132782.e001){ref-type="disp-formula"} can be written as $$\begin{array}{r} \begin{array}{cl} & {\left( U_{l + 1,m} - 2U_{l,m} + U_{l - 1,m} \right) + \frac{h^{2}}{12}\left\lbrack {\overline{U}}_{yyl + 1,m} + {\overline{U}}_{yyl - 1,m} + 10{\overline{U}}_{yyl,m} \right\rbrack} \\ = & {\frac{h^{2}}{12}\left\lbrack D_{l + 1}{\overline{\overline{U}}}_{xl + 1,m} + D_{l - 1}{\overline{\overline{U}}}_{xl - 1,m} + 10D_{l}{\hat{U}}_{xl,m} \right\rbrack + \frac{h^{2}}{12}\left\lbrack g_{l + 1,m} + g_{l - 1,m} + 10g_{l,m} \right\rbrack + T_{l,m}} \\ \end{array} \\ \end{array}$$ where *T* ~*l*,\ *m*~ is the local truncation error. Substituting the above approximations ([13](#pone.0132782.e014){ref-type="disp-formula"})--([15](#pone.0132782.e020){ref-type="disp-formula"}) into ([16](#pone.0132782.e026){ref-type="disp-formula"}), it results $$\begin{array}{r} \begin{array}{cl} & \left\{ - 2 + 12\lambda^{2} - \lambda^{2}h\left( D_{l + 1} + 5D_{l} \right) + \frac{\lambda^{2}h^{2}}{12}\left\lbrack \left( 5D_{l} - 2D_{l - 1} \right)D_{l - 1} - 3\left( 2D_{l + 1} - 5D_{l} \right)D_{l + 1} \right. \right. \\ & {\left. - \left( D_{l + 1} - D_{l - 1} \right)D_{l} \right\rbrack\left. - \frac{h}{3}\left( 2D_{l + 1} - 5D_{l} \right) \right\} U_{l + 1,m}} \\ & {+ \left\{ - 20 - 24\lambda^{2} - \lambda^{2}h\left( D_{l - 1} - D_{l + 1} \right) + \frac{\lambda^{2}h^{2}}{3}\left\lbrack \left( 2D_{l + 1} - 5D_{l} \right)D_{l + 1} - \left( 5D_{l} - 2D_{l - 1} \right)D_{l - 1} \right\rbrack \right.} \\ & {\left. - \frac{h}{3}\left( D_{l + 1} - D_{l - 1} \right) \right\} U_{l,m}} \\ & {+ \left\{ - 2 + 12\lambda^{2} + \lambda^{2}h\left( 5D_{l} + D_{l - 1} \right) + \frac{\lambda^{2}h^{2}}{12}\left\lbrack 3\left( 5D_{l} - 2D_{l - 1} \right)D_{l - 1} - \left( 2D_{l + 1} - 5D_{l} \right)D_{l + 1} \right. \right.} \\ & {\left. + \left( D_{l + 1} - D_{l - 1} \right)D_{l} \right\rbrack\left. - \frac{h}{3}\left( 5D_{l} - 2D_{l - 1} \right) \right\} U_{l - 1,m}} \\ & {+ \left\lbrack 1 + \frac{h}{6}\left( 2D_{l + 1} - 5D_{l} \right) \right\rbrack\left( U_{l + 1,m + 1} + U_{l + 1,m - 1} \right)} \\ & {+ \left\lbrack 1 + \frac{h}{6}\left( 5D_{l} - 2D_{l - 1} \right) \right\rbrack\left( U_{l - 1,m + 1} + U_{l - 1,m - 1} \right)} \\ & {+ \left\lbrack 10 + \frac{h}{6}\left( D_{l + 1} - D_{l - 1} \right) \right\rbrack\left( U_{l,m + 1} + U_{l,m - 1} \right)} \\ = & {k^{2}\left\{ \left\lbrack 1 + \frac{h}{6}\left( 2D_{l + 1} - 5D_{l} \right) \right\rbrack g_{l + 1,m} + \left\lbrack 1 + \frac{h}{6}\left( 5D_{l} - 2D_{l - 1} \right) \right\rbrack g_{l - 1,m} \right.} \\ & {+ \left\lbrack 10 + \frac{h}{6}\left( D_{l + 1} - D_{l - 1} \right) \right\rbrack\left. g_{l,m} \right\} + T_{l,m}} \\ \end{array} \\ \end{array}$$ where *λ* is the mesh ratio, denoted by *λ* = (*k*/*h*). If the singular terms like $\frac{1}{x}$ appear in the functions *D*(*x*) and/or *g*(*x*, *y*), which is unable to evaluate at *x* = 0. The following approximations are considered $$\begin{matrix} & {D_{l \pm 1} = D_{00} \pm hD_{10} + \frac{h^{2}}{2}D_{20} \pm O\left( h^{3} \right)} \\ \end{matrix}$$ $$\begin{array}{ll} & {g_{l \pm 1,m} = g_{00} \pm hg_{10} + \frac{h^{2}}{2}g_{20} \pm O\left( h^{3} \right)} \\ \end{array}$$ where $$\begin{matrix} & {W_{ab} = \frac{\partial^{a + b}W\left( x_{l},y_{m} \right)}{\partial x^{a}\partial y^{b}}, W\; = \; D\;\text{and}\; g} \\ \end{matrix}$$ Thus, neglecting the higher order terms and local truncation error, [Eq (17)](#pone.0132782.e027){ref-type="disp-formula"} can be written as $$\begin{array}{r} \begin{array}{cl} & {\left\{ - 2 + 12\lambda^{2} - \frac{1}{2}\lambda^{2}h\left( 12D_{00} + h^{2}D_{20} \right) - \lambda^{2}h^{2}\left( D_{10} - D_{00}D_{00} \right) + hD_{00} \right\} U_{l + 1,m}} \\ + & {\left\{ - 20 - 24\lambda^{2} + 2\lambda^{2}h^{2}\left( D_{10} - D_{00}D_{00} \right) - \frac{2}{3}h^{2}D_{10} \right\} U_{l,m}} \\ + & {\left\{ - 2 + 12\lambda^{2} + \frac{1}{2}\lambda^{2}h\left( 12D_{00} + h^{2}D_{20} \right) + \lambda^{2}h^{2}\left( D_{00}D_{00} - D_{10} \right) - hD_{00} \right\} U_{l - 1,m}} \\ + & {\left( 1 - \frac{h}{2}D_{00} \right)\left( U_{l + 1,m + 1} + U_{l + 1,m - 1} \right) + \left( 1 + \frac{h}{2}D_{00} \right)\left( U_{l - 1,m + 1} + U_{l - 1,m - 1} \right)} \\ + & {\left( 10 + \frac{h^{2}}{3}D_{10} \right)\left( U_{l,m + 1} + U_{l,m - 1} \right)} \\ = & {k^{2}\left\lbrack 12g_{00} + h^{2}\left( g_{20} + D_{10}g_{00} - D_{00}g_{10} \right) \right\rbrack = G_{l,m}} \\ \end{array} \\ \end{array}$$ This modified equation retains its order of accuracy everywhere throughout the solution region, moreover in the vicinity of the singularity. Note that, this proposed scheme ([19](#pone.0132782.e032){ref-type="disp-formula"}) is of *O*(*k* ^2^ + *k* ^2^ *h* ^2^ + *h* ^4^) and applicable to both singular and non-singular elliptic equations of the form ([1](#pone.0132782.e001){ref-type="disp-formula"}). Spline Explicit Group Method {#sec003} ============================ We apply [Eq (19)](#pone.0132782.e032){ref-type="disp-formula"} to any group of four points on the solution domain (as shown in [Fig 1](#pone.0132782.g001){ref-type="fig"}). Then, a (4 × 4) system as below, can be obtained $$\begin{array}{r} {\begin{bmatrix} a_{1} & a_{2} & a_{3} & a_{4} \\ a_{5} & a_{1} & a_{4} & a_{6} \\ a_{6} & a_{4} & a_{1} & a_{5} \\ a_{4} & a_{3} & a_{2} & a_{1} \\ \end{bmatrix}\begin{bmatrix} U_{l,m} \\ U_{l + 1,m} \\ U_{l + 1,m + 1} \\ U_{l,m + 1} \\ \end{bmatrix} = \begin{bmatrix} {rhs_{l,m}} \\ {rhs_{l + 1,m}} \\ {rhs_{l + 1,m + 1}} \\ {rhs_{l,m + 1}} \\ \end{bmatrix}} \\ \end{array}$$ where $$\begin{array}{cl} & {a_{1} = - 20 - 24\lambda^{2} + 2\lambda^{2}h^{2}\left( D_{10} - D_{00}D_{00} \right) - \frac{2}{3}h^{2}D_{10}} \\ & {a_{2} = - 2 + 12\lambda^{2} - \frac{1}{2}\lambda^{2}h\left( 12D_{00} + h^{2}D_{20} \right) - \lambda^{2}h^{2}\left( D_{10} - D_{00}D_{00} \right) + hD_{00}} \\ & {a_{3} = 1 - \frac{h}{2}D_{00}} \\ & {a_{4} = 10 + \frac{h^{2}}{3}D_{10}} \\ & {a_{5} = - 2 + 12\lambda^{2} + \frac{1}{2}\lambda^{2}h\left( 12D_{00} + h^{2}D_{20} \right) + \lambda^{2}h^{2}\left( D_{00}D_{00} - D_{10} \right) - hD_{00}} \\ & {a_{6} = 1 + \frac{h}{2}D_{00}} \\ \end{array}$$ and $$\begin{array}{cl} & {rhs_{l,m} = - a_{5}U_{l - 1,m} - a_{3}U_{l + 1,m - 1} - a_{6}\left( U_{l - 1,m + 1} + U_{l - 1,m - 1} \right) - a_{4}U_{l,m - 1} + G_{l,m}} \\ & {rhs_{l + 1,m} = - a_{2}U_{l + 2,m} - a_{3}\left( U_{l + 2,m + 1} + U_{l + 2,m - 1} \right) - a_{6}U_{l,m - 1} - a_{4}U_{l + 1,m - 1} + G_{l + 1,m}} \\ & {rhs_{l + 1,m + 1} = - a_{2}U_{l + 2,m + 1} - a_{3}\left( U_{l + 2,m + 2} + U_{l + 2,m} \right) - a_{6}U_{l,m + 2} - a_{4}U_{l + 1,m + 2} + G_{l + 1,m + 1}} \\ & {rhs_{l,m + 1} = - a_{5}U_{l - 1,m + 1} - a_{3}U_{l + 1,m + 2} - a_{6}\left( U_{l - 1,m + 2} + U_{l - 1,m} \right) - a_{4}U_{l,m + 2} + G_{l,m + 1}} \\ \end{array}$$ [Eq (20)](#pone.0132782.e033){ref-type="disp-formula"} can be inverted and written in explicit forms $$\begin{array}{r} {\begin{bmatrix} U_{l,m} \\ U_{l + 1,m} \\ U_{l + 1,m + 1} \\ U_{l,m + 1} \\ \end{bmatrix} = \frac{1}{denom}\begin{bmatrix} b_{1} & b_{2} & b_{3} & b_{4} \\ b_{5} & b_{1} & b_{4} & b_{6} \\ b_{6} & b_{4} & b_{1} & b_{5} \\ b_{4} & b_{3} & b_{2} & b_{1} \\ \end{bmatrix}\begin{bmatrix} {rhs_{l,m}} \\ {rhs_{l + 1,m}} \\ {rhs_{l + 1,m + 1}} \\ {rhs_{l,m + 1}} \\ \end{bmatrix}} \\ \end{array}$$ where $$\begin{array}{cl} {denom =} & {a_{1}^{4} - 2a_{1}^{2}a_{4}^{2} + a_{4}^{4} - 2a_{1}^{2}a_{2}a_{5} + 4a_{1}a_{3}a_{4}a_{5} - 2a_{2}a_{4}^{2}a_{5} + a_{2}^{2}a_{5}^{2} - a_{3}^{2}a_{5}^{2} - 2a_{1}^{2}a_{3}a_{6}} \\ & {+ 4a_{1}a_{2}a_{4}a_{6} - 2a_{3}a_{4}^{2}a_{6} - a_{2}^{2}a_{6}^{2} + a_{3}^{2}a_{6}^{2}} \\ \end{array}$$ and $$\begin{array}{cl} & {b_{1} = a_{1}^{3} - a_{1}a_{4}^{2} - a_{1}a_{2}a_{5} + a_{3}a_{4}a_{5} - a_{1}a_{3}a_{6} + a_{2}a_{4}a_{6}} \\ & {b_{2} = - a_{1}^{2}a_{2} + 2a_{1}a_{3}a_{4} - a_{2}a_{4}^{2} + a_{2}^{2}a_{5} - a_{3}^{2}a_{5}} \\ & {b_{3} = - a_{1}^{2}a_{3} + 2a_{1}a_{2}a_{4} - a_{3}a_{4}^{2} - a_{2}^{2}a_{6} + a_{3}^{2}a_{6}} \\ & {b_{4} = - a_{1}^{2}a_{4} + a_{4}^{3} + a_{1}a_{3}a_{5} - a_{2}a_{4}a_{5} + a_{1}a_{2}a_{6} - a_{3}a_{4}a_{6}} \\ & {b_{5} = - a_{1}^{2}a_{5} - a_{4}^{2}a_{5} + a_{2}a_{5}^{2} + 2a_{1}a_{4}a_{6} - a_{2}a_{6}^{2}} \\ & {b_{6} = 2a_{1}a_{4}a_{5} - a_{3}a_{5}^{2} - a_{1}^{2}a_{6} - a_{4}^{2}a_{6} + a_{3}a_{6}^{2}} \\ \end{array}$$ The Gauss-Seidel technique is employed to accelerate the convergence process. Iterations are generated in groups of four points over the entire spatial domain until the convergence test is satisfied. Once the approximations *U* ~*l*,*m*~ had been calculated, the value of *M* ~*l*,*m*~ can be easily obtained by solving the system generated by ([12](#pone.0132782.e013){ref-type="disp-formula"}). Then, the piecewise polynomial of the function can be obtained from [Eq (3)](#pone.0132782.e003){ref-type="disp-formula"}. Finally, the approximate solution at any point at *m*-th mesh row can be easily calculated. {ref-type="disp-formula"}.](pone.0132782.g001){#pone.0132782.g001} Applying [Eq (21)](#pone.0132782.e036){ref-type="disp-formula"} to each of the group in natural row ordering ([Fig 2](#pone.0132782.g002){ref-type="fig"}) will lead to a linear system $$AU = b$$ where the matrix of coefficient *A* is given by $$\begin{array}{r} {A = \begin{bmatrix} D & V & \\ L & D & V \\ & L & D \\ \end{bmatrix}} \\ \end{array}$$ with $$D = \begin{bmatrix} R_{0} & R_{2} & \\ R_{5} & R_{0} & R_{2} \\ & R_{5} & R_{0} \\ \end{bmatrix}, V = \begin{bmatrix} R_{4} & R_{3} & \\ R_{6} & R_{4} & R_{3} \\ & R_{6} & R_{4} \\ \end{bmatrix}, L = \begin{bmatrix} R_{4}^{\prime} & R_{3}^{\prime} & \\ R_{6}^{\prime} & R_{4}^{\prime} & R_{3}^{\prime} \\ & R_{6}^{\prime} & R_{4}^{\prime} \\ \end{bmatrix}$$ The submatrices are given by $$R_{0} = \begin{bmatrix} a_{1} & a_{2} & a_{3} & a_{4} \\ a_{5} & a_{1} & a_{4} & a_{6} \\ a_{6} & a_{4} & a_{1} & a_{5} \\ a_{4} & a_{3} & a_{2} & a_{1} \\ \end{bmatrix}, R_{2} = \begin{bmatrix} 0 & 0 & 0 & 0 \\ a_{2} & 0 & 0 & a_{3} \\ a_{3} & 0 & 0 & a_{2} \\ 0 & 0 & 0 & 0 \\ \end{bmatrix}, R_{3} = \begin{bmatrix} 0 & 0 & 0 & 0 \\ 0 & 0 & 0 & 0 \\ a_{3} & 0 & 0 & 0 \\ 0 & 0 & 0 & 0 \\ \end{bmatrix},$$ $$R_{4} = \begin{bmatrix} 0 & 0 & 0 & 0 \\ 0 & 0 & 0 & 0 \\ a_{6} & a_{4} & 0 & 0 \\ a_{4} & a_{3} & 0 & 0 \\ \end{bmatrix}, R_{5} = \begin{bmatrix} 0 & a_{5} & a_{6} & 0 \\ 0 & 0 & 0 & 0 \\ 0 & 0 & 0 & 0 \\ 0 & a_{6} & a_{5} & 0 \\ \end{bmatrix}, R_{6} = \begin{bmatrix} 0 & 0 & 0 & 0 \\ 0 & 0 & 0 & 0 \\ 0 & 0 & 0 & 0 \\ 0 & a_{6} & 0 & 0 \\ \end{bmatrix},$$ $$R_{3}^{\prime} = \begin{bmatrix} 0 & 0 & 0 & 0 \\ 0 & 0 & 0 & a_{3} \\ 0 & 0 & 0 & 0 \\ 0 & 0 & 0 & 0 \\ \end{bmatrix}, R_{4}^{\prime} = \begin{bmatrix} 0 & 0 & a_{3} & a_{4} \\ 0 & 0 & a_{4} & a_{6} \\ 0 & 0 & 0 & 0 \\ 0 & 0 & 0 & 0 \\ \end{bmatrix}, R_{6}^{\prime} = \begin{bmatrix} 0 & 0 & a_{6} & 0 \\ 0 & 0 & 0 & 0 \\ 0 & 0 & 0 & 0 \\ 0 & 0 & 0 & 0 \\ \end{bmatrix}$$ In order to derive the explicit formulae, the matrix *A* is transformed into *A* ^*E*^ and vector *b* is modified into *b* ^*E*^, where, $$\begin{matrix} & {A^{E} = diag\left\{ R_{0}^{- 1} \right\} A} \\ & {b^{E} = diag\left\{ R_{0}^{- 1} \right\} b} \\ \end{matrix}$$ The block structure of *A* ^*E*^ is the same as matrix *A* with the nonzero block *R* ~0~ replaced by identity matrices, *I* and the blocks *R* ~*i*~ and $R_{j}^{\prime}$, replaced by $R_{0}^{- 1}R_{i}$, *i* = 0, 2, 3, 4, 5, 6 and $R_{0}^{- 1}R_{{}^{{}_{j}}}^{\prime}$, *j* = 3,4,6 respectively. Since the coefficient matrix ([22](#pone.0132782.e040){ref-type="disp-formula"}) is block tridiagonal with non-vanishing diagonal element, it is *π*-consistently ordered and has property-*A* ^(*π*)^ \[[@pone.0132782.ref022]\]. Thus, the theory of block S.O.R. is also applicable to the SEG iterative method and therefore, is convergent. {#pone.0132782.g002} Computational Complexity Analysis {#sec004} ================================= In order to show the efficiency of the proposed method, computational complexity of the SEG iterative method is examined. Assume that the solution domain is discretized into even intervals, *N* ~*x*~ and *N* ~*y*~ in *x*- and *y*-directions, respectively. Therefore, we have (*n* ~*x*~ − 1)(*n* ~*y*~ − 1) grouped points and (*n* ~*x*~ + *n* ~*y*~ − 1) ungrouped points, where *n* ~*x*~ = *N* ~*x*~ − 1 and *n* ~*y*~ = *N* ~*y*~ − 1. This can be shown as in [Fig 3](#pone.0132782.g003){ref-type="fig"}. {#pone.0132782.g003} The estimation on this computational complexity is based on the arithmetic operations performed at each iteration for the additions/substractions (Add/Sub) and multiplications/divisions (Mul/Div) operations. Therefore, the number of operations required for SEG is given as in [Table 1](#pone.0132782.t001){ref-type="table"}. The total number of arithmetic operations can be obtained by multiplying the number of arithmetic operations for each iteration with the number of iterations. 10.1371/journal.pone.0132782.t001 ###### The number of arithmetic operations per iteration for SEG iterative method. {#pone.0132782.t001g} Internal Points Add/Sub Mul/Div ------------------ -------------------------------- --------------------------------- ----------------------------------------------------- Grouped Points (*n* ~*x*~ − 1)(*n* ~*y*~ − 1) 8(*n* ~*x*~ − 1)(*n* ~*y*~ − 1) 8(*n* ~*x*~ − 1)(*n* ~*y*~ − 1) Ungrouped Points (*n* ~*x*~ + *n* ~*y*~ − 1) 8(*n* ~*x*~ + *n* ~*y*~ − 1) 6(*n* ~*x*~ + *n* ~*y*~ − 1) Total *n* ~*x*~ *n* ~*y*~ 8*n* ~*x*~ *n* ~*y*~ 8*n* ~*x*~ *n* ~*y*~ − 2(*n* ~*x*~ + *n* ~*y*~ − 1) Numerical Results {#sec005} ================= In this section, two benchmark test problems, whose exact solutions are known are solved by the proposed combination of cubic spline and explicit group iterative method. The results are then compared with those obtained by the Combination of cubic spline with block Gauss-Seidel iterative method (SBGS)Combination of central difference scheme with explicit group iterative method (CDEG) where the CDEG scheme can be derived by substituting the partial derivative in [Eq (1)](#pone.0132782.e001){ref-type="disp-formula"} by the central difference approximation. In all cases, we assume that *u* ^(0)^ = 0 as the initial guess and the iterations were stopped when the estimated error was below tolerance, that is when ∣*u* ^(*s*+1)^ − *u* ^(*s*)^∣ ≤ 10^−12^ was achieved. Example 1 {#sec006} --------- Consider the convection-diffusion equation $$\begin{matrix} & {\frac{\partial^{2}u}{\partial x^{2}} + \frac{\partial^{2}u}{\partial y^{2}} = \beta\frac{\partial u}{\partial x}, 0 < x,y < 1} \\ \end{matrix}$$ The exact solution for the problem is given by $$u\left( x,y \right) = e^{\frac{\beta x}{2}}\frac{\sin\pi y}{\sinh\sigma}\left\lbrack 2e^{\frac{- \beta}{2}}\sinh\sigma x + \sinh\sigma\left( 1 - x \right) \right\rbrack$$ where $\sigma^{2} = \pi^{2} + \frac{\beta^{2}}{4}$ and *β* \> 0. The boundary conditions can be obtained from the exact solution. The (4 × 4) matrix system can be obtained by substituting *D* ~00~ = *β*, *D* ~10~ = *D* ~20~ = 0 and *G* ~*l*,\ *m*~ = *G* ~*l*+1,\ *m*~ = *G* ~*l*+1,\ *m*+1~ = *G* ~*l*,\ *m*+1~ = 0 in [Eq (20)](#pone.0132782.e033){ref-type="disp-formula"}. The maximum absolute errors are tabulated in [Table 2](#pone.0132782.t002){ref-type="table"} and the number of arithmetic operations are shown in [Table 3](#pone.0132782.t003){ref-type="table"}. 10.1371/journal.pone.0132782.t002 ###### Computational errors for proposed method, SEG compared with CDEG \[[@pone.0132782.ref018]\] and SBGS \[[@pone.0132782.ref023]\] by using *β* = 10 and *k*/*h* ^2^ = 64. {#pone.0132782.t002g} *h* **CDEG** **SBGS** **SEG** ------ ------------- ---------- ------------- ------- ------------- ------- 1/16 1.27722E-02 0.03 1.63610E-02 0.02 1.63610E-02 0.01 1/32 3.56699E-03 0.21 1.02672E-03 0.23 1.02672E-03 0.22 1/64 1.03777E-03 18.40 6.42077E-05 25.16 6.42077E-05 18.40 10.1371/journal.pone.0132782.t003 ###### Total arithmetic operations needed to generate the above results for CDEG \[[@pone.0132782.ref018]\], SBGS \[[@pone.0132782.ref023]\] and the proposed method, SEG (*β* = 10, *k*/*h* ^2^ = 64). {#pone.0132782.t003g} *h* **CDEG** **SBGS** **SEG** ------ ---------- ------------ --------- --------------- ------ ------------- 1/16 119 47,481 16 26,832 123 78,843 1/32 376 1,697,640 163 5,239,635 373 2,568,105 1/64 2245 88,262,175 2261 1,194,099,669 2159 127,996,315 Example 2 {#sec007} --------- Given the following Poisson's equation in polar cylindrical coordinates in *r* − *z* plane. $$\begin{matrix} & {\frac{\partial^{2}u}{\partial r^{2}} + \frac{\partial^{2}u}{\partial z^{2}} + \frac{1}{r}\frac{\partial u}{\partial r} = \cosh z\left( 5r\cosh r + 2\left( 2 + r^{2} \right)\sinh r \right), 0 < r,z < 1} \\ \end{matrix}$$ The exact solution is *u*(*r*, *z*) = *r* ^2^ sinh *r* cosh *z*. The solutions can be approximated by replacing the variables (*x*, *y*) by (*r*, *z*) and substituting $D(r) = - \frac{1}{r}$ and *g*(*r*, *z*) = cosh *z*(5*r* cosh *r* + 2(2 + *r* ^2^) sinh *r*) into scheme ([20](#pone.0132782.e040){ref-type="disp-formula"}). The corresponding errors and the number of arithmetic operations are tabulated in Tables [4](#pone.0132782.t004){ref-type="table"} and [5](#pone.0132782.t005){ref-type="table"}, respectively. 10.1371/journal.pone.0132782.t004 ###### Maximum absolute errors of proposed method, SEG compared with CDEG \[[@pone.0132782.ref018]\] and SBGS \[[@pone.0132782.ref023]\] compared to the exact solution (*k*/*h* = 0.8). {#pone.0132782.t004g} *h* **CDEG** **SBGS** **SEG** ------- ------------- ---------- ------------- --------- ------------- --------- 1/16 1.73383E-03 0.52 6.90966E-05 0.92 6.37611E-05 0.23 1/32 4.59371E-04 3.61 1.05767E-05 14.34 9.51966E-06 3.61 1/64 1.20177E-04 92.87 1.55798E-06 271.72 1.36015E-06 91.43 1/128 3.12015E-05 3329.25 2.23341E-07 7330.74 1.87608E-07 3302.78 10.1371/journal.pone.0132782.t005 ###### Total arithmetic operations needed to generate the above results for CDEG \[[@pone.0132782.ref018]\], SBGS \[[@pone.0132782.ref023]\] and the proposed method, SEG for *k*/*h* = 0.8. {#pone.0132782.t005g} *h* **CDEG** **SBGS** **SEG** ------- ---------- --------------- --------- ----------------- ------- --------------- 1/16 456 1,384,416 550 5,998,300 439 1,972,866 1/32 1724 22,570,608 2094 177,541,884 1658 31,843,548 1/64 6524 354,409,776 7930 5,291,165,620 6263 496,969,050 1/128 24579 5,438,546,172 29849 157,937,088,498 23561 7,598,846,598 Conclusions {#sec008} =========== In this paper, a new method namely, the SEG iterative method was formulated for solving the elliptic boundary value problems. The presented results show that the proposed method is capable of approximating the solution very well in terms of accuracy and execution time. It can be seen that the computation cost is reduced substantially compared to those obtained by the cubic spline block Gauss-Seidel iterative method \[[@pone.0132782.ref023]\], especially when the grid size increases. Furthermore, in terms of accuracy, the proposed method is superior to the original central difference explicit group iterative method \[[@pone.0132782.ref018]\]. In conclusion, the proposed method is a viable alternative approximation tool for solving the elliptic partial differential equations. The authors gratefully thank to the reviewers for their valuable suggestions which definitely help to improve the quality of the paper. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: JG NHMA. Performed the experiments: JG NHMA. Analyzed the data: JG NHMA. Contributed reagents/materials/analysis tools: JG NHMA. Wrote the paper: JG NHMA.

In late December of 2019, authorities from the Peoples Republic of China (PRC) announced an epidemic of pneumonia ([@R33]). A novel coronavirus (Severe Acute Respiratory Syndrome coronavirus 2, SARS-CoV-2) was identified as the etiologic agent and the disease has been named "coronavirus disease 2019" (COVID-19). The virus spread rapidly, first to Thailand, Japan, Korea, and Europe, and now to over 188 countries across all continents except Antarctica. The global total of infected individuals now exceeds six million (<https://coronavirus.jhu.edu/>). Public health professionals around the world are working to limit the spread of SARS-CoV-2, and "flatten the curve", which requires a reduction in cases from one day to the next. However, SARS-CoV-2 containment has been outpaced by viral spread and limited resources for testing. Moreover, mounting evidence suggests that the virus is not only spread by aerosols but may also be transmitted via feces. Both viral RNA and infectious virus have been detected in the stool of COVID-19 patients ([@R11]; [@R16]; [@R29]; [@R32]; [@R36]; [@R38]; [@R39]). This has important implications for the spread of the virus and suggests that wastewater may be used to monitor progression or abatement of viral spread at the community level ([@R17]). To test this hypothesis, we collected samples of untreated wastewater from the municipal wastewater treatment plant in Bozeman, Montana (USA). Samples were collected on 12 different days over the course of a 52-day period, using two different collection methods. The samples were filtered and concentrated prior to RNA extraction. Isolated RNA was used as a template for one-step reverse transcription quantitative polymerase chain reaction (RT-qPCR), performed according to CDC guidelines (<https://www.fda.gov/media/134922/download>). Each RT-qPCR reaction was performed using two primer pairs (i.e., N1, and N2), which target distinct regions of the nucleocapsid (N) gene from SARS-CoV-2 ([Fig 1](#F1){ref-type="fig"} and [Supplemental Fig S1](#SD3){ref-type="supplementary-material"}). The first two influent samples were collected manually at the headworks, using a sampling stick on the mornings of March 23^rd^ and 27^th^, respectively. Both samples tested positive for SARS-CoV-2 RNA ([Fig 1A](#F1){ref-type="fig"}). To minimize inherent variability that comes with collecting a single sample from a large heterogeneous volume at any given point in time, subsequent sampling was performed using an autosampler that collects a volume proportional to flow for 24-hours. This composite sample reflects average characteristics of the wastewater over the previous day. Similar to the first two timepoints, composite samples collected in late March and early April tested positive for SARS-CoV-2, though concentrations of viral RNA steadily declined and then dropped below the limit of detection ([Fig 1A](#F1){ref-type="fig"} and [Supplemental Table S1](#SD3){ref-type="supplementary-material"}). To better understand how SARS-CoV-2 RNA concentrations measured in the wastewater compare to individual SARS-CoV-2 infections in the community, we gathered symptom onset data from patients that tested positive for SARS-CoV-2 in Bozeman. This comparison was performed using a cross-correlation analysis, which measures similarity as a function of displacing one dataset relative to the other. We compared concentration of SARS-CoV-2 RNA determined using the N1 or N2 primer pairs, to symptom onset data that was collected by a retrospective survey of individuals that tested positive for SARS-CoV-2. N1 has a maximum positive correlation (0.736) with symptom onset when we account for a of 5-day lag, while N2 has a maximum positive correlation (0.589) with symptom onset with a 7-day lag ([Fig 1B](#F1){ref-type="fig"}). We hypothesized that the number of COVID-19 cases should have a linear relationship with viral RNA copies in wastewater. To test this hypothesis, we performed a regression analysis. In agreement with the cross-correlation analysis, linear regression reveals that changes in SARS-CoV-2 RNA concentrations are best predicted by symptom onset dates with a 5-to 8-day lag ([Fig 1C](#F1){ref-type="fig"}). However, an 8-day lag for N1 (R~2~ = 0.93) and a 7-day lag for N2 (R~2~ = 0.9) result in the best fits for the linear models ([Fig 1D](#F1){ref-type="fig"}, [E](#F1){ref-type="fig"}). This observation agrees with clinical data showing a delay between symptom onset, and detection of SARS-CoV-2 in respiratory and stool samples of COVID-19 patients ([@R5]; [@R36]). To verify that the RT-qPCR results reflect bona fide detection of SARS-CoV-2 rather than priming from an unintended template, we repeated the PCR using 10 primer pairs that tile across the SARS-CoV-2 genome ([@R22]). These primers were designed to target conserved regions of the genome that flank polymorphic sites that have been used to trace viral ancestry and geographic origins ([@R2]; [@R22]) ([Supplemental Fig S2A](#SD3){ref-type="supplementary-material"}). RNA isolated from the Bozeman waste stream on March 27th was used as a template for these RT-PCR reactions, and all 10 primer pairs produced PCR products of the expected sizes ([Supplemental Table S2](#SD3){ref-type="supplementary-material"} and [Fig S2B](#SD3){ref-type="supplementary-material"}). PCR products were sequenced using Sanger methods and the reads were aligned to the reference genome using MUSCLE ([@R6]; [@R35]). We observed no sequence heterogeneity in redundant reads derived from each location of the genome ([Supplemental Fig S2C](#SD3){ref-type="supplementary-material"}), which suggests the predominance of a single SARS-CoV-2 genotype in the waste stream at the time of sampling. We then used this same RNA sample to determine a near complete (98.5%) SARS-CoV-2 genome sequence using a long read sequencing platform ([@R22]). Efforts to understand the origins and evolution of SARS-CoV-2 have resulted in \~35,000 genome sequences from 86 countries as of May 30^th^, 2020 (<https://www.gisaid.org/>). Phylogenetic analyses of these sequences have enabled molecular tracking of viral spread ([@R4]; [@R7]; [@R9]; [@R28]; [@R31]; [@R41]). To determine the ancestry of the predominant SARS-CoV-2 strain circulating in Bozeman's wastewater on March 27th, we determined the genome sequence using Oxford Nanopore. Sequencing resulted in \~700,000 reads. Quality control and base calling were performed with MinKNOW v19.06.8 in High Accuracy mode (Oxford Nanopore Technologies) and the sequences were assembled using the bioinformatic pipeline from ARTIC Network (<https://artic.network/ncov-2019>). This approach resulted in a single viral contig with 6,875X average sequencing depth that covered 98.5% of the SARS-CoV-2 reference genome. Unsequenced regions of the genome include the 5'- and 3'-ends, and a stretch of 170 bases (22,346 -- 22,515 in the reference genome), that likely had too few reads for basecalling due to PCR bias ([@R12]). This genome was aligned to 14,970 SARS-CoV-2 genomes from 74 different countries (Global Initiative on Sharing All Influenza Data; <https://www.gisaid.org/>). The resulting alignment was used to build a phylogenetic tree ([Fig 2A](#F2){ref-type="fig"}), which indicates that the predominant SARS-CoV-2 genotype in Bozeman's wastewater is most closely related to genomes from California (USA) and Victoria (Australia) ([Fig 2B](#F2){ref-type="fig"}). In total, 11 mutations distinguish the Bozeman wastewater SARS-CoV-2 sequence from the Wuhan-Hu-1/2019 reference sequence, 10 of these 11 mutations are also present in sequences from California and nine of these 11 mutations are also in an isolate from Victoria ([Fig 2B](#F2){ref-type="fig"} and [Supplemental Fig S3A](#SD3){ref-type="supplementary-material"}). To determine how these sequence variations may have accumulated over space and time, we mapped each mutation onto the phylogenetic tree of SARS-CoV-2 sequences ([Supplemental Fig 3B](#SD3){ref-type="supplementary-material"}). Mutations that do not confer a fitness defect are preserved in viral progeny, and thus serve as genetic landmarks that can be used to trace viral ancestry ([Supplemental Fig 3B](#SD3){ref-type="supplementary-material"}). This analysis indicates that the predominate viral stain circulating in Bozeman is most closely related to a strain circulating in California and that the Bozeman isolate acquired an additional mutation (A23122T) that does not co-occur with the other ten mutations. Thus, mutation A23122T may provide a unique genetic signature that can be linked to Bozeman. While this sequencing approach reveals the genetic history of the dominant SARS-CoV-2 variant circulating in Bozeman wastewater at the time of sampling, it does not measure the fitness of this or any of the mutations associated with distinct geographic locations. We anticipate that temporal genome sequencing will help identify dominant strains of the virus circulating in a specific community over time. Collectively, the results presented here demonstrate that wastewater monitoring for SARS-CoV-2 RNA by RT-qPCR correlates with prevalence of viral infections in the community ([Fig 1](#F1){ref-type="fig"}). Our data indicate that symptom onset precedes detection of SARS-CoV-2 RNA in wastewater by 5--8 days. However, patient testing or hospital admission typically occurs 3--9 days after symptoms onset ([@R8]). Thus, an increase of SARS-CoV-2 RNA in wastewater may coincide or even precede by a day or two the detection of new COVID-19 cases in a community. Furthermore, wastewater surveillance may capture mild and asymptomatic infections that are actively shedding virus, which could be used to and alert Public Health officials about emerging undetected transmission events ([@R30]). While this paper was under review, several preprints and publications have reported similar findings from wastewater system around the world ([@R1]; [@R14]; [@R18]; [@R25]; [@R26]; [@R34]; [@R37]). Despite SARS-CoV-2 RNA being present in the wastewater, and viral RNA and infectious virus found feces, infectious virus has not been isolated directly from wastewater ([@R26]). The study presented here complements the rapidly emerging body of work by providing an important link between sewage surveillance, COVID-19 epidemiology and tracing SARS-CoV-2 spread patterns with genome sequencing. METHODS {#S1} ======= Wastewater sampling {#S2} ------------------- Wastewater samples were collected at the Bozeman Water Reclamation Facility (BWRF) that receives and treats domestic, commercial, and industrial wastewater from the City of Bozeman, Montana (USA). Wastewater is sourced from the city limits (\~60 km^2^, 49'831 population) with an average flow rate of \~2.31 × 10^4^ m^3^ / d. Grab samples were collected in triplicates with 15 second intervals from the influent, immediately downstream of the 6-mm screen and grit washer, which remove large solids and heavy inorganic particles. Composite samples were collected from raw influent with automatic flow proportional sampler Liquistation CSF34 (Endress+Hauser) located at the entrance to the facility downstream of a rock trap. Autosampler was set to collect 150 mL of influent per 150'000 gal of flow (\~ 5.68 × 10^5^ L) 7 AM to 7 AM. During collection temperature was kept +2 to +6°C, and samples were stored at +4°C before processing (2--3 h). The composite sample was subsampled in three 500 mL aliquots. Samples were collected three times a week during the outbreak, and once a week after April 8th. Wastewater sample processing and RNA extraction {#S3} ----------------------------------------------- Each wastewater sample (500 mL) was sequentially filtered through 20 μM, 5 μM (Sartorius Biolab Products) and 0.45 μM (Pall Corporation) membrane filters and concentrated down to 150--200 μL using Corning Spin-X UF concentrators with 100 kDa molecular weight cut-off. Total RNA from concentrated samples was extracted with RNeasy Mini Kit (Qiagen) and eluted with 40 \|μL of RNase free buffer. This RNA was used as a template for RT-qPCR. Reverse Transcription quantitative PCR (RT-qPCR) {#S4} ------------------------------------------------ RT-qPCR was performed using two primers pairs (N1 and N2) and probes from 2019-nCoV CDC EUA Kit (IDT\#10006606). SARS-CoV-2 in wastewater was detected and quantified using one-step RT-qPCR in ABI 7500 Fast Real-Time PCR System according to CDC guidelines and protocols (<https://www.fda.gov/media/134922/download>). 20 μL reactions included 8.5 μL of Nuclease-free Water, 1.5 μL of Primer and Probe mix, 5 μL of TaqPath 1-Step RT-qPCR Master Mix (ThermoFisher, A15299) and 5 μL of the template. Nuclease-free water was used as negative template control (NTC). Amplification was performed as follows: 25°C for 2 min, 50°C for 15 min, 95 °C for 2 min followed by 45 cycles of 95 °C for 3 s and 55 °C for 30 s. To quantify viral genome copy numbers in the samples, standard curves for N1 and N2 were generated using a dilution series of a positive template control (PTC) plasmid (IDT\#10006625) with concentrations ranging from 10 to 10,000 copies per reaction. Three technical replicates were performed at each dilution. The limit of detection was 10 copies of the control plasmid. The NTC showed no amplification over the 40 cycles of qPCR. Run data was analyzed in SDS software v1.4 (Applied Biosystems). Threshold cycle (Ct) values were determined by manually adjusting the threshold to fall within exponential phase of the fluorescence curves and above any background signal. Ct values of PTC dilutions were plotted against log~10~(copy number) to generate standard curves. Linear regression analysis was performed in RStudio v1.2.1335 and the trend line equation (Ct = \[*slope*\] × \[log~10~(copy number)\] + b) was used to calculate copy numbers from mean Ct values of technical replicates for each biological replicate. Primer efficiencies calculated as E = (10^(−1/\[*slope*\])^ − 1) × 100% were 150.36 ± 12.13% for N1 and 129.45 ± 25.5% for N2 (n = 7 runs, mean ± sd). RT-PCR and SARS-CoV-2 genome sequencing {#S5} --------------------------------------- Reverse transcription was performed with 10 μL of RNA from SARS-CoV-2 positive wastewater sample using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific) according to the supplier's protocol. The amplicon library for SARS-CoV-2 whole genome sequencing on Oxford Nanopore was generated as described in protocol developed by ARTIC Network (<https://artic.network/ncov-2019>) ([@R22]). Briefly, V3 primer pools containing 110 and 108 primers were used for the multiplex PCR (<https://artic.network/ncov-2019>). PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs) with the following thermocycling conditions: 98°C for 2min, 35 cycles of 98°C for 15 s and 65°C for 5 min, 35 cycles. Two resulting amplicon pools were combined and used for library preparation pipeline that included end preparation and Nanopore adaptors ligation. 20 ng of final library DNA was loaded onto the MinlON flowcell for sequencing. A total of 304.77 Mb of raw sequencing data was collected. PCR products used for Sanger sequencing were generated with a subset of primers from ARTIC V3 pools ([Supplemental Table S2](#SD3){ref-type="supplementary-material"}). PCR reactions were performed as described above. PCR products were analyzed on 1% agarose gels stained with SYBR Safe (Thermo Fisher Scientific), remaining DNA was purified using DNA Clean & Concentrator™ kit (Zymo Research) and sent to Psomagen for Sanger sequencing. Each PCR product was sequenced with both forward and reverse primers used for PCR. SARS-CoV-2 genome assembly {#S6} -------------------------- Nanopore raw reads (304.77 Mb) were basecalled with MinKNOW software in high-accuracy mode. Successfully basecalled reads (273.8 Mb) were further analyzed using the ARTIC bioinformatic pipeline for COVID-19 (<https://artic.network/ncov-2019>). Consensus sequence was generated with minimap2 and single nucleotide variants were called with nanopolish (both integrated in the pipeline) relative to Wuhan-Hu-1/2019 reference genome ([@R15]; [@R23]; [@R35]). The resulting assembly had nearly complete genome coverage (98.51%) with 6,875X average sequencing depth. Regions of the genome that were not captured by this sequencing method include 5' and 3' ends of the genome and a stretch of 170 nucleotides (22,346 -- 22,515 nucleotide positions in reference genome), presumably due to amplicon dropout. Consensus sequence was upload to GISAID (<https://www.gisaid.org/>), accession ID: EPI_ISL_437434. Phylogenetic and Position-specific Mutation Analysis {#S7} ---------------------------------------------------- Phylogenetic analysis was performed by aligning the consensus sequence to 14,970 SARS-CoV-2 genomes retrieved from GISAID on 5/5/2020, 8:25:22 AM (<https://www.gisaid.org/>), using the FFT-NS-2 setting in MAFFT v7.429 ([@R13]; [@R27]). Columns composed of more than 70% gaps were removed with trimAl v1.2rev59 ([@R3]). A maximum-likelihood phylogenetic tree was constructed from this alignment using IQTree in the Augur utility of Nextstrain ([@R10]; [@R19]). The APE v5.3 package in R was used to re-root the tree relative to RaTG13 bat coronavirus genome sequence ([@R21]), and the tree was plotted using ggtree v3.10 package in R ([@R40]). The subtree, visualized in [Figure 2B](#F2){ref-type="fig"}, was rendered in FigTree v1.4.4 ([@R24]). Position specific mutation analysis was conducted in R using the BioStrings package ([@R20]), and chromatograms of Sanger sequencing reads were rendered in SnapGene (GSL Biotech; available at [snapgene.com](http://snapgene.com)). Symptoms onset and contact tracing data collection {#S8} -------------------------------------------------- Suspect cases of COVID-19 were tested in a CLIA lab and instructed to self-quarantine until notified of the RT-qPCR test results. All laboratory confirmed positive cases of COVID-19 were contacted via telephone by local public health nurses to complete contact tracing. During this interview, the nurses collected recorded symptoms, symptom onset date, travel history, contact with other known laboratory confirmed cases, close contacts and activities on the two days before symptom onset up until notification of a positive test. Data collection was conducted as part of a public health response. The study was reviewed by the Montana State University Institutional Review Board (IRB) For the Protection of Human Subjects (FWA 00000165) and was exempt from IRB oversight in accordance with Code of Federal regulations, Part 46, section 101. All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Quantification and Statistical Analyses {#S9} --------------------------------------- All statistical analyses were performed in RStudio v1.2.1335. Data in figures are shown as mean of three biological replicates (each with two technical replicates) ± standard error of mean (sem). Estimated copy numbers in RT-qPCR reactions were used to calculate titers per liter of wastewater for each biological replicate. Viral RNA concentrations in the composite samples were normalized (\[SARS-CoV-2 concentration\]~Normalized~ = \[SARS-CoV-2 concentration\] × (Daily flow / Average flow)). Cross correlation analysis was performed in RStudio using ccf function from stats R package. Linear regression analysis was performed by shifting the SARS-CoV-2 viral RNA data along the axis (see [Fig 1](#F1){ref-type="fig"}) in one day increments and plotting against symptom onset. Data was plotted and analyzed using geom_smooth function from ggplot2 R package, with method = 'lm'. Supplementary Material {#SM1} ====================== Research in the Wiedenheft lab is supported by the National Institutes of Health (1R35GM134867), the Montana State University Agricultural Experimental Station, the MJ Murdock Charitable Trust, the Gianforte Foundation and the MSU Office of the Vice President for Research. We are grateful to Josh French, Justin Roberts, and the other dedicated wastewater technicians that made this work possible. We gratefully acknowledge the authors, originating and submitting laboratories of the sequences from GISAID's EpiFlu™ Database ([Supplemental Table S3](#SD3){ref-type="supplementary-material"}). Declaration of interests B.W. is the founder of SurGene, LLC, and is an inventor on patent applications related to CRISPR-Cas systems and applications thereof. {ref-type="sec"}). Temporal dynamics of SARS-CoV-2 RNA (line graph) superimposed on the epidemiological data (bar plot). Symptom onset data was collected by interviewing COVID-19 patients. Bars represent number of patients who reported symptom onset on the specified day. **B)** Cross-correlation of epidemiological data and SARS-CoV-2 RNA levels in wastewater. Values of cross-correlation function (CCF) are offset by 0- to 14-days. The 95% confidence interval is shown with dashed blue line. CCFs above this line are statistically significant. **C)** The relationship between symptom onset and SARS-CoV-2 RNA measured in wastewater was modeled using linear regression. Goodness-of-fit (R-squared values) metrics are shown for displacing RT-qPCR data relative to symptom onset by 0- to 14-days. **D and E)** Linear regression between symptom onset and total SARS-CoV-2 RNA in wastewater adjusted for 8-day and 7-day lags for N1 and N2, respectively. Pink and blue shadows define the 95% confidence interval.](nihpp-2020.04.15.20066746-f0001){#F1} ). Mutations that occurred over space and time are shown in red. **B)** Inset from panel B: sequences isolated from Bozeman wastewater clade with sequences of American and Australian origin (left). Sequences are named according to geographic origin and viral isolation date. Comparison of mutations in sequences shown in inset (right). The Wuhan reference sequence for each of the positions where mutations occur is shown across the top. Mutated positions and bases present in Bozeman wastewater (WW) sequence are shown in red, bases matching Wuhan reference sequence are shown in white, and mutations not present in Bozeman WW sequence are shown in blue.](nihpp-2020.04.15.20066746-f0002){#F2} [^1]: These authors contributed equally.

INTRODUCTION {#S1} ============ Extracellular vesicles (EVs) that carry extracellular RNAs (ex-RNAs) are generated from different intracellular origins. Microvesicles are assumed to be released directly from budding of the plasma membrane, whereas exosomes originate from the endosomal compartment and are released upon fusion of multivesicular bodies (MVBs) with the plasma membrane ([@R11]). The encapsulation of exRNAs within vesicles protects them from degradation, making them stable constituents of body fluids. Moreover, exosome-associated CD47 inhibits phagocytosis, increasing retention in circulation ([@R28]). These properties of exRNAs and their carriers have been exploited for biomarker discovery, and they allow exRNAs to mediate communication between exosome secreting cells and recipient cells ([@R52]). In addition, the exosome biogenesis pathway modulates microRNA (miRNA) silencing activity through the association of miRNA effector proteins with MVBs ([@R24]). T cells are a robust source of EVs containing small RNAs. T-cell-expressed miRNAs are associated with EVs and increase in the serum of immunized mice and humans ([@R13], [@R14]), while cellular miRNAs are globally downregulated upon T cell activation ([@R6]). Exosome secretion is important for proper immune function, as Rab27 deficiency modulates inflammatory responses and inhibits chronic inflammation in mice ([@R1]; [@R43]). Target cell killing by cytotoxic T cells involves the activation-induced fusion of Rab7-containing cytotoxic granules with the plasma membrane in a Rab27-dependent manner ([@R12]; [@R15]). The fusion of MVBs with the plasma membrane in may be regulated in a similar manner to control exRNA release in exosomes. For these reasons, T cells are a good model for investigating signal-regulated mechanisms of RNA packaging into exosomes and how this process affects their biological activity in source and recipient cells. tRNA fragments (tRFs) are generated through endonucleolytic cleavage of tRNAs ([@R23]). They are among the most prevalent small RNA species detected in exRNA, and in cells they rank second in abundance only to miRNAs ([@R31]). Early studies detected tRFs in the urine of cancer patients ([@R5]; [@R48]), raising the possibility that tRFs may be oncogenic and that they may be actively released into body fluids. tRFs can be transferred from epididymosomes to sperm, epigenetically transmitting information about paternal diet and metabolism to offspring ([@R46]). tRFs also impact a number of functions in somatic cells, including cell proliferation, cancer progression, and the activity of endogenous retroelements ([@R25]; [@R39]; [@R45]). However, their secretion and biological effects in T cells remain unexplored. In the present study, we analyzed EVs rigorously separated from ribonucleoprotein aggregates in cell culture supernatants of activated T cells. RNA sequencing showed that compared with cellular RNAs, tRFs were enriched in EVs more than any other class of RNA, which is consistent with studies in cell lines ([@R2]; [@R29]; [@R33]; [@R34]; [@R53]). We further identified specific sets of tRFs whose release via EVs is enhanced by T cell activation and showed that blocking tRF release by neutral sphingomyelinase (nSMase) inhibitor increased the cellular levels of these activation-induced EV-enriched tRFs but not other activation-independent EV-enriched tRFs. Subcellular fractionation further showed that nSMase inhibitor treatment specifically led to the accumulation of these activation-induced EV-enriched tRFs within the Rab7-containing compartments, suggesting that these tRFs were released via MVB sorting. T cells transfected with antisense oligonucleotides against these tRFs displayed enhanced T cell activation. These results indicate that activated T cells utilize EV biogenesis pathways to selectively secrete tRFs that can repress T cell activation. RESULTS {#S2} ======= Purified T Cell EVs Contain Intact Extracellular Small RNA Species {#S3} ------------------------------------------------------------------ To establish an experimental system for investigating the effects of activating signals on exRNA biogenesis, we isolated EVs secreted by primary T cells stimulated with antibody agonists of antigen and costimulatory receptors (anti-CD3 and anti-CD28) ([Figure S1A](#SD1){ref-type="supplementary-material"}). After 3 days in these culture conditions, approximately 60% of *in vitro* activated T cells remain alive, and they uniformly upregulate CD44 and downregulate CD62L ([Figure S1B](#SD1){ref-type="supplementary-material"}). Since so many T cells die during activation, we expected that intracellular contents passively released from dead cells would contaminate EVs secreted by live cells. To separate EVs from contaminating particles, we used a two-stage purification procedure to isolate EVs. First, large contaminating cells and cellular debris were removed from the media by successive low and medium speed centrifugation, and EVs and other small particles were pelleted by ultracentrifugation at 100,000 × *g*. Second, to eliminate non-vesicle contaminants, the 100,000 × *g* pellet was resuspended in 80% sucrose buffer, overlaid with lower concentrations of sucrose buffer, and ultracentrifuged again to float vesicles into the less dense sucrose layers ([Figure 1A](#F1){ref-type="fig"}). Fractions of these gradients were collected for further analysis. Several independent lines of evidence indicated that fraction 3 contained EVs, while fraction 6 was dominated by degraded RNA and/or protein aggregates. First, fraction 3 was composed of 34% to 52% sucrose, corresponding to a density of 1.15 to 1.20 g/ml, the known density range of exosomes ([@R50]). Second, fraction 3 was highly enriched in the microvesicle and exosome protein markers Arf6 and Tsg101 ([Figure 1B](#F1){ref-type="fig"}). Ago2 and GW182 proteins involved in miRNA function were present but not enriched in EVs. The total protein quantity in fraction 3 was similar to that of T cell lysates made from 100-fold fewer cells ([Figure 1B](#F1){ref-type="fig"}, bottom; compare fraction 3 of 100,000 × *g* pellet from 12 × 10^6^ cells versus cell lysate from 0.12 × 10^6^ cells), indicating that activated T cells secreted at least 1% of their cellular protein content in an EV-rich exudate. Fraction 6 contained even more total protein and was non-selectively enriched for all proteins tested by immunoblotting, suggesting that it was dominated by dense protein aggregates that remained at the bottom of the sucrose gradient. Finally, RNA analyses showed that fraction 3 contains intact exRNA species. Bioanalyzer analysis detected intact 28S and 18S rRNA in fraction 3 but not in fraction 6, despite the latter's higher overall RNA content ([Figure 1C](#F1){ref-type="fig"}). Compared with cells, both fractions were enriched in smaller RNA species. PAGE separation of 50--300 nt RNAs further demonstrated that fraction 3 contained intact 5.8S and 5S rRNAs and tRNAs, whereas fraction 6 was dominated by apparently randomly fragmented RNAs. Although full-length rRNAs were detected in EV fractions, they were not enriched in EV fractions compared to cellular RNA, consistent with previous reports ([@R56]). qPCR showed that intact miRNAs were also much more abundant in fraction 3 than in fraction 6 ([Figure 1D](#F1){ref-type="fig"}). These results suggest that the various exRNA species in fraction 3 are protected from degradation by encapsulation within EVs. To test this possibility, we treated fraction 3 or fraction 6 with RNase A in the presence or absence of the vesicle-disrupting detergent Triton X-100. For fraction 3, RNase treatment did not dramatically alter miRNA or other exRNA species detection unless Triton X-100 was present ([Figure 1E](#F1){ref-type="fig"}, top). By contrast, RNase treatment of fraction 6 with RNase A dramatically reduced the detection of exRNAs even in the absence of Triton X-100 ([Figure 1E](#F1){ref-type="fig"}, bottom). All together, these data show that *in vitro* activated T cells release EVs that contain and protect exRNAs and that our purification procedure separates these EVs from RNA and/or protein aggregates that also accumulate in the culture media. tRFs Are Enriched in T Cell EVs {#S4} ------------------------------- To identify specific small RNAs that were enriched in bona fide T cell EVs free from aggregate contamination, we performed sequencing of small (\~15--31 nt) RNA libraries prepared from EVs (F3), aggregates (F6), and activated T cells ([Figure 1B](#F1){ref-type="fig"}). To reduce bias in small RNA library preparation, we modified the 5′ and 3′ ligation adaptors with five random bases added to the 3′ end and 5′ end, respectively ([@R22]). Greater than 70% of sequence reads from cell libraries mapped to the genome, compared to 62% from EVs and 55% from aggregates ([Figure S2A](#SD1){ref-type="supplementary-material"}), consistent with lower mapping rates for EV libraries in the previous study ([@R7]). Classifying the sequenced RNAs revealed several distinct features of the composition of small RNAs and RNA fragments in T cells, EVs, and aggregates. First, RNA fragments derived from larger RNA classes, including protein coding mRNAs and long intergenic noncoding RNAs (lincRNAs), accounted for about 50% of the total reads in all libraries, and their composition was similar among all samples ([Figure 2A](#F2){ref-type="fig"}). Second, miRNAs were depleted compared to cellular RNA, slightly in EVs but greatly in aggregates ([Figure 2A](#F2){ref-type="fig"}). This is consistent with qPCR results that detected much higher miRNA abundance in EVs than in aggregate fractions ([Figure 1D](#F1){ref-type="fig"}). Third, EVs and aggregates differ greatly in their composition of other RNA classes. In particular, tRFs were specifically enriched in EVs, whereas aggregates contained more small nucleolar RNA (snoRNAs) and small nuclear RNA (snRNAs) ([Figure 2B](#F2){ref-type="fig"}). We further classified tRFs into five groups derived from different parts of full-length tRNAs: the 5′ end (5′tRF), 3′ end (3′CCA-tRF), 5′-internal region (5′i-tRF), 3′-internal regions (3′i-tRF), and central internal region (i-tRF) ([Figure 2C](#F2){ref-type="fig"}). In each group, longer tRFs (26 to 31 nt) and shorter tRFs (14 to 25 nt) were classified as L-tRF and tRF, respectively. We found that 5′tRFs and 3′i-tFRs were enriched, but 3′CCA-tFRs were depleted in EVs compared to cellular small RNAs ([Figure 2D](#F2){ref-type="fig"}). Analysis of tRF read length revealed that 18--21 nt 5′tRFs were dominantly enriched in EVs, while 17--18 and 22 nt 3′CCA-tRF reads were significantly depleted ([Figure S2B](#SD1){ref-type="supplementary-material"}). These data indicate that specific tRNA cleavage events generate tRFs that are preferentially released or retained in cells. Detailed comparison of all RNA species in EVs and cells further elucidated the specificity ofTcell release of tRFs within EVs. As a fraction of all sequenced RNAs, only one miRNA (miR-150) exhibited EV enrichment (EE) as measured by the ratio of RNA reads in EVs versus cells. In contrast, 45% of tRFs showed at least 1.5-fold EE ([Figure 2E](#F2){ref-type="fig"}; [Table S1](#SD2){ref-type="supplementary-material"}). Among the subgroups of tRFs, 5′tRFs were the most uniformly and highly enriched in EVs. 98% of all 5′tRFs displayed at least 1.5-fold EE, with some enriched as much as 10-fold ([Figure 2F](#F2){ref-type="fig"}). In comparison, EE greater than 1.5-fold was observed for just 2%, 26%, 29%, and 36% of 3′L-tRF/i-tRF, 5′i-tRF, 5′L-tRF, and 3′i-tRF, respectively. Although 53% of 3′CCA-tRFs exhibited at least 1.5-fold EE ([Figure 2F](#F2){ref-type="fig"}), the 3′CCA-tRF derived from tRNA-Ala-TGC that accounted for 10% of all tRF reads were depleted in EVs, resulting in fewer total reads of 3′CCA-tRFs in EVs compared to cells ([Figure 2D](#F2){ref-type="fig"}). Taken together, our results revealed that tRFs were more significantly enriched in EVs than were miRNAs and other small RNAs and that 5′tRFs and select other tRFs were preferentially exported into EVs secreted from activated T cells. Selective Signal-Regulated tRF Release by T Cells {#S5} ------------------------------------------------- To determine whether T cell activation modulates the export of tRFs, we isolated EVs secreted from resting T cells using the same two-step procedure ([Figure S3A](#SD1){ref-type="supplementary-material"}). Resting T cells were much smaller than stimulated T cells ([Figure S3B](#SD1){ref-type="supplementary-material"}), and 3.3-fold more resting cells were required to obtain the same yield of EVs (by protein content) as was obtained from stimulated cells ([Figure 3A](#F3){ref-type="fig"}; compare 40 million resting versus 12 million stimulated T cells). However, the efficiency of EV production was similar under resting and stimulated conditions. Once again, approximately 1% of the protein yield from cell lysates was released into the EV fraction during culture under resting conditions ([Figure 3B](#F3){ref-type="fig"}; compare EVs from 40 × 10^6^ cells to cell lysate from 0.4 × 10^6^ cells). Small RNAs under 150 nt and the exosome protein markers Tsg101 andCD81 were less enriched in EV fractions under resting conditions (Figures [3B](#F3){ref-type="fig"} and [S3C](#SD1){ref-type="supplementary-material"}), whereas EV fractions contained similar amounts of β-actin and total protein under resting conditions and stimulated conditions. This indicates that fewer exosomes were secreted and other types of co-purifying EVs that contained lower levels of exosomal markers and small RNAs may be more predominantly released under resting conditions. Small RNA sequencing showed that miRNAs were similar, but tRFs showed less EE under resting conditions ([Figures S3D and S3E](#SD1){ref-type="supplementary-material"}). In particular, 5′tRF and 3′i-tRF reads were less enriched, and 3′CCA-tRF reads were less depleted from EV fractions under resting conditions compared to stimulated conditions ([Figure 3C](#F3){ref-type="fig"}). To identify the specific tRFs that exhibit activation-induced EE, we directly compared sequencing data for EVs and cells under stimulated and resting conditions. Among the 5′tRFs, 5′L-tRFs, and 5′-i-tRFs with ≥ 1.5-fold EE in stimulated conditions, large majorities (77%, 98%, and 69%, respectively) exhibited at least 1.5-fold greater EE in stimulated T cell cultures as compared to their EE in resting T cells. In contrast, 3′CCA-tRFs displayed uniformly higher EE in resting conditions, and only 48% of 3′i-tRFs were more EV enriched in stimulated conditions ([Figure 3D](#F3){ref-type="fig"}; [Table S2](#SD3){ref-type="supplementary-material"}). There are two structural types of 3′i-tRF, as follows: hairpin fragments (3′i-tRF-H) and linear fragments (3′i-tRF-L) that derive from tRNAs with and without variable loops, respectively ([Figure 2C](#F2){ref-type="fig"}). 63% of the linear 3′i-tRF-L, but none of the haripin 3′i-tRF-H fragments, showed greater EE in stimulated conditions ([Figure 3E](#F3){ref-type="fig"}). These results indicate that the positions within parent tRNAs and the secondary structures of internal tRFs correlate with selective release into EVs in response to T cell activation. tRFs derived from the 5′ end of tRNAs and from internal regions without hairpin structures exhibited EE that is enhanced upon T cell activation. Examination of read alignments to individual representative tRNAs illustrate these regional patterns of EE and revealed that tRFs derived from the same tRNA can display different types of EE (Figures [4A](#F4){ref-type="fig"} and [S4A](#SD1){ref-type="supplementary-material"}). For simplicity, we classified tRFs into the following three categories: activation-induced EE, activation-independent EE, and not enriched. 5′tRF derived from the tRNAs Leu-TAA and Leu-TAG showed activation-induced EE, whereas 3′i-tRF-H from tRNA Leu-TAA exhibited activation-independent EE. Oligo(dT) qRT-PCR detected both tRFs and full-length tRNA in Poly(A) polymerase treated cellular RNA, as shown by gel electrophoresis of PCR products in the exponential phase of amplification ([Figure 4B](#F4){ref-type="fig"}). However, since EV did not contain detectable full-length tRNA, this assay could be used to measure the relative abundance of tRFs in EVs from resting and activated T cells. Activation-induced EV-enriched tRFs were increased by 4- to 6-fold in EVs following T cell stimulation, whereas activation-independent EV-enriched tRFs were only \~2-fold more abundant in EVs from resting versus stimulated T cells ([Figure 4C](#F4){ref-type="fig"}). RNase treatment with and without Triton X-100 indicated that these tRFs were encapsulated within EVs ([Figure 4D](#F4){ref-type="fig"}). We designed stem-loop qRT-PCR assays that reliably distinguish tRFs from their corresponding full-length tRNAs ([Figures S4B and S4C](#SD1){ref-type="supplementary-material"}) and used them to further confirm that all three of these EV-enriched tRFs were enriched in EV compared to cellular RNA ([Figure 4E](#F4){ref-type="fig"}). Moreover, the difference in tRF abundance in EV and cellular RNA was greater in stimulated conditions than resting conditions for activation-induced EV-enriched 5′tRFs Leu-TAA and Leu-TAG but not for the activation-independent EV-enriched 3′i-tRF Leu-TAA. These data validate specific abundant tRFs that were differentially enriched in EVs produced by T cells in response to activating antigen and costimulatory receptor signals. Activation-Induced nSMase-Dependent MVB Formation and tRF Downregulation {#S6} ------------------------------------------------------------------------ We hypothesized that activation-induced EV release may lead to the downregulation of cellular tRFs through MVB-mediated EV biogenesis pathways. Exosomes are released when cellular MVBs fuse with the plasma membrane, releasing intraluminal vesicles and their associated cargo. This process is regulated by ESCRT (endosomal sorting complexes required for transport) complexes and the activity of nSMase ([@R11]). The nSMase inhibitor GW4869 ([@R37]) repressed EV production by activated T cells, as indicated by dose-dependent reductions in the recovery of EV proteins and RNA in EV fractions ([Figures 5A--5C](#F5){ref-type="fig"}). Total EV RNA recovery was reduced to about 50% of control in GW4869 treated cells, similar to the decrease of TfR and Arf6, but less than the 95% reduction of the restricted exosomal marker Tsg101 (compare [Figure 5B](#F5){ref-type="fig"} and [Figure 5C](#F5){ref-type="fig"}). These data indicate that GW4869 repressed the release of exosomes more than other types of EVs produced by activated T cells. We compared the small RNA species in cells and EV fractions from T cells treated with GW4869 and DMSO vehicle alone by sequencing. GW4869 treatment did not significantly alter the overall distribution of broad classes of small RNAs in cells or EV fractions ([Figures S5A--S5C](#SD1){ref-type="supplementary-material"}). However, the EE of tRFs was altered by GW4869 treatment. 40% of all activation-induced EV-enriched tRFs and 63% of activation-independent EV-enriched tRFs exhibited 1.5-fold or more reduction in EE in GW4869 treated cells, compared with just 13% of non-enriched tRFs ([Figure 5D](#F5){ref-type="fig"}; [Table S2](#SD3){ref-type="supplementary-material"}). Oligo(dT) qRT-PCR confirmed that GW4869 reduced the abundance of EV-enriched tRFs in EVs by 20%--50% at low concentration (10 μM) and by 60%--80% at high concentration (20 μM) ([Figures 5E and 5F](#F5){ref-type="fig"}). Quantifying gel bands corresponding to tRF qRT-PCR products from cellular RNA indicated that activation-induced EV-enriched tRFs accumulated in cells upon GW4869 treatment more than activation-independent EV-enriched tRFs (Figures [5E](#F5){ref-type="fig"} and [S5D](#SD1){ref-type="supplementary-material"}). Stem-loop qRT-PCR confirmed that GW4869 treatment reduced both activation-induced and activation-independent EV-enriched tRF abundance in EVs while selectively increasing the cellular abundance of activation-induced EV-enriched tRFs ([Figure 5G](#F5){ref-type="fig"}). Thus, nSMase activity is required for EV-enriched tRF enrichment in EVs and to prevent accumulation of activation-induced EV-enriched tRFs in activated T cells. GW4869 represses not only the release of exosomes via MVB but also microvesicles that bud directly from the plasma membrane ([@R41]). To further probe the pathway of tRF release by T cells, we analyzed their subcellular localization by cellular organelle fractionation. Previous studies have reported that the 15,000 × *g* pellet of homogenized cells contains active MVBs that can sort proteins or RNAs into intraluminal vesicles ([@R47]). We obtained 15,000 × *g* pellets from homogenized T cells and then further fractionated them by Opti-prep gradient centrifugation ([Figure 6A](#F6){ref-type="fig"}). In resting T cells, the late endosome protein Rab7 was enriched in fractions (6 and 7) that contained no detectable CD81 ([Figure 6B](#F6){ref-type="fig"}). CD81 is an exosome marker that can appear on the plasma membrane and endomembranes in lymphocytes. By contrast, in activated T cells, Rab7 was enriched in fractions (4,5, and 6) with substantial CD81 protein. These results indicate that CD81 may be more actively incorporated into Rab7-containing MVBs in activated T cells. Notably, we also observed greater enrichment of CD81 in EV fractions from activated compared with resting T cell supernatants ([Figures 3A and 3B](#F3){ref-type="fig"}). The lower-density Rab7- and CD81-containing membrane fractions may represent MVBs that are intracellular intermediates of exosome biogenesis. Consistent with this interpretation, GW4869 treatment of stimulated T cells shifted the enrichment of Rab7 back to the higher-density fractions 6 and 7. GW4869 reduces the formation of intraluminal vesicles in MVBs, which may lead to increased MVB density ([@R54]). These high-density Rab7-containing fractions also contained CD81, suggesting that GW4869 blocked MVB maturation after incorporation of CD81 and potentially RNA cargo as well. Oligo(dT) RT-PCR detected both full-length tRNAs and tRFs in key cellular Optiprep gradient fractions ([Figure S6](#SD1){ref-type="supplementary-material"}). Therefore, we used stem-loop qRT-PCR to quantify tRFs ([Figure 6C](#F6){ref-type="fig"}). Under resting conditions, both activation-independent and activation-induced EV-enriched tRFs were concentrated in cytosolic and high-density Rab7-containing fractions ([Figure 6C](#F6){ref-type="fig"}, left panel). These tRFs were mainly restricted to the cytosolic fractions from stimulated T cells ([Figure 6C](#F6){ref-type="fig"}, middle panel). Increased MVB formation and EV release may efficiently reduce the burden of EV-enriched tRFs in Rab7-containing endomembrane compartments in activated T cells. Consistent with this possibility, GW4869 treatment induced dramatic accumulation of activation-induced EV-enriched tRFs (and to a lesser degree, the activation-independent EV-enriched 3′i-tRF from Leu-TAA) in high-density Rab7- and CD81-containing fractions as compared to cytosolic fractions ([Figure 6C](#F6){ref-type="fig"}, right panel). Taken together, these results indicate that T cell activating signals induce functional MVB formation and constitutive removal of activation-induced EV-enriched tRFs. Activation-Induced EV-Enriched tRFs Inhibit T Cell Activation {#S7} ------------------------------------------------------------- We hypothesized that signal-regulated release of tRFs in EVs may serve as a means to export cellular tRFs that could alter T cell activation. To test the functional impact of these tRFs, we transfected T cells with 2′-o-methylated oligonucleotides antisense to specific abundant tRFs (Table S2) on day 1 of activation and then analyzed transfected cells on day 3. Activated T cells upregulate CD44, downregulate the lymph node homing molecule CD62L, and produce cytokines such as interleukin-2 (IL-2). Antisense oligos against the activation-independent EV-enriched 3′i-tRF derived from tRNA Leu-TAA and tRNA Ser-GCT did not affect the downregulation of CD62L or the upregulation of CD44 ([Figures 7A--7C](#F7){ref-type="fig"}). However, antisense oligos against the activation-induced EV-enriched 5′tRFs derived from both tRNA Leu-TAA and tRNA Leu-TAG enhanced cell activation, as shown by decreased CD62L^hi^ cells and a corresponding increase in the frequency of fully activated CD44^hi^CD62L^lo^ cells ([Figures 7A--7C](#F7){ref-type="fig"}). We measured cytokine production by flow cytometry following restimulation with PMA (phorbol 12-myristate 13-acetate) and ionomycin ([Figures 7D and 7E](#F7){ref-type="fig"}). IL-2 production was enhanced in T cells transfected with antisense oligo against either activation-induced EV-enriched tRF but neither of the activation-independent EV-enriched tRFs. These transfected T cells expressing more IL-2 and other activation markers also displayed lower viability in culture ([Figure S7](#SD1){ref-type="supplementary-material"}). These observations may be linked, as IL-2 sensitizes T cells to activation-induced apoptotic cell death ([@R38]). These results suggest that T cells engage EV biogenesis pathways to dispose of tRFs that can inhibit their activation and cytokine production. DISCUSSION {#S8} ========== Our results show that T cells release specific sets of tRFs within EVs through their association with MVBs in a signal-regulated manner. By rigorously separating contaminating RNA and/or protein aggregates from EVs, we found that tRFs, but not miRNAs, are enriched in T-cell-derived EVs relative to all other cellular small RNA species. Specific classes of tRFs distinguished by their location in the parent tRNA and by tRNA-specific structural features were preferentially released in EVs. For some of these classes, their release was regulated during T cell activation. tRFs enriched in EVs in an activation-induced manner were mostly derived from tRNA 5′ ends, 5′-internal regions, and 3′-internal regions without variable loop hairpins. In contrast, tRFs derived from tRNA 3′ ends and 3′-internal regions with variable loop hairpins were frequently enriched in EVs from activated T cells similarly or even less than those produced by resting T cells. These patterns of EV release corresponded with differences in the accumulation of tRFs within activated T cells treated with the nSMase inhibitor GW4869, which inhibits EV biogenesis pathways. GW4869-treated activated T cells accumulated activation-induced EV-enriched tRFs, but not activation-independent EV-enriched tRFs within high-density fractions containing Rab7 and the exosome marker CD81, suggesting that these tRFs are released through an MVB-mediated EV biogenesis pathway. EVs released from activated T cells also contained more CD81 than EVs from resting T cells, further indicating that signal-regulated exosome production contributes to tRF secretion. Antisense oligos directed against activation-induced tRFs increased T cell activation. Taken together, these findings imply that secretion via exosomes and other EVs is a mechanism whereby T cells rapidly remove tRFs through endomembrane compartments to prevent them from engaging cytosolic targets that mediate T cell activation. Global miRNA turnover and selective downregulation also occurs during T cell activation ([@R6]). However, in contrast to tRFs, miRNAs were present but not enriched in EVs released by T cells. This finding is consistent with previous analyses of the RNA composition of exosomes secreted by B cells, stem cells, and glioblastoma, esophageal, and breast cancer cells ([@R2]; [@R29]; [@R33]; [@R34]; [@R53]). Ago2 protein was also not enriched in T cell EVs compared to cellular protein, indicating that Ago2-miRNA complexes are not selected for sorting into EVs. In isogenic cell lines differing only in KRAS status, MVB association and exosomal release of Ago2 and miRNAs was decreased by an activating KRAS mutation ([@R40]). Since Kras is activated by T cell receptor signaling ([@R17]), it is possible that activated Kras represses exosomal release of miRNAs associated with Ago2 in T cells and in a variety of stem and cancer cell lines. This possibility is consistent with the report that few miRNAs are enriched in exosomes secreted by the Jurkat T cell line and that these exosomal miRNAs are not associated with Ago2 but instead interact with another RNA binding protein, hnRNPA2/B1 ([@R55]). Coordinated mechanisms of cellular small RNA repertoire remodeling may be an important feature of T cell activation and immune responses. Among small RNAs, tRFs are second only to miRNAs in their abundance, and they have been shown to regulate critical cellular functions ([@R31]; [@R51]). Although tRF species have been shown to be abundant in exosomes in several studies, their biogenesis and function as exRNAs are relatively understudied. Our comprehensive analysis of tRFs in T cells and their secreted EVs showed that almost all 5′tRFs, but only a select few derived from other regions of tRNAs, are enriched in EVs. Three types of tRFs derived the 5′ terminus of tRNAs have been classified according to their length: 5′halves (31--33 nt), 5′L-tRFs (26--31 nt), and 5′tRF (less than 26 nt). Specific 5′halves repress protein translation by targeting eukaryotic translation initiation factors ([@R27]) and modulate the formation of stress granules to inhibit protein synthesis ([@R18]). 5′L-tRFs derived from tRNA Gly-GCC are transferred from the epididymis to sperm to regulate endogenous retroelements ([@R46]). In contrast to 5′halves and 5′L-tRFs, 5′tRFs have no hairpin structures. Their specific and prominent activation-induced EE suggest that 5′tRFs may regulate different cellular processes that are associated with membrane compartments, allowing their rapid removal through EV-biogenesis pathways. The positional and structural patterns apparent in tRFs that undergo activation-induced EV release indicates that mechanisms that sort specific tRFs into EVs may be coupled to tRNA processing pathways. Dicer and Angiogenin (ANG) cleave the D-loops, T-loops, and anticodon loops of mature tRNAs ([@R10]; [@R21]). ANG is a candidate mediator of tRF processing and sorting into EVs, possibly in collaboration with the RNA binding protein YBX1. ANG is a secreted protein present at high concentration in blood plasma ([@R3]) and is itself enriched in exosomes ([@R56]). YBX1 associates with miR-223 and packages this miRNA into exosomes ([@R20]; [@R47]). YBX1 also interacts with a specific set of tRFs ([@R25]). Some of these tRFs could be derived from ANG cleavage. Most YBX1-associated tRFs are derived from 5′ internal regions or central regions that include the anticodon loops of tRNAs ([@R25]). Both miR-223 and some of the known YBX1-associated tRFs were enriched in EVs in an activation-induced manner in T cells, making YBX1 a candidate for tRF sorting into EVs upon T cell activation. tRNA synthetases that associate with ceramide or the lysosome may also play a role in the observed selectivity of tRF processing and secretion ([@R4]; [@R9]; [@R26]). Our findings indicate that removal of cellular tRFs via sorting into MVBs and EVs may be an important mechanism to avert inhibitory functions of tRFs during T cell activation. However, tRFs in secreted EVs may be actively transferred among cells, allowing them to mediate intercellular RNA communication. These uniquely regulated exRNA species may also have utility as biomarkers of immune activity and disease processes. Compared with miRNAs, which are ubiquitous in almost all cell types, 5′tRFs as a class appear to be selectively abundant in hematopoietic tissues ([@R16]). In summary, our data show that tRFs are functionally active small RNAs that are dynamically regulated by differential intracellular compartmentalization and release within EVs in response to T cell activating signals. STAR★METHODS {#S9} ============ CONTACT FOR REAGENT AND RESOURCE SHARING {#S10} ---------------------------------------- Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, K. Mark Ansel (<mark.ansel@ucsf.edu>). EXPERIMENTAL MODEL AND SUBJECT DETAILS {#S11} -------------------------------------- ### Mice {#S12} C57BL/6J were purchased from Jackson Laboratory and bred in specific pathogen-free conditions in the Animal Barrier Facility at the University of California, San Francisco. Male and female mice were euthanized with CO~2~ at 4-7 weeks of age for spleen and lymph node removal. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco. METHOD DETAILS {#S13} -------------- ### CD4^+^ T cell stimulation and culture {#S14} Naive CD4^+^ T cells from spleen and lymph nodes of mice were purified by magnetic bead selection (Invitrogen) and used in all experiments. For stimulated conditions, naive T cells were stimulated with plate-bound anti-CD3 (2.0 μg/ml) and soluble anti-CD28 (0.5 μg/ml) in the presence of 10 units/mL IL2 (National Cancer Institute) for \~60 hr at initial cell density of \~1 × 10^6^ cells/ml. After stimulation, the cell density of live cells was similar to the initial density of the naive cells (\~1 × 10^6^ cells/ml) since not only cell proliferation but also cell death occurs during stimulation. For resting conditions, T cell cultures that were stimulated for \~48 hr were resuspended and diluted with 3 volumes of media containing 10 units/ml IL2. After expansion for \~24 hr, cells were spun down and resuspended in EV-free media containing 10 units/ml IL2 for 48 hr at initial cell density of \~2 × 10^6^ cells/ml, which then becomes \~4 × 10^6^ cells/ml after 48 hr-resting in IL2-containing media. Cells grown for exosome production were incubated in EV-free medium produced by ultracentrifugation at 100,000 × g (28,000 RPM) for 18 hr using an SW-28 rotor (Beckman Coulter). All T cell culture was done in DMEM high-glucose media supplemented with 10% FBS and other chemicals as previously described ([@R49]). ### Exosome/EV purification {#S15} Cell culture supernatant and T cells were harvested together by gently pipetting T cells sediment in the bottom of the plate with the conditioned medium. The collected medium containing both T cells and cell culture supernatant was centrifuged for 10 min at 300 × g. The resulting cell pellet was used for cell lysate, RNA preparation or subcellular fractionation and the resulting supernatant was further centrifuged for 20 min at 2000 × g to remove cellular debris. To remove microvesicles, the supernatant collected from 2000 × g spin was centrifuged for 30 min at 10,000 × g. The resulting supernatant containing both exosomes, other types of EVs and RNA and/or protein/membrane complexes or aggregates was further subjected to ultracentrifugation at 100,000 × g for 2 hr. To separate EVs from the contaminating aggregates, the resulting crude EV pellet was resuspended in 100 μL PBS and 1 mL 90% sucrose buffer (final sucrose concentration = 82%), which was then overlaid with 11 layers of 1 mL sucrose buffer in order from high to low concentrations, including buffers containing 70%, 64%, 58%, 46%, 40%, 34%, 28%, 22%, 16%, and 10% of sucrose. The resulting 12 mL sucrose gradient was subjected to ultracentrifugation at 100,000 × g for 18 hr. Six fractions, each of which is 2 ml, were collected from top to bottom and resuspended in 9 mL PBS, which were then further centrifuged at 100,000 × g for 1 hr. The resulting supernatants were poured out and the pellets were resuspended in 50 μL PBS. The third fraction from the top, which contains the interface of the 34% and 40% sucrose solutions, should be enriched for EVs. The sixth fraction, which contains the interface of the 70% and 82% sucrose solutions, contains protein/RNA/membrane aggregates. All sucrose buffers were made with sucrose and PBS. ### Qligo(dT) RT-qPCR {#S16} Total RNA was extract from EVs by using Trizol LS (Life Technologies). For RNase protection assays, EVs were left untreated, treated with 50 μg/ml RNase A or treated with RNase A in the presence of 0.1% Triton X-100 (Sigma) for 15 min at room temperature. The relative levels of miRNAs or tRFs in EVs were quantified by miScript II RT Kit and miSYBR Green PCR Kit (QIAGEN). The miScript II RT Kit added poly (A) to the 3′ end of RNAs followed by using oligo(dT) adaptor for the RT reactions. The qPCR universal reverse primer was included in the miSYBR Green PCR Kit. The qPCR forward primers for miRNAs, U6 snRNA, and 18S rRNA were previously described ([@R6]). The qPCR forward primers for all tRFs were the full-length tRF sequences provided in [Figure S4](#SD1){ref-type="supplementary-material"}. ### Stem loop RT-qPCR {#S17} The design of primers, probes and reaction conditions were as previously described with the following modifications ([@R8]; [@R42]). TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems) was used for reverse transcription reactions. The 7.5 μL RT reactions contained 20 ng of total RNA, 1 × RT buffer, 1 mM dNTP, 3.33 U/ìl MultiScribe reverse transcriptase, 0.25 U/ìl RNase inhibitor and 50 nM stem-loop RT primer. RT reaction conditions: 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min. The 10 μL pre-PCR reactions contained 3 μL RT-product, 1 × TaqMan Universal PCR master Mix with no AmpErase UNG (Applied Biosystems), 50 nM forward primer and 50 nM universal reverse primer. Pre-PCR conditions: 95°C for 10 min, followed by 8 cycles of 95°C for 10 s, and 65°C for 1 min. The 10 μL qPCR reactions included 2 μL pre-PCR product, 1 × TaqMan Universal PCR master Mix with no AmpErase UNG (Applied Biosystems), 0.2 ìM TaqMan probe (IDT), 1.5 ìM forward primer, and 0.7 ìM universal reverse primer. qPCR reaction conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, and 65°C for 1 min. The universal reverse primer sequence is GTGTCGTGGAGTCGGC. The stem-loop RT primer sequence is CTCAACTGGTGTCGTG GAGTCGGCAATTCAGTTGAG(X)~8~. The forward primer sequence is ACACTCCAGCTGGGTGGATTTATATC(X)~15-16~. The TaqMan probe sequence is /56-FAM/TTCAGTTGA/ZEN/G(X)~8~/3IABkFQ/ (PrimeTime qPCR probes, IDT). In all sequences, X indicates the tRF-specific sequence shown in [Figure S4C](#SD1){ref-type="supplementary-material"}. ### Subcellular fractionation {#S18} 10^7^ cells T cells were washed 3 times in PBS and resuspended in 2 volumes of homogenization buffer (400 mM sucrose, 20 mM HEPES, pH 7.5, 1mM EDTA) with Complete Mini protease inhibitor (Roche) and 1U RNasin (Promega). The activated T cells in homogenization buffer were passed \~50 times through a 22 gauge needle, and resting T cells were passed \~80 times through a 25 gauge needle until 50%--80% of cells were disrupted, as assessed by trypan blue staining. The homogenized cells were then centrifuged at 1,500 × g for 5 min to remove nuclei and the resulting supernatant was centrifuged at 15,000 × g for 20 min to spin down large cellular membranes, which were then subjected to OptiPrep gradient centrifugation. The OptiPrep gradient was made by overlaying 10 layers of 0.4 mL buffer in order from high to low concentration (33%, 30%, 27%, 24%, 21%, 18%, 15%, 12%, 9% and 6%) of OptiPrep. All OptiPrep solutions were made in buffer containing 50 mM HEPES, 78 mM NaCl, 4 mM MgCl~2~ and 8.4 mM CaCl~2~ and 10 mM EDTA. The 15,000 × g pellet fraction was resuspended in 100 μL PBS and added on the top of the OptiPrep gradients, and centrifuged at 40,000 rpm for 45 min using a SW55 rotor (Beckman Coulter). Seven fractions of 600 μL each were collected from top to bottom. 200 μL of each fraction was extracted in 700 μL Trizol (Invitrogen) for RNA purification and 400 μL of each fraction was further concentrated by StrataClean Resin (Agilent Genomics) for western blot analysis. Briefly, 10 μL of StrataClean Resin was added to each fraction followed by incubation at 4°C for 1 hr on a rotator mixer. After spinning down resin and removing supernatant, 30 μL of 1.5 × Laemmli buffer was added to each sample, which was then frozen for at −80°C until use. ### Small RNA cDNA library preparation {#S19} Barcoded small RNA cDNA libraries were prepared as previously described with modifications ([@R57]). Briefly, to reduce ligation bias, 5 random nucleotides were added into the 3′ end of 5′-adaptor and the 5′end of 3′-adaptors during the oligo synthesis. Barcoded 3′-adaptors were adenylated by 5′DNA adenylation kit (NEB). 19-nt and 24-nt synthetic oligoribonucleotides that were used as size markers were radiolabeled at the 5′ end by T4 PNK (NEB). 10 ng RNA from each EV or cell sample and the radiolabeled size markers were ligated separately to 10 μM adenylated 3′-adaptor using T4 RNA ligase 2, truncated K227Q (NEB) at 4°C for overnight. The 3′-ligation reactions containing EV or cell samples were pooled, ethanol-precipitated and loaded into a single lane of a 15% denaturing polyacrylamide gel. The ligation reactions containing radiolabeled 19-nt and 24-nt oligos were loaded into the nearby lane to be the size markers for cutting the band containing 3′-ligated products of pooled EV and cell samples from gels. The RNAs within the cut gel pieces were eluted at 4°C for overnight followed by ethanol precipitation. The purified 3′-ligated products were further ligated to 10 μM 5′-adaptor using T4 RNA ligase 1 (NEB) at 37°C for 1 hr. Ligated small RNAs were again cut and purified from a 12% polyacrylamide gel based on the radiolabeled size markers. The purified ligated small RNAs were reverse-transcribed using SuperScript III (Invitrogen), and amplified by PCR. The PCR products that were in the exponential phase of amplification, which is 8-12 cycles in this study, were purified from 2.5% agarose gel (Semkem LE agarose, Lonza) using a gel extraction kit (QIAGEN). The gel purification step was performed twice to increase the removal of adaptor dimers from ligated small RNAs. Except for the five random nucleotides within the 3′ end of 5′-adaptor and the 5′ end of 3′-adaptors, all the sequences including 5′-adaptors, barcoded 3′-adaptors, PCR primers were the same as those published in the previous studies ([@R19]). ### Annotation of tRFs from RNA-seq data {#S20} The obtained sequence files were trimmed for the constant regions of the adaptor sequences by FASTQ/A Trimmer. The resulting files were split into the separate samples according to the barcode sequences in the 3′ end of the read by FASTQ/A Barcode splitter. The reads in the resulting files contain 5 random nucleotides in the 5′ end and barcoded sequences followed by 5 random nucleotides in the 3′ end, which were both trimmed by FASTQ/A Trimmer. Extracted reads were mapped by Bowtie2 v2.2.4 (default settings) to mouse genome reference sequence (GRCm38/mm10). The mapped reads were then intersected with gene annotation (Ensemble GRCm38 release 91) supplemented with tRNA annotation from the Genomic tRNA Database ([@R36]). The numbers of mapped reads that overlap with each annotations were counted by bedtools coverage. For annotating tRNA fragments, a bed file that contains the coordination of different tRF groups, as described in [Figure 2C](#F2){ref-type="fig"}, within 472 annotated tRNAs was created. The coverage of tRNA reads in EV or cell samples for different tRF groups annotated in this bed file was calculated by bedtools coverage. The tRNA read was assigned to a specific tRF group that has the largest coverage by the read. More then 90% of tRNA reads were assigned to specific tRFs with more then 70% coverage. tRNA reads were annotated as ambigious when their coverage for all tRF groups was less than 70%. ### tRF antisense oligonucleotides transfection {#S21} Antisense 2′-o-methyl oligonucleotides (IDT) were transfected using a Neon transfection kit and device (Invitrogen). A total of 5 × 10^5^ T cells that have been stimulated for 1 day were washed two times with PBS before suspension in 10 μL of buffer T (Neon kit, Invitrogen). Antisense oligos (1 μL of 125 μM) were added to the cell suspension to a final volume of 11 μL and mixed. 10 μL of the suspension was electroporated with a Neon electroporation device (Invitrogen; 1,600 V, 10 ms, three pulses). The transfected T cells were then cultured in 0.5 mL media so that the final concentration of tRF antisense oligos is 250 nM. The 0.5 mL media for culturing transfected T cells were in 24-well plates with plate-bound anti-CD3 (2.0 μg/ml), soluble anti-CD28 (0.5 μg/ml) and 10 units/mL IL2. After 2 days of culture, the transfected T cells were harvested and analyzed by flow cytometry for surface CD44 and CD62L expression. The sequence of antisense oligo for Leu-TAA:5′tRF was CCACUCGGCCAUUCUG, for Leu-TAG:5′tRF was CCGCUCGGCCACGCUA, for Ser-GCT:3′i-tRF was CCCUCGCGUGCAAAGCACA, and for Leu-TAA:3′i-tRF was ACGCGGAUAUAAAUCC. ### Intracellular cytokine staining {#S22} After 2 days of culture, T cells transfected with antisense oligos were restimulated for 3 hours with 10 nM PMA and 1 μM Ionomycin in the presence of 5 μg/ml brefeldin A. The harvested cells were stained with cell viability dye (eFluor 780, Invitrogen) followed by fixation, permeabilization and IL-2 staining as previously described ([@R49]). QUANTIFICATION AND STATISTICAL ANALYSIS {#S23} --------------------------------------- All statistical information pertaining to the number of replicates and significance can be found in the figure legends. All experiments were performed at least three times independently where possible. In instances where the experiments were performed two times due to limited availability of primary cells or reagents, independent techniques were used to validate the findings. ### RNA-seq data analysis {#S24} The 'Deseq2′ package ([@R35]) was used for analysis of differential expression. 'ggplot2′ was used to produce scatterplots. EV enrichment (EE) in Figures [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, and [5](#F5){ref-type="fig"} was the fold-change of read counts in EVs versus cells. Deseq2 was used for analyzing the fold-changes of all annotated genes in EVs versus in cells and then the fold-changes of miRNAs and tRFs were subset from all genes. Thus, the EE of miRNAs and tRFs is a relative value that is compared to small RNAs derived from all genes. Deseq2 was also used for analyzing the fold-changes of the EE of all annotated genes in stimulated versus resting conditions or in stimulated conditions with DMSO versus with GW4869. The fold-changes of the EE of tRFs were subset from all genes. Thus, the fold-change of the EE of tRFs is also a relative value that is compared to small RNAs derived all genes. DATA AND SOFTWARE AVAILABILITY {#S25} ------------------------------ RNA sequencing data are deposited at GEO under the accession number GEO: GSE121724. Supplementary Material {#SM1} ====================== This publication is part of the NIH Extracellular RNA Communication Consortium paper package and was supported by the NIH Common Fund's exRNA Communication Program. We thank Darryl Mar, Priti Singh, and Annie Wang for help with mouse colony maintenance. This work was supported by the NIH (U19CA179512) and the Sandler Asthma Basic Research Center. DECLARATION OF INTERESTS The authors declare no competing interests. SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures and two tables and can be found with this article online at <https://doi.org/10.1016/j.celrep.2018.11.073>. {ref-type="supplementary-material"}.](nihms-1517606-f0002){#F1} {ref-type="supplementary-material"} and [Table S1](#SD2){ref-type="supplementary-material"}.](nihms-1517606-f0003){#F2} {ref-type="fig"}.\ (D) EV enrichment (EE) for the indicated tRF classes from T cells cultured under resting (EE~R~, x axis) and stimulated (EE~S~, y axis) conditions. Dotted black lines and arrows indicate thresholds at 1.0 EE~S~ (no enrichment) and 1.5 EE~S~. Solid black line demarcates equal EE in resting and stimulated cells (EE~S~/EE~R~ = 1). Red dotted line indicates threshold at 1.5-fold increased EE in stimulated cells compared to resting cells (EE~S~/EE~R~ ≥ 1.5). tRFs exhibiting activation-induced EV enrichment (EEs ≥ 1.5 and EE~S~/EE~R~ ≥ 1.5) are marked by red circles (adjusted p \< 0.05) or pink circles (adjusted p \< 0.5). Black circles (·) mark tRFs with activation-independent EV enrichment in stimulated cells (EE~S~ ≥ 1.5; adjusted p \< 0.05). The percentage of tRFs with activation-induced EE (union of red and pink circles) among all tRFs enriched in EVs in stimulated cells (union of black, red, and pink circles; total number indicated in parentheses after tRF class label in each panel) is indicated in each plot.\ (E) 3′i-tRF-L (linear) and 3′i-tRF-H (hairpin) subsets of the total 3′i-tRF shown in (D), lower left panel.\ see also [Figure S3](#SD1){ref-type="supplementary-material"} and [Table S2](#SD3){ref-type="supplementary-material"}.](nihms-1517606-f0004){#F3} {ref-type="supplementary-material"}.](nihms-1517606-f0005){#F4} {ref-type="fig"}.\ (F) Oligo(dT) qRT-PCR measurement of tRF abundance in EVs from cells stimulated with DMSO (black bars) and the indicated concentrations of GW4869 (gray bars).\ (G) tRF abundance in cells (black bars) and in EVs (gray bars) under resting and stimulated conditions as determined by stem-loop qRT-PCR.\ Statistical significance is measured using a one-tailed t test: \*p \< 0.05, \*\*p \< 0.01, and \*\*\*p \< 0.001. Error bars indicate SD of the mean.\ See also [Figure S5](#SD1){ref-type="supplementary-material"} and [Table S2](#SD3){ref-type="supplementary-material"}.](nihms-1517606-f0006){#F5} {ref-type="supplementary-material"}.](nihms-1517606-f0007){#F6} {ref-type="supplementary-material"}.](nihms-1517606-f0008){#F7} ###### KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER ---------------------------------------------------------------- ----------------------------------------------------- --------------------------------------------- Antibodies GW182 Bethyl Laboratories Cat\# A302-329A; RRID: AB_1850240 Ago2 (C34C6) Cell Signaling Cat\# 2897S; RRID: AB_2054446 Tsg101 (C-2) Santa Cruz Biotechnology Cat\# sc-7964; RRID: AB_671392 CD81 (D5O2Q) Cell Signaling Cat\# 10037S; RRID: AB_2714207 Arf6 (3A-1) Santa Cruz Biotechnology Cat\# sc-7971; RRID: AB_2289810 Transferrin Receptor Abcam Cat\# ab84036; RRID: AB_10673794 Beta-actin (AC-74) Sigma Cat\# A5316; RRID: AB_476743 Lamp1 Abcam Cat\# ab24170; RRID: AB_775978 Rab7 (D95F2) Cell Signaling Cat\# 9367S; RRID: AB_1904103 TIA1 (D-9) Santa Cruz Biotechnology Cat\# sc-48371; RRID: AB_628358 Rabbit IgG (H+L) Bio-Rad Cat\# 170-6515; RRID: AB_11125142 Mouse IgG (H+L) Bio-Rad Cat\# 170-6516; RRID: AB_11125547 CD62L-BV605 (MEL-14) Biolegend Cat\# 563252; RRID: AB_11125577 CD44-A700 (IM7) eBioscience Cat\# 56-0441-80; RRID: AB_494012 IL2-PerCP-Cy5.5 (JES6-5H4) eBioscience Cat\# 503821; RRID: AB_961403 Chemicals, Peptides, and Recombinant Proteins GW4869 Sigma Cat\# D1692 StrataClean Resin Agilent Technologies Cat\# 400714 Deposited Data Raw and processed small RNA sequencing data This paper GEO: GSE121724 Experimental Models: Organisms/Strains C57BL/6 Jackson Laboratory JAX:000664 Oligonucleotides Leu-TAA-5RF: ACCAGAATGGCCGAGTGGTTA This paper N/A Leu-TAG-5RF: GGTAGCGTGGCCGAGCGGTC This paper N/A Leu-TAA-3RF: GGATTTATATCCGCGTGGG This paper N/A LeuTAA5_FP_2: ACACTCCAGCTGGGACCAGAATGGCCGA This paper N/A LeuTAA3_FP_2: ACACTCCAGCTGGGTGGATTTATATCCG This paper N/A LeuTAG5_FP_2: ACACTCCAGCTGGGGGTAGCGTGGCCGA This paper N/A LeuTAA5_RP_2: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACCACTCG This paper N/A LeuTAA3_RP_2: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCACGCGG This paper N/A LeuTAG5_RP_2: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACCGCTCG This paper N/A LeuTAA3_Pr_2: 5′- /56-FAM/TTCAGTTGA/ZEN/GCCACGCGG/3IBkFQ/ --3′ This paper N/A LeuTAG5_Pr_2: 5′- /56-FAM/TTCAGTTGA/ZEN/GACCGCTCG/3IBkFQ/ --3′ This paper N/A LeuTAA5_Pr_2: 5′- /56-FAM/TTCAGTTGA/ZEN/GACCACTCG/3IBkFQ/ --3′ This paper N/A Universal RP: GTGTCGTGGAGTCGGC This paper N/A Software and Algorithms Bowtie2 v2.2.4 [@R30] <http://bowtie-bio.sourceforge.net/bowtie2> Samtools v1.7 [@R32] <https://github.com/samtools/samtools> Bedtools v2.27.1 [@R44] <https://github.com/arq5x/bedtools2> DEseq2 v1.18.1 [@R35] <https://bioconductor.org/biocLite.R> FASTX-Toolkit v0.0.14 Gregory Hannon lab at cold spring harbor laboratory <http://hannonlab.cshl.edu/fastx_toolkit> ###### Highlights - Specific tRFs are enriched in extracellular vesicles released by T cells - T cell activating signals regulate tRF secretion - Activation enhances multivesicular body formation and cellular tRF downregulation - Activation-induced, extracellular-vesicle-enriched tRFs inhibit T cell activation [^1]: AUTHOR CONTRIBUTIONS N.-T.C. performed and analyzed all of the experiments. R.K. established the bioinformatic pipeline for small RNA sequencing analysis. K.M.A. and N.-T.C. designed experiments, interpreted the data, and wrote the manuscript. All authors discussed the results and approved the manuscript.

Introduction {#s1} ============ Naïve CD4^+^ T cells can differentiate into distinct effector helper T cell (Th) subsets, including Th1, Th2, and Th17 cells, as well as regulatory T cells (Treg) ([@B1]--[@B3]). Th1, Th17, and Treg cell subsets have been regarded as major players in immunopathology of autoimmune diseases. Th1 and Th17 cells are pro-inflammatory subsets that promote the development of autoimmunity and tissue damage, while Treg cells maintain immunotolerance and prevent autoimmunity. Thus, maintaining the balance of anti-inflammatory Treg cells and pro-inflammatory Th1 and Th17 cells has significant implication in preventing and/or attenuating autoimmunity and chronic inflammation. Protective, nonpathogenic Th1 and Th2 cells can be generated *in vitro* from naïve T cells by using IL-12 and IL-4 which is regulated by their specific transcription factors T-bet and GATA3, respectively ([@B4], [@B5]).The cytokine TGF-β drives the conversion of naïve T cells into induced Treg (iTreg) cells, while TGF-β, together with pro-inflammatory cytokines, in particular IL-6, drives naïve CD4^+^ T cell differentiation toward Th17 ([@B3], [@B6]). Mechanistically, TGF-β alone can activate its downstream transcription factors Smad2 and Smad3 to induce expression of Treg-specific marker Foxp3, which control the generation and function of Treg. In contrast, IL-6 induces activation of STAT3 to promote expression of Th17 cell-specific transcription factor RORγt critical for IL-17 expression. Furthermore, TGF-β-induced Foxp3 suppressed RORγt function partly via their interaction ([@B7]). Therefore, the fate of naïve CD4^+^ T cells upon stimulation by antigens to turn into Th17 or Treg cells for a significant part depends on the micro-environmental cytokine-regulated balance of RORγt and Foxp3. Naringenin, a major flavanone in grapefruits, has a wide range of anti-inflammatory and neuro-protective properties ([@B8]). We recently reported that dietary naringenin supplementation ameliorated experimental autoimmune encephalomyelitis (EAE) in mice, which was associated with the decrease in Th1 and Th17 cell populations and pro-inflammatory cytokine IL-6 production, which promotes CD4^+^ T cells differentiation into Th17 cells ([@B9]). In addition, our *in vitro* study showed that naringenin directly inhibited effector T cell functions, including T cell proliferation, cell division, and production of cytokines IL-6, IFN-γ, and IL-17, in normal and EAE mice ([@B10]). These data suggest that naringenin may affect CD4^+^ T cell differentiation process. However, there was no direct evidence to substantiate this hypothesis and furthermore, if it is the case, it would be important to know through what molecular mechanisms naringenin exerts its such effect. Thus, in the present study, using *in vitro* model, we characterized (1) which type of T cells (CD4^+^ or CD8^+^) are affected by naringenin, and (2) how naringenin modulates CD4^+^ T cell differentiation into effector lineages (Th1, Th17, and Treg), and (3) what regulating networks are involved in the effects of naringenin on regulating CD4^+^ T cell differentiation. Materials and methods {#s2} ===================== Animals ------- Specific pathogen-free C57BL/6 female mice (6--8 wk) were purchased from Nanjing Biomedical Research Institution of Nanjing University (Nanjing, China). Mice were maintained at a controlled environment with a 12 h light:dark cycle and provided *ad libitum* access to water and mouse chow. Mice were killed by CO~2~ asphyxiation followed by exsanguination and tissues were collected post-mortem. All conditions and handling of the animals were approved by the Institutional Animal Care and Use Committee of Huaihe Hospital at Henan University. T cell division --------------- After mice were euthanized, inguinal lymph node (LN) cells were collected and single cells suspension was prepared for evaluation of CD4^+^ and CD8^+^ T cell proliferation using tracking dye fluorescein diacetatesuccinimidyl ester (CFSE, Molecular Probes, Eugene, OR, USA) method as previously described ([@B10]). A stock solution of naringenin (Sigma-Aldrich, St. Louis, CA) dissolved in DMSO at 400 mM was stored at −80°C and diluted with culture medium to the appropriate working concentrations immediately prior to use. Briefly, after LN cells were labeled with 1 μM of CFSE, they were added to a 24-well plate at 2 × 10^6^/well and stimulated with immobilized anti-CD3 Ab at 5 μg/ml and soluble anti-CD28 Ab at 1 μg/ml (anti-CD3/CD28) (both from Biolegend, San Jose, CA) in the presence of different levels of naringenin for 48 h. At the end of incubation, cells were collected, washed, and stained with fluorochrome conjugated anti-CD3, anti-CD4, and anti-CD8 (eBioscience). Fluorescence signals of stained cells were acquired by an Accuri C6 (Ann Arbor, MI) flow cytometer and data were analyzed with FlowJo7.6 software (Treestar Inc., OR, USA). Intracellular cytokine measurement ---------------------------------- After spleen cells were stimualted with anti-CD3/CD28 in the presence of naringenin for 48 h, they were re-stimulated during the last 4 h with 50 ng/ml PMA and 500 ng/ml ionomycin (both from Sigma-Aldrich) in the presence of monensin (GolgiStop, BD Pharmingen, San Jose, CA), and then the frequency and intensity of IFN-γ and IL-4 in CD4^+^ and CD8^+^ T cells were performed using flow cytometry method as described above. CD4^+^ T cell differentiation ----------------------------- Naïve CD4^+^ T cells were isolated from spleens using a CD4^+^CD62L^+^ T cell isolation kit II (Miltenyi Biotec, Auburn, CA) and incubated at 2 × 10^6^ cells/ml completed RPMI-1640 medium containing 5%FBS in 24-well plate. Cells were activated with anti-CD3/CD28 in all the experiments described below and T cell differentiation was induced as described previously ([@B11]). Briefly, the cultures were supplemented with IL-12 (10 ng/ml) (R&D systems, Inc., Minneapolis, MN) and anti-IL-4 (10 μg/ml) (BD Pharmingen) for Th1 differentiation, with IL-4 (10 ng/ml) (R&D systems, Inc.) and anti-IFN-γ (10 μg/ml) (BD Pharmingen) for Th2 differentiation, and with IL-6 (20 ng/ml), TGF-β (5 ng/ml), IL-23 (20 ng/ml) (all from R&D system), anti-IFN-γ, and anti-IL-4 (each 10 μg/ml) for Th17 differentiation. For Treg differentiation, naïve CD4^+^ T cells were incubated in the presence of TGF-β (5 ng/ml) for 72 h. To determine if naringenin (80 μM) affects the reciprocal effect between Treg and Th17, IL-6 was also added during Treg differentiation. Intracellular levels of Th1 (IFN-γ), Th2 (IL-4, IL-10, and IL-13), Th17 (IL-17A), and Treg (Foxp3) (all from eBioscience) were determined by flow cytometry as previously described ([@B11]). In addition, differentiated cells were stained with fluorochrome-conjugated anti-STAT1 (pY701/p-STAT1), anti-STAT3 (pY705/p-STAT3), anti-STAT4 (pY693/p-STAT4), T-bet (all from BD Pharmingen), and RORγt (R&D systems, Inc.) following standard protocols as described previously ([@B11]). Isotype Controls were used as negative control. Cells were analyzed using flow cytometry as described above. Western blot ------------ Naive CD4^+^ T cells were cultured with anti-CD3/CD28 and TGF-β with/without IL-6 in the presence/absence of naringenin for the time as indicated in the result section. Cells were harvested at 3 × 10^6^ cells/50 μl into RIPA cell lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 1 × protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN), and 1 × phosphatase inhibitor cocktail (Sigma-Aldrich), and incubated on ice for 15 min. Total cell protein extract was resolved in 7.5% acrylamide gels and then transferred to nitrocellulose membranes. The membrane was blocked with 5% non-fat milk in Tris-buffered saline before being incubated, respectively with specific primary antibodies for the following proteins: STAT-3(1:1000), Smad2/3 (1:1000), phosphorylated Smad2/3 (p-Smad2/3) (1:1000), Acetyl-STAT3 (Lys685, Ac-STAT3) (1:1000), phosphorylated STAT3 (p-STAT3) (1:1000) (all from Cell Signaling Technologies, Danvers, MA), and β-actin (1:5000, Sigma-Aldrich). The membranes were next incubated with horseradish peroxides (HRP)-conjugated secondary antibodies followed by exposure to enhanced chemiluminescent reagents (Millipore, Burlington, MA). Induction and evaluation of EAE ------------------------------- Mice were fed a diet supplemented with 0.5% naringenin and then immunized to induce EAE as described before ([@B9]). At day 42, mice were killed, and plasma was collected to measure soluble IL-6Rα (sIL-6R) and soluble gp130 (sgp130) as described below. Detection of mIL-6R, mgp130, sIL-6Rα and sgp130 ----------------------------------------------- After differentiation, one set of cells was stained with fluorochrome-conjugated anti-CD4, anti-CD126 (mIL-6R), and anti-CD130 (mgp130) (all from eBioscience). Cells were analyzed using flow cytometry as described above in "T cell division." The concentrations of sIL-6Rα and sgp130 in the plasma samples mentioned above or supernatants from differentiated CD4^+^ T cells were quantified using the IL-6R and gp130 ELISA kit (Sino Biological Inc., Beijing) following the manufacturer\'s instruction. IL-6-induced phosphorylation and acetylation of STAT3 ----------------------------------------------------- After naïve CD4^+^ T cells were incubated with/without 80 μM naringenin for 2 h, recombinant IL-6 (20 ng/ml) was added to the cultures and incubated at 37°C in a water bath for 15 min. Total cell protein was extracted and the p-STAT3 and Ac-STAT3 levels were determined as described above in "Western blot." Statistical analysis -------------------- Results are expressed as means ± SD. Statistical analysis was conducted using SYSTAT 12 statistical software. Differences were determined using one-way ANOVA followed by Tukey\'s HSD *post-hoc* test for multiple comparisons, or non-paired Student\'s *t*-test. Significance was set at *P* \< 0.05. Results {#s3} ======= Naringenin impacts CD4^+^ T cell functions ------------------------------------------ We in a previous study found that naringenin inhibited T cell proliferation in anti-CD3/CD28-activated lymphocytes ([@B10]). However, it is still unclear how different types of T cell populations (CD4^+^ and CD8^+^) may be affected by naringenin. Thus, we first evaluated CD4^+^ and CD8^+^ T cell proliferation from anti-CD3/CD28-activated lymphocytes treated by naringenin. As shown in Figures [1A,B](#F1){ref-type="fig"}, naringenin dose-dependently inhibited CD4^+^ T cell division and proliferation index, but did not significantly affect CD8^+^ T cells. Furthermore, naringenin inhibited CD4^+^ T cell production of IFN-γ (Th1 response) (Figure [1C](#F1){ref-type="fig"}), but not IL-4 (Th2 response) in a dose-dependent manner (Figure [1D](#F1){ref-type="fig"}). While the high concentration of naringenin (80 μM) decreased IFN-γ production in CD8^+^ T cells stimulated by anti-CD3/CD28 (Figure [1E](#F1){ref-type="fig"}). These data suggest that naringenin mainly affects CD4^+^ rather than CD8^+^ T cells; within CD4^+^ T cells, it appears that Th1 rather than Th2 response is affected by naringenin. {#F1} Naringenin inhibits Th1, but not Th2 differentiation ---------------------------------------------------- Our previous *in vivo* study showed that EAE mice receiving naringenin had smaller Th1 cell population, but similar Th2 cell population compared to those fed control diet ([@B9]). However, since the magnitude of a given cell population in the body may be affected by multiple factors including proliferation, differentiation, and shrinking, as well as interaction among different cell population, we speculated but could not convincingly conclude that naringenin directly affects CD4^+^ T cell differentiation. Thus to seek direct answer to this issue, in the current study we used an *in vitro* differentiation model, in which naive CD4^+^ T cells cultured under standard Th1 or Th2 polarization condition and production of IFN-γ and IL-4 was used as hallmark for Th1 and Th2, respectively. We found that Th1 polarization was inhibited by naringenin (80 μM) compared to the control (21 vs. 40%) (Figure [2A](#F2){ref-type="fig"}); while Th2 polarization was not significantly affected by naringenin (Figures [3A,B](#F3){ref-type="fig"}). These results are in agreement with those in the *in vivo* study. Additionally, we found that in Th2-polarized CD4^+^ T cells, IL-13^+^ population was marginally decreased (*P* = 0.05) (Figure [3C](#F3){ref-type="fig"}) and IL-10^+^ (Figure [3D](#F3){ref-type="fig"}) and IL-10^+^IL-13^+^ (Figure [3E](#F3){ref-type="fig"}) populations were significantly decreased by naringenin. {#F2} {#F3} To further investigate how naringenin modulated regulation mechanism upstream to Th1 differentiation, we determined expression of T-bet, a transcriptional factor known to be the master regulator in Th1 cell differentiation. Consistent with naringenin\'s inhibitory effect on Th1 differentiation, naringenin was found to decrease T-bet expression in differentiating CD4^+^ T cells (Figure [2B](#F2){ref-type="fig"}). It has been shown that T cell differentiation toward Th1 subset can be triggered by IL-12 and IFN-γ signaling via their transducers STAT4 and STAT1, respectively, which induce T-bet expression and drive Th1 cell differentiation ([@B12], [@B13]). Thus, we next determined involvement of STAT activation. Indeed, STAT1 and STAT4 activation (phosphorylation) in CD4^+^ T cells cultured under Th1 polarization condition was inhibited by naringenin treatment (Figures [2C,D](#F2){ref-type="fig"}). Naringenin inhibits Th17 cell differentiation --------------------------------------------- Th17 cells, which are commonly defined as IL-17-producing CD4^+^ T cells, are present at low level in naïve T cells. In the autoimmune disorders such as EAE, this population can be greatly increased. Th17 cells are believed to play a critical role in the development of autoimmunity. Under the *in vitro* polarizing conditions, naïve CD4^+^ T cells can be driven to develop into Th17 cells, usually by TCR stimulation in the presence of IL-6 and TGF-β. In such an experimental setting, we found that naringenin prohibited differentiation of naïve CD4^+^ T cells into IL-17-producing Th17 cells (Figure [4](#F4){ref-type="fig"}A). {#F4} RORγt is a specific transcription factor driving Th17 cell differentiation, and STAT3 is the key signal transducer which mediates action of IL-6, IL-21, and IL-23 ([@B14], [@B3]). Consistent with its effect on Th17 differentiation, nairngenin inhibited RORγt expression (Figure [4B](#F4){ref-type="fig"}) as well as its upstream event, STAT3 phosphorylation (Figure [4C](#F4){ref-type="fig"}). Naringenin promotes iTreg development and prevents IL-6-induced suppression of treg development ----------------------------------------------------------------------------------------------- Our *in vivo* study showed that dietary naringenin did not affect Treg cells in EAE mice ([@B9]). Varied results have been reported regarding how naringenin affects Treg development ([@B15], [@B16]). It is noted that those results were generated with use of different culture conditions, which often makes data interpretation difficult. Thus, we next determined whether naringenin affects iTreg development under standard iTreg-polarized condition. Although naringenin promoted Treg cell differentiation driven by TGF-β (Figure [5](#F5){ref-type="fig"}A), it only moderately reduced activation of Smad2/3 (3.45 vs. 2.90), a transducer of TGF-β-mediated Foxp3 induction (Figure [6](#F6){ref-type="fig"}A). {#F5} {#F6} IL-6 has been shown to inhibit Treg cell generation induced by TGF-β which has been demonstrated in this study and our previous study ([@B11]). This IL-6-induced inhibition of Treg development was prevented by naringenin (Figure [5A](#F5){ref-type="fig"}). This is consistent with reported decrease in Th17 differentiation by naringenin because the presence of IL-6 can switch TGF-β-induced differentiation in favor of Th17, and there is reciprocal modulation between Treg and Th17 differentiation. To further confirm this, we also directly determined Th17 population in the same cultures in which Treg were quantified and found that naringenin caused a significant reduction of Th17 population as manifested by decreased the level of both RORγt and IL-17 (Figures [5B,C](#F5){ref-type="fig"}). As a further support, it was also found that naringenin decreased p-STAT3 and Ac-STAT3 expression under TGF-β plus IL-6 co-cultured condition (Figure [6B](#F6){ref-type="fig"}). Naringenin interferes with IL-6/IL-6R signaling to affect treg cell development ------------------------------------------------------------------------------- It has been known that IL-6 conveys signals through STAT3 to promote Th17 and inhibit Treg lineage commitment. Classical IL-6R signaling and IL-6 *trans*-signaling have been involved in inflammatory diseases ([@B17], [@B18]). Naïve T cells have high mIL-6R expression and then are lost during an immune response that becomes an important source of sIL-6R ([@B19], [@B20]). IL-6 or IL-6R cannot bind to gp130 alone. A complex of IL-6-IL-6R is necessary for binding to gp130 to form a high-affinity, signaling-competent hexamer that activates STAT3 induces RORγt expression but not Foxp3 expression induced by TGF-β ([@B21], [@B22]). We thus hypothesized that naringenin may influence Th17/Treg balance through modulating IL-6 signaling. To address this, we first evaluated IL-6R expression under Th17/Treg polarized conditions in the absence of neutralized conditions. In accordance with our and other previous reports ([@B11], [@B20]), naïve unstimulated CD4^+^ T cells had high mIL-6R expression and sIL-6R was undetected in cultured medium alone in the absence of anti-CD3/CD28 Abs. The mIL-6R expression (Figure [7](#F7){ref-type="fig"}A) decreased and the levels of sIL-6R increased upon activation (Figure [7B](#F7){ref-type="fig"}), both of which were partially prevented by naringenin. We further showed that expression of another IL-6R, mgp130, was not affected by naringenin treatment (Figure [7C](#F7){ref-type="fig"}). Finally, we found that naringenin diminished IL-6-induced STAT3 phosphorylation and acetylation, the indicator downstream events in IL-6 signaling (Figure [7D](#F7){ref-type="fig"}). Together, these data suggest that naringenin-induced alteration in IL-6 signaling may be an important mechanism for its effect on Th17/Treg balance. {#F7} Results from dietary supplementation study support naringenin\'s inhibitory effect on IL-6 signaling ---------------------------------------------------------------------------------------------------- To test whether the *in vitro* study results were relevant to the *in vivo* situation, we measured plasma sIL-6R and sgp130 concentrations utilizing the samples collected from naringenin-fed mice in our previous study which showed the attenuated EAE mice by naringenin. We found that sIL-6R was higher and sgp130 was lower in plasma from EAE mice compared to that from the naïve (unimmunized normal) mice, and these changes were partially prevented by dietary naringenin supplementation (Figure [8](#F8){ref-type="fig"}). These *in vivo* results validate our *in vitro* results in terms of naringenin\'s effect on IL-6 signaling. {ref-type="fig"} Naringenin reduces level of sIL-6R while increasing sgp130 level in the plasma of EAE mice. C57BL/6 mice were fed a diet containing 0.5% naringenin for 30 day before they were immunized to induce EAE as previously described ([@B10]). On day 14 post-immunization, mice were euthanized, and plasma was collected to determinesIL-6R **(A)** and sgp130 **(B)** in the plasma levels. Values are mean ± SD (12/group). Means without a common letter significantly differ at least at *P* \< 0.05. Con, control; NAR, naringenin.](fimmu-09-02267-g0008){#F8} Discussion {#s4} ========== A delicate balance between effector T cells with different functions, in particular pro-inflammatory and pro-tolerance, plays a crucial role for eliciting protective immune response to pathogens without losing immune tolerance to self-antigens. Failure to maintain this balance is an important mechanism responsible for the development of many autoimmune diseases. Therefore, exploring the new strategies targeting this factor should have significant clinical potential in dealing with autoimmune diseases. Targeted drug therapy has made impressing progress; however, the efficacy vs. side effect is still a major issue limiting unrestricted application. Nutritional intervention through consuming bioactive food components has become a desirable alternative and complementary strategy for this purpose. Several major categories of dietary flavonoids are known to have immune-modulating property, which implies their potential application in preventing and/or mitigating autoimmune diseases. We recently showed that dietary supplementation with naringenin, a flavonoid compound found abundant in citrus fruits, particularly in grapefruit, attenuated EAE symptoms and pathology via favorably modulating effector T cell functions involved in T cell-mediated autoimmunity ([@B9]). In an *in vitro* study we further demonstrated that naringenin could directly suppress effector T cell functions including total T cell proliferation, and production of cytokines ([@B10]). In this study, we demonstrated that naringenin primarily affected functions of effector CD4^+^ cells, which is based on the following observations: (1) T cell proliferation and IFN-γ production in CD4^+^ T cells were inhibited by naringenin; (2) under Th1 differentiation condition, naringenin not only diminished Th1differentiation, but also decreased Th1-specific transcription factor T-bet and transducer STAT4 (for IL-12) and STAT1 (for IFN-γ) activation; (3) naringenin impaired Th17 differentiation which might be mediated by the down-regulation of RORγt, p-STAT3, and Ac-STAT3 under Th17 differentiation condition; (4) naringenin promoted iTreg cells under iTreg polarization condition via down-regulating Smad2/3 phosphorylation; (5) under iTreg polarization condition in the presence of IL-6, naringenin prevented IL-6-induced iTreg suppression through suppressing IL-6/IL-6R signaling, and STAT3 phosphorylation and acetylation. Together, these results suggest that naringenin plays a crucial role in maintaining the balance between Treg and pro-inflammatory T helper cells, which sheds light on the mechanistic insight to its beneficial effect observed in our previous study. The immune system has evolved several mechanisms to control activated T cell expansion and differentiation, including anergy, death, and regulation ([@B23]). One level of control resides in the function of CD4^+^and CD8 ^+^ regulatory cells ([@B24], [@B25]). While CD4^+^ T cells are primary cells in mediating adaptive immunity to a variety of pathogens, they are also a key player implicated in regulation of autoimmunity by their pro-inflammatory and pro-tolerance functions ([@B2]). Likewise, CD8^+^ T cells are important in effective vaccination and vial clearance as well as participant in maintaining the immune-tolerance ([@B26]--[@B28]), On the flip side, however, CD8^+^ T cells are the effector cells contributing to the disease of autoimmunity ([@B29]--[@B31]). Therefore, altered control of CD4^+^ and CD8^+^ T cells in their response to self-Ag is expected to significantly impact outcomes of autoimmune diseases. We recently reported that naringenin was an inhibitor of effector function of T cells ([@B10]). In this study, we expanded the research along this line by investigating how naringenin affected T cell sub-populations because of their unique function and implication in the development of autoimmune disease. We showed, for the first time, that naringenin mainly affected CD4^+^ T cell proliferation, among which naringenin inhibited Th1 response, but had no effect on Th2 response. Although CD8^+^ T cell proliferation was not significantly inhibited by naringenin with the concentrations used, IFN-γ production from CD8^+^ T cells appeared to be dose-dependently inhibited and this effect was clearly significant at high level of naringenin (80 μM). Given this, whether naringenin-induced change in CD8^+^ T cell functions has significant contribution to its beneficial effect in autoimmunity remains to be further investigated. Although CD8 ^+^ T cells have been shown to be involved in the development of autoimmune diseases such as MS and EAE ([@B31], [@B26]), it is generally accepted that over-activation of self-Ag pathogenic CD4^+^ T cells is the direct cause of these diseases ([@B3], [@B32]). IFN-γ-secreting Th1 cells ([@B33]) and IL-17-secreting Th17 cells ([@B3]) are first primed in the periphery, migrate into central nervous system (CNS), and then cause demyelization and neurological disability ([@B34]). Th1 and Th17 cells can also help recruit other inflammatory cells into CNS to exacerbate the disease process ([@B3]). Thus, the agents which target to pro-inflammatory Th1 and Th17 cell populations should be taken as potential candidates for preventing and/or treating autoimmune diseases like MS. Our recent *in vivo* study demonstrated that dietary naringenin reduced immune cell infiltration, and attenuated demyelination in CNS, and these changes were associated with decreased Th1 and Th17 cells, which were, in turn, associated with down-regulation of their respective transcription factors, T-bet and RORγt. However, anti-inflammatory Treg cells were not found to be affected by naringenin ([@B9]). These results suggest that naringenin may modulate CD4^+^ T cell subset balance via directly impacting their differentiation processes. Indeed, in the current study, we provided direct evidence supporting this hypothesis as we found that naringenin decreased differentiation of naive CD4^+^ T cells into Th1 and Th17 cells, while increased Treg cell differentiation. In addition, naringenin-induced alteration in CD4^+^ T cell subsets might be due in part to its specific effect on the reduction in abundance or activity of the corresponding regulators for each sunset. These findings reinforce our understanding of beneficial effect of naringenin for the management of autoimmune diseases, which contribute to developing the effective preventive and/or therapeutic approach to combat T-cell mediated autoimmune response. Treg cells play an important role in maintaining immune tolerance against self-tissues. Some compounds such as TGF-β ([@B7]), retinoic acid ([@B35]), and estrogen ([@B36]) can drives CD4^+^CD25^−^ naïve T cells developing to CD4^+^CD25^+^ iTreg cells. Naringenin has been shown to induce iTreg cells from CD4^+^ T cells independent of TGF-β ([@B37]). In accordance with this, current study demonstrated that naringenin dose-dependently induced iTreg cells from anti-CD3/CD28 activated T cells (Supplemental Figure [1](#SM1){ref-type="supplementary-material"}). In the presence of TGF-β, naringenin could further potentiate naïve CD4^+^ T cell conversion into iTreg cells. The mechanism of TGF-β-induced generation of Foxp3 is partly due to Smad proteins, such as Smad2 and Smad3 phosphorylation, activation, nuclear translocation, and finally, binding to the Foxp3 locus and causing Treg polarization ([@B38], [@B39]). Naringenin has been regarded as the Smad3 specific inhibitor via suppressing TGF-β ligand-receptor interaction ([@B40], [@B41]). Indeed, naringenin *in vitro* slightly inhibited Smad2 and Smad3 phosphorylation which results in decreased generation of Foxp3. However, naringenin promotes, rather than inhibits, iTreg cell differentiation. These contradictory observations remain to be further elucidated. Notably, TGF-β enables naïve CD4^+^ T cells to become Th17 cells when co-cultured with pro-inflammatory cytokines, such as IL-6 ([@B3]). Increased IL-6 could redirect TGF-β-induced Treg differentiation toward Th17 cells and as such, tilts the Th17 and Treg balance. Since we found that naringenin inhibited Th17 differentiation and also diminished IL-6-induced suppression in iTreg development, we addressed whether naringenin exerted these effects by affecting IL-6 signaling. IL-6 signaling is mediated via binding to its two receptors: mIL-6R and sIL-6, which elicit classical IL-6R signaling and IL-6 *trans*-signaling, respectively. Naïve T cells have high mIL-6R expression that will be lost during inflammation ([@B19]). Of note, naringenin partly prevented the reduction of mIL-6R in activated T cells, followed a decrease in sIL-6R levels in cultured mediums, which could be generated by activated T cells through shedding of mIL-6R ([@B20]). These studies justifies our results given that, after naïve T cells were polarized under Th17 differentiated condition, naringenin prevented a decrease in mIL-6R, while decreased sIL-6R in cultured supernatants. Since we did not observe any difference in mgp130 between naringenin and control, it may be suggested that naringenin might inhibit IL-6 *trans*-signaling. This inhibited IL-6 signaling by naringenin was further verified to be functionally relevant as we showed that naringenin suppressed IL-6-induced STAT3 phosphorylation. In addition to phosphorylation, STAT3 activation can be regulated by acetylation on lysine 685, which promotes Th17 development ([@B42]). Our observation that STAT3 acetylation was inhibited by naringenin further support involvement of altered STAT activation in naringenin\'s effect. Elevated sIL-6R by auto-reactive CD4^+^ T cells contributes to autoimmune disease development via conferring IL-6 responsiveness ([@B20]) as well as blocking Treg development ([@B43]). Combination with the observed impact of naringenin on IL-6 signaling in CD4^+^ T cell differentiation, we speculated that naringenin\'s benefits on EAE might be partly due to naringenin\'s effect on IL-6 signaling. To confirm this, we conducted relevant analysis using the samples from our *in vivo* studies. Consistent with our previous study, EAE mice had two-fold higher plasma sIL-6R levels compared to the healthy control mice and this increase in plasma sIL-6R was prevented by dietary naringenin, which is in agreement with the findings in the current *in vitro* study. Together with the observation in that *in vivo* study that naringenin reduced plasma IL-6 levels in EAE mice, these results suggest that the results from the current *in vitro* study are relevant to the *in vivo* situation and that naringenin may block IL-6 *trans*-signaling, at least in part by reducing IL-6 and sIL-6R levels. Next we further analyzed the plasma sgp130, a natural inhibitor of IL-6 *trans*-signaling, from naringenin-treated EAE mice. Naringenin prevented the decrease in plasma sgp130 in EAE mice. This is in agreement with the decreased plasma IL-6 and sIL-6R levels in naringenin-treated EAE mice because reduced IL-6/sIL-6R complex formation in the trans-signaling would be assumed to spare some sgp130. In addition, IL-2, a T cell growth factor, has been demonstrated to inhibit Th17 development while promote Treg development ([@B44], [@B45]). Our previous study has shown that naringenin inhibited IL-2/IL-2R signaling pathway ([@B10]), which indicates that naringenin might promote Th17 generation and inhibit Treg development via modulating IL-2/IL-2R signaling in differentiating CD4^+^ T cells. However, naringenin actually inhibited Th17 generation while promoted iTreg development in the current study. Furthermore, Blimp-1, a key regulator of terminal differentiation in B cells and T cell linage, can repress IL-2, IFN-γ, and IL-17 and maintain Treg cell function ([@B46]--[@B48]). The underlying mechanisms are mediated by binding to their regulatory factors such as ifnγ, tbx21, bcl6, stat3, stat5, il17. However, whether these genes are involved in the effects of naringenin on CD4^+^ T cell differentiation is still unclear. Thus, we will plan a specific in depth study in the soon future to determine the role of IL-2/IL-2R signaling and these regulatory genes in naringenin\'s effect on CD4^+^ T cell differentiation involving altered Treg/Th17 balance and Th1 differentiation. In summary, this study demonstrated that naringenin inhibited Th1 and Th17 development; while naringenin did not affect Th2 cells in IL-4 production, it decreased IL-10 and IL-13 production. In addition, naringenin promoted iTreg development and prevented IL-6-induced suppression on iTreg development, which may be associated inhibition of Th17 differentiation. To our knowledge, this is the first comprehensive study reporting that naringenin modulates functions of effector CD4^+^ T cell subsets via targeting their respectively transcription and/or transducer factors. Especially, inflammatory cytokine IL-6 signaling appears to be a key factor through which naringenin favorably influences the balance between Th17 and Treg cells, leading to an alleviated autoimmunity. These novel observations allow us to gain a better understanding for the mechanisms underlying the naringenin\'s beneficial effect in attenuating T-cell mediated autoimmune disorders. We propose that these effects of naringenin may have translational value in potential clinical application to prevent/mitigate T cell-mediated autoimmune diseases. Author contributions {#s5} ==================== JW and DW designed the research. XN, CW, and JW conducted research and analyzed data and interpreted the data. JW and DW wrote the paper. All authors reviewed the manuscript. Conflict of interest statement ------------------------------ The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was supported by the National Natural Science Foundation of China (81402677), the Natural Science Foundation of Projects of Henan (182300410325), the Science and Technology Development of Henan (162102410008, 172102410036, 162300410116, & 172102310151)), and the U.S. Department of Agriculture (USDA)-Agriculture Research Service (ARS) under agreement \#58-1950-0-014. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the USDA. Supplementary material {#s6} ====================== The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fimmu.2018.02267/full#supplementary-material> ###### Click here for additional data file. [^1]: Edited by: Thea Magrone, Università degli Studi di Bari, Italy [^2]: Reviewed by: Yanfei Zhang, Geisinger Health System, United States; Jianlin Geng, University of Alabama at Birmingham, United States [^3]: This article was submitted to Nutritional Immunology, a section of the journal Frontiers in Immunology

Introduction {#Sec1} ============ In eutherian mammals, X-linked gene dosage compensation between females and males is achieved by the transcriptional inactivation of one X in female cells (XCI) during embryonic development^[@CR1]^. In mice, the process starts at 2- to 4-cell stage female embryos, where the long non-coding *Xist* RNA is first observed coating the paternal X *in cis* leading to imprinted XCI. At the blastocyst stage, cells from the epiblast reactivate the paternal X, and random XCI subsequently takes place, resulting in random inactivation in the embryo proper and imprinted XCI in mouse placenta^[@CR2],\ [@CR3]^. In contrast, the dynamics of XCI during human embryonic development is still much unknown, with reports presenting conflicting results^[@CR4]--[@CR6]^. While accumulation of *XIST* RNA in one X exclusively in female embryos was shown at the morula and blastocyst stage, along with inactivation of one gene in the *XIST*-coated region in blastocysts^[@CR4]^, another report employing equivalent *in situ* analysis detected *XIST* RNA accumulation in both Xs of female and in the single X of male morulas and blastocysts, but no transcriptional silencing of three X-linked genes^[@CR5]^, arguing against XCI during human preimplantation development. These conflicting results may be due to limitations of *in situ* methods and to the small number of X-linked genes assayed, but they indicate important differences in XCI between mouse and humans. More recently, using single-cell RNA-sequencing (scRNA-seq) from human preimplantation embryos, Petropoulos *et al*.^[@CR7]^ showed that dosage compensation between males and females is achieved by day 7 (E7) blastocysts. However, since no sign of monoallelic transcriptional silencing of X-linked genes was observed, the authors concluded that, instead of XCI, dosage compensation during human preimplantation development occurs by downregulation of both Xs in females, a phenomenon named *dampening* of X expression. Here, we used a novel pipeline to analyze XCI in human preimplantation embryos using data from Petropoulos *et al*.^[@CR7]^ (embryos from 8-cell to the E7 blastocyst -- dataset-1); from previous scRNA-seq study of human preimplantation embryos^[@CR8]^ (from oocytes to the E6 blastocyst stage, and male human embryonic stem cells -- dataset-2), and of female adult fibroblast^[@CR9]^; and from RNA-seq of whole blastocysts^[@CR10]^ and female human embryonic stem cells (hESCs, H9 cell line)^[@CR11]^, Table [1](#Tab1){ref-type="table"}. Starting from oocytes, we give a complete panorama of dosage compensation during the initial stages of embryo development, showing decrease of biallelic and concomitant increase of monoallelic X-linked gene expression along development, consistent with random XCI in human preimplantation embryos rather than dampening of X expression. In addition, we address for the first time the issue of dosage compensation between the active X (X~a~) and autosomes during human embryonic development, and show that primed hESCs have an upregulated X~a~.Table 1Number of embryos and cells analyzed in each dataset.StageEmbryonic dayDataset \#^17^Dataset \#^28^EmbryocellEmbryocellOocyte------33Zygote------332-cell------364-cell------3128-cell(E3)Male64018Female425212Morula(E4)Male797212Female788------Blastocyst(E5)Male13204------Female9160------Blastocyst(E6)Male6119222Female1023418Blastocyst(E7)Male7175------Female10290------ Results {#Sec2} ======= Initiation of the X inactivation process in human development {#Sec3} ------------------------------------------------------------- Single-cell RNA-seq data were screened for informative SNPs, generating an RNA-based genotype of each embryo and cell line. While Petropoulos *et al*.^[@CR7]^ used a minimum of 3 reads as SNP call threshold, possibly generating a high rate of false positive and negative calls of biallelic expression, our strategy combined a more stringent threshold for SNP calling (number of reads ≥20 for SNP call), and the use of several filters to avoid artifacts (see Methods). To determine the fraction of X-linked mono and biallelically expressed genes per cell in each embryo, we looked for informative transcribed SNPs outside of the pseudoautosomal region. To consider a gene as informative in an embryo, it should either show biallelic expression in at least one cell; or have at least two cells of the same embryo expressing just one, but different, alleles (*e.g. A* and *A'*), which was considered monoallelic expression. Therefore, an imprinted X-linked gene, which would express the same allele (*e.g. A'*) in all cells of an embryo, would be considered a non-informative gene rather than monoallelic. Thus, imprinted XCI would lead to no informative monoallelically expressed X-linked genes in latter stage female embryos (similar to male embryos, Supplementary Fig. [S1](#MOESM1){ref-type="media"}). Importantly, to avoid a bias in the number of informative SNPs in embryos with more sequenced cells, leading to a false increase in monoallelically expressed genes, we randomly picked four cells of each sample for monoallelic expression analyses (similar results were obtained using all cells from each embryo - Supplementary Fig. [S1](#MOESM1){ref-type="media"}). Exploring dataset-1, we found consistent low coverage of aligned sequences to genes, SNP calls and heterozygous gene detection that affect analysis of allele-specific gene expression (Supplementary Fig. [S2](#MOESM1){ref-type="media"}). Nevertheless, we were able to find a significant decrease in the fraction of X-linked biallelically expressed genes in female embryos from 8-cell to E7 blastocyst stage (Pearson's r = −0.2137; *P*-value = 1.538 × 10^−9^), consistent with XCI (Fig. [1A](#Fig1){ref-type="fig"}, left panel). This decrease cannot be attributed to lower sequencing depth of latter staged embryos (Supplementary Fig. [S2](#MOESM1){ref-type="media"}). Male embryos showed decreased biallelic expression of X-linked gene consistent with degradation of maternal RNA (Fig. [1A](#Fig1){ref-type="fig"}, middle panel), while, in contrast, no significant negative correlation was found for autosomal genes (Fig. [1A](#Fig1){ref-type="fig"}, right panel). When analyzed individually, only 2 out of 22 autosomes in females showed significant decrease in biallelic expression (Supplementary Fig. [S4](#MOESM1){ref-type="media"}).Figure 1Allelic expression during preimplantation development. Analyses of changes in fractions of biallelically and monoallelically expressed genes during development in male and female embryos. Fractions of biallelically (black) and monoallelically (red) expressed genes per total of expressed genes in each cell for each developmental stage are shown. (**A**) Dataset-1; (**B**) Dataset-2. Pearson's r values (corr) are depicted in each panel. Non-paired Wilcoxon test was performed to compared the fractions of biallelically expressed X-linked genes between two stages in dataset-2. *P*-value (\*) \< 0.05; (\*\*\*) ≤0.001; (ns) not significant. Numbers of genes analyzed and similar analysis of individual autosomes and are shown in Supplementary Fig. [S3](#MOESM1){ref-type="media"} and Supplementary Fig. [S4](#MOESM1){ref-type="media"}, respectively. In order to further investigate XCI, we performed the same analysis in a different data set of scRNA-seq from human preimplantation embryos^[@CR8]^, spanning from oocytes to E6 blastocyst stage (dataset-2) (Fig. [1B](#Fig1){ref-type="fig"}). Embryos were sexed based on expression of Y-linked genes, using oocytes and male human embryonic stem cells at passage 0 (hESp0) as female and male controls, respectively. We identified two 8-cell embryos and one blastocyst as female; and one 8-cell embryo, two morulas, and two blastocysts as male (Supplementary Fig. [S5](#MOESM1){ref-type="media"} and Supplementary Table [S1](#MOESM2){ref-type="media"}). Sexing zygotes, 2- and 4-cell stage embryos was not possible because they have not undergone full embryonic genome activation (EGA)^[@CR8],\ [@CR12],\ [@CR13]^. Analysis of *XIST* expression in single cells from dataset-2 shows that at the 8-cell and blastocyst stages *XIST* expression in female was significantly higher than in pre-EGA or male embryos (Supplementary Fig. [S6](#MOESM1){ref-type="media"}, Supplementary Table [S2](#MOESM3){ref-type="media"}). Since no informative SNP in *XIST* was found, we could not distinguish mono from biallelic *XIST* expression using dataset-2 (Supplementary Note, Supplementary Fig. [S6](#MOESM1){ref-type="media"}). To test whether the process of X-linked gene silencing had initiated in female embryos, we analyzed allele-specific gene expression in scRNA-seq dataset-2. Four aneuploid cells in male morula \#2 were excluded from subsequent analyses (see Methods, Supplementary Fig. [S7](#MOESM1){ref-type="media"} and Supplementary Table [S3](#MOESM4){ref-type="media"}). We found that the mean number of monoallelically and biallelically expressed X-linked genes per cell was 25 and 2 times higher, respectively, than those found in dataset-1 (Supplementary Table [S3](#MOESM4){ref-type="media"}). As in dataset-1, the number of reads aligned to genes in dataset-2 was not lower in latter stage embryos (Supplementary Fig. [S2](#MOESM1){ref-type="media"}). In female embryos of dataset-2, the proportion of X-linked genes biallelically expressed decreased with development (Non-paired Wilcoxon test *P*-value = 3.175 × 10^−5^) (Fig. [1B](#Fig1){ref-type="fig"}, left panel), corroborating the results we obtained from the scRNA-seq dataset-1 (Fig. [1A](#Fig1){ref-type="fig"}). However, female, but not male, embryos showed a significant decrease also in the proportion of biallelically expressed autosomal genes (Supplementary Fig. [S4](#MOESM1){ref-type="media"}). Due to the greater coverage of this set of scRNA-seq (Supplementary Fig. [S2](#MOESM1){ref-type="media"}), we were able to analyze monoallelic expression from the X chromosome. We detected a significant positive correlation between the fractions of monoallelically expressed X-linked genes and the developmental stage in female embryos (Pearson's r = 0.4918; *P*-value = 0.0146), indicating an increase in monoallelic expression from the X consistent with random XCI (Fig. [1B](#Fig1){ref-type="fig"}, left panel; see Fig. [S4B](#MOESM1){ref-type="media"} for Bonferroni's correction when considering all chromosomes analyzed separately). In contrast, no significant correlation between monoallelic expression of autosomal genes and development was seen in male or female embryos (Fig. [1B](#Fig1){ref-type="fig"}, right panel). Figure [2](#Fig2){ref-type="fig"} depicts graphically the expression pattern of seven X-linked genes in dataset-2 E6 blastocysts. Although some female cells still exhibit biallelic expression, a considerable proportion of them already present monoallelic expression, a signature of random XCI. As expected, male cells express predominantly the same allele reflecting the single X chromosome (Fig. [2](#Fig2){ref-type="fig"}). The same analysis with informative genes common among female 8-cell embryos and blastocyst indicates initiation of the transition from biallelic to monoallelic expression (Supplementary Fig. [S8](#MOESM1){ref-type="media"}).Figure 2Allele-specific expression of X-linked genes at the blastocyst stage. Cells are grouped by embryo where each bar represents a single cell. Allelic expression pattern was detected by informative SNPs in the female blastocyst \#3. M, male; F, female. Allelic relative expression ratios ≤0.2 or ≥0.8 were considered as monoallelic expression (white dotted line). Relative expression of reference and alternative alleles in blue and orange, respectively. Arrows point to cells with random XCI, *i.e*., cells from the same embryo presenting monoallelic expression from different alleles. Y axis: relative expression ratio. Genes in red and green were described as subjected to and escaping XCI, respectively^[@CR36]^. Expression pattern of gene *APOO* detected by rs8680; *ATRX*, rs3088074; *HNRNPH2*, rs41307260; *RBMX2*, rs142885112; *VBP1*, rs11887; *SYAP1*, rs144608858 and *EIF2S3*, rs5949273. All blastocysts from dataset-2. No evidence of dampening of X expression {#Sec4} ---------------------------------------- We then looked at global expression level of biallelically expressed X-linked genes during development in scRNA-seq datasets-1 and -2 (Fig. [3](#Fig3){ref-type="fig"}). In both datasets we observed a decrease in the sum of expression levels of biallelically expressed X-linked genes, which could be due to dampening (decrease in the expression level of each biallelically expressed gene) or to XCI (decrease in the number of genes biallelically expressed) (Fig. [3A and B](#Fig3){ref-type="fig"}). To discern between the two mechanisms, we looked at the median of the expression of biallelically expressed X-linked genes, which, in the case of dampening, should also decrease during development (Fig. [3A](#Fig3){ref-type="fig"}). As shown in Fig. [3C](#Fig3){ref-type="fig"}, although presenting considerable variation among cells, in both datasets the median of the expression levels of biallelically expressed X-linked genes does not decrease during development.Figure 3Expression of biallelically expressed X-linked genes during female development. (**A**) Predictions of sum, median and female to male ratio of expression of biallelically expressed X-linked genes for X dampening and XCI hypotheses. Colored asterisks represent expression levels of X-linked genes. Dotted lines highlight biallelically expressed genes. X~d~: dampened X; X~a/i~: X chromosome undergoing XCI. X dampening would lead to decreased expression levels of both alleles, while XCI to a decrease in the number of biallelically expressed genes. Both lead to a decrease in the expression sum of biallelically expressed genes (sum). However, the median expression of biallelically expressed genes (median) should decrease with X dampening and not change with XCI. The female to male ratio of genes biallelically expressed in females should approach 1 in the case of X dampening and 2 in the case of XCI; (**B**) Distributions of sums (green) and (**C**) medians (orange) of biallelically expressed X-linked genes in each developmental stage for dataset-1 and -2. (**D**) Distributions of female to male ratio of expression of X-linked genes biallelically expressed in females (dataset-1). Numbers of genes (green), of female (red) and of male (blue) cells analyzed are indicated for each stage. Differences between males and females in each stage are indicated above the respective boxplots. Differences in female to male ratios between stages are shown. Wilcoxon tests: *P*-value (\*) \< 0.05; (\*\*) ≤0.01; (\*\*\*) ≤0.001; (ns) not significant. Expression levels differ between females and males at morula and blastocysts stages. Despite the borderline difference between E4 and E6 (*P*-value = 0.045), there are no differences on female-to-male expression ratio among developmental phases, indicating no down-regulation of female X-linked biallelically expressed genes. To corroborate these results, we analyzed the female-to-male relative expression level of each biallelically expressed X-linked gene for each developmental stage from E3 to E7. X dampening would lead to a ratio of biallelically expressed genes between females and males close to 1, while in the case of XCI this ratio would be around 2 (only genes that have not undergone XCI have biallelic expression) (Fig. [3A](#Fig3){ref-type="fig"}). Figure [3D](#Fig3){ref-type="fig"} shows that in E6 and E7 the expression level of biallelically expressed X-lined genes in females is on average two times higher than in males, again arguing against X dampening. Moreover, female-to-male relative expression levels of biallelically expressed X-linked genes do not change from E4 to E7. Therefore, we conclude that, rather than the expression levels, it is the number of genes biallelically expressed that diminish in females from the 8-cell to the blastocyst stage, consistent with an ongoing establishment of XCI (see also Fig. [1](#Fig1){ref-type="fig"}). X-linked dosage compensation between males and females {#Sec5} ------------------------------------------------------ In order to evaluate whether the increase of X-linked monoallelic expression was accompanied by dosage compensation, we compared the ratio of X to autosomal gene expression levels (X:A) in male and female embryos from dataset-2, which should be equivalent once XCI is complete (Fig. [4](#Fig4){ref-type="fig"}). Female 8-cell stage embryos \#1 and \#2 showed similar X:A ratios (mean 0.988 and 0.887, respectively), while male 8-cell embryo \#3 had a significantly lower X:A ratio (mean 0.605; Wilcoxon test, *P*-value = 0.0004), consistent with lack of dosage compensation. Likewise, female blastocyst \#3 and twin ICMs and trophectoderm showed a significantly higher X:A ratio when compared to male blastocysts \#1 and \#2 (non-paired Wilcoxon test, *P*-value 0.003018, Fig. [4](#Fig4){ref-type="fig"}). Thus, our data show that, despite *XIST* expression (Supplementary Fig. [S6](#MOESM1){ref-type="media"}) and increase in X-linked monoallelically expressed genes (Fig. [1B](#Fig1){ref-type="fig"}, left panel), the latter is still not sufficient to lead to detectable dosage compensation of the X chromosome between males and females by the E6 blastocyst stage (Fig. [4](#Fig4){ref-type="fig"} and Supplementary Fig. [S1](#MOESM1){ref-type="media"}).Figure 4X chromosome dosage compensation. Scatter plots of the X:A expression ratio (mean ± s.e.m. in gray) in single cells of 8-cell embryos and blastocysts from dataset-2; Twin corresponds to ICMs and trophectoderm of a female blastocyst with two ICMs^[@CR10]^. *P*-values: (\*) \<0.05, (\*\*) ≤0.01, (\*\*\*) ≤0.001 non-paired Wilcoxon test. Looking at the total sum of X chromosome rpkm during development, Petropoulos *et al*.^[@CR7]^ detected equivalent X-linked expression levels in male and female embryos only at the E7 blastocyst stage. Analyzing their scRNA-seq data (dataset-1), we observed similar X:A ratios in male and female embryos only at E7 blastocysts (Supplementary Fig. [S9](#MOESM1){ref-type="media"}), corroborating that dosage compensation in humans is achieved at that latter embryonic stage. Taken together, the analyses of both datasets indicate that in humans random XCI progresses through early embryonic development, leading to dosage compensation just before implantation. X-Autosomes dosage compensation {#Sec6} ------------------------------- Interestingly, our analyses of the female fibroblast cell line, which has undergone complete random XCI^[@CR9]^, showed an X:A ratio close to 1, similar to those of the pre-XCI female embryos with two X~a~'s (Fig. [5](#Fig5){ref-type="fig"}). This is consistent with dosage compensation between the X chromosome and autosomes achieved by transcriptional upregulation of the single X~a~ in adult cells, as proposed by Ohno^[@CR14]^. In addition, the X:A ratio in the male hESp0 cells was not different from that in female fibroblasts, but significantly higher than in male blastocysts (Fig. [5](#Fig5){ref-type="fig"}, *P*-value = 0.0005 non-paired Wilcoxon test); and the H9 female hESCs line, which has undergone XCI^[@CR11],\ [@CR15]^, also presented an X:A ratio close to 1 and equivalent to pre-XCI female embryos with two X~a~ (Fig. [5](#Fig5){ref-type="fig"}). These results show that upregulation of the human X~a~ does not take place by the E6 blastocyst stage, but can be detected in male and female pluripotent cells *in vitro*. Together with the observation of X-linked dosage compensation at E7^[@CR7]^, these results indicate intense epigenetic modifications of the X chromosomes during this narrow window of development. Finally, pre-EGA embryos also displayed X:A ratios similar to female pre-XCI embryos (Fig. [5](#Fig5){ref-type="fig"}). Since RNAs present in pre-EGA embryos are those transcribed from maternal cells with two X~a~, these data suggest that, in addition to reactivating the inactive X (X~i~)^[@CR16]^, oogenesis includes erasing X~a~ upregulation.Figure 5X-autosomes dosage compensation during development. Mean X:A expression ratio per embryo/sample (mean ± s.e.m. in red)*. P*-values: (\*\*) ≤0.01, (\*\*\*) ≤0.001 non-paired Wilcoxon test; sex of post-EGA stages is indicated and highlighted by background color: females (F, pink), males (M, blue). Embryos and male hESC (hESCp0) from dataset-2; twin corresponds to ICMs and trophectoderm of a female blastocyst with two ICMs^[@CR10]^; Fibrob. is a female fibroblast cell line^[@CR9]^; H9, female hESC line^[@CR11]^. See Supplementary Table [S4](#MOESM5){ref-type="media"}, for single-cell X:A expression ratios. Below, the activity of the X chromosomes in the respective embryos/cells. mat(X~a~X~a~): maternal mRNA from primordial germ cells with two active X chromosomes; X~a/i~: X chromosome undergoing XCI; X~2a~: upregulated active X. Discussion {#Sec7} ========== Single-cell RNA-seq technology has allowed more detailed analysis of gene expression during human preimplantation development, leading to better characterization of lineage segregation and of the *in vivo* pluripotent state^[@CR7],\ [@CR8]^. In this study, we used those data to investigate the process of XCI. By globally analyzing X chromosome expression during human embryonic development (dataset-1^7^ and dataset-2^8^), we were able to detect decrease of biallelic and increase of monoallelic X-linked gene expression from the 4-cell to the blastocyst stage, indicating that, contrary to the findings originally published for dataset-1^7^, initiation of XCI does take place during human preimplantation development. Analyzing scRNA-seq from dataset-1, Petropoulos *et al*.^[@CR7]^ found no evidence of XCI, despite observing dosage compensation between male and female E7 blastocysts. Therefore, they proposed that at that preimplantation stage dosage compensation was achieved by expression dampening of both X's in the female, albeit not formally testing this hypothesis. Indeed, due to the relatively low coverage, we did not have enough statistical power to rigorously analyze allele-specific gene expression in their data. Nevertheless, we could show decrease of the fraction of biallelically expressed X-linked genes with development, indicative of XCI. In addition to showing indications of an ongoing process of XCI, we present compelling evidence against X dampening in human preimplantation embryos. Specifically, we found no decrease in the median expression of biallelic X-linked genes from the 8-cell to the blastocyst stage in females, which would be expected if dosage compensation were due to dampening of X-linked expression in both chromosomes. Rather, we show that the lower expression sum of biallelically expressed X-linked genes in latter stage embryos results from the decrease in their number, another hallmark of XCI. Finally, we show that at the E7 blastocyst stage, expression of biallelically expressed X-linked genes is significantly higher in females than in males, again arguing against X dampening. Our results reveal additional differences between human and murine preimplantation development. We place induction of *XIST* expression at the 8-cell stage, while in mice *Xist* expression is first detected at the 2- to 4-cell embryo in an imprinted pattern, both coinciding with the respective time of EGA^[@CR2],\ [@CR3]^. However, we show that in human males the maternal *XIST* allele is also expressed in preimplantation embryos, corroborating lack of imprinted XCI^[@CR17]^. Moreover, using stringent statistical analysis, we detect progression of random XCI from the 8-cell to the E6 blastocyst stages in dataset-2. In contrast, mice undergo rapid random XCI only in the epiblast and upon differentiation^[@CR2],\ [@CR3]^. Finally, we investigated the issue of X to autosomes dosage compensation for the first time during human embryonic development. It has been recently shown by scRNA-seq that upregulation of the murine X~a~ initiates at the 4-cell stage, leading to X:A dosage compensation by E4.5, with the onset of random XCI in females^[@CR18]^. Our results are consistent with the establishment of human X:A dosage compensation after the E6 blastocyst stage, as shown *in vitro* in male and female embryonic stem cells and in female fibroblasts. Thus, in addition to inactivation of a single X in females, the human peri-implantation stage also involves upregulation of the X~a~ in both sexes. Finally, we show evidence that X~a~ upregulation is erased during oogenesis, reflected in the observed X:A dosage compensation of maternal RNAs in X~a~X~a~ oocytes and pre-EGA embryos. Our observations help understand the differences between human and mouse blastocyst-derived pluripotent stem cells (PSCs) in regards to the epigenetic state of the X chromosome. While the murine cells model well the *in vivo* pre-XCI state and undergo random XCI upon differentiation, human PSCs show variable states of XCI (reviewed in ref. [@CR19]). However, specific culture conditions shift human PSCs into a naïve state, more similar to mouse PSCs, which includes the presence of two X~a~'s (reviewed in ref. [@CR20]). Nevertheless, our findings show that in order to recapitulate the epigenetic state of the X chromosome in the human epiblast, naïve human PSCs must be in the process of establishing XCI. Thus, to capture the human pre-XCI state and develop an *in vitro* model of initiation of XCI, conditions to culture morulas must be established. Recently, X chromosome dampening has been reported in naïve hESCs, where the transition of primed lines of hESCs to the naïve state lead to reactivation of the X~i~ and an overall decrease in X-linked gene expression, albeit not sufficient to achieve dosage compensation^[@CR21],\ [@CR22]^. However, in their analysis of global X-linked expression levels, the authors did not take into account the possibility of X-upregulation, as we show in male and female primed hESC. Indeed, the unexpected decrease of X-linked gene expression in male naïve compared to male primed hESCs observed by Theunissen *et al*.^[@CR21]^ may be due to erasure of upregulation of the X~a~ in the naïve cells. Therefore, the epigenetic remodeling of the X chromosomes in the transition from the primed to the naïve state is more complex than previously anticipated, and it should be reanalyzed in light of the findings reported in this work that primed male and female hESC have an upregulated X~a~. In conclusion, our study takes a step further in understanding the dynamics of the X chromosome during human development and the naïve state of human PSCs (Fig. [6](#Fig6){ref-type="fig"}). The recent capability of cultivating human embryos *in vitro* until day 13^[@CR23],\ [@CR24]^ will allow the investigation of the subsequent development of dosage compensation in the X chromosome beyond the blastocyst stage.Figure 6Model of X chromosome activity during female human embryonic development. *XIST* expression is induced biallelically at the 8-cell stage, concomitant with EGA^[@CR8],\ [@CR12],\ [@CR13]^, and is upregulated in female early blastocysts, where early signs of XCI are detected (X~a~X~a/i~). XCI is mostly complete in late blastocyst (X~a~X~a/i~), approaching dosage compensation between male and female embryos^[@CR7]^. Upon implantation, XCI is complete and the single X~a~ is upregulated, leading to X-autosome dosage compensation. Primordial germ cells (PGC) repress *XIST*, reactivate the X~i~ ^[@CR16]^ and erase X~a~-upregulation, thus becoming X~a~X~a~. Therefore, pre-EGA embryos have maternal RNAs from X~a~X~a~/*XIST* ^−^ cells \[mat.(X~a~X~a~)\]. Primed hESCs derived from blastocysts have variable and unstable states of XCI^[@CR37]^, but after many passages most of them undergo XCI, repress *XIST* and upregulate the X~a~ (male lines only upregulate the X~a~). Transition to the naïve state leads to *XIST* expression at levels equivalent to those of female blastocysts and monoallelic in most cells^[@CR15],\ [@CR21],\ [@CR22]^, and loss of X~a~-upregulation in male and female cells. It remains to be further understood the mechanisms behind the decreased X-linked expression in female naïve versus primed cells -- is X~i~ reactivation incomplete in the naïve cells, or are they in an early stage of XCI? Methods {#Sec8} ======= Data acquisition {#Sec9} ---------------- Dataset-1 containing 1,529 single-cell RNA-seq (scRNA-seq) from 88 human embryos was acquired at ArrayExpress -- functional genomics data (EMBL-EBI - <http://www.ebi.ac.uk/>) under the accession number E-MTAB-3929^[@CR7]^. The data had been previously filtered by the authors to exclude outlier cells based on gene expression^[@CR7]^. For the present study we considered embryos with at least four cells sequenced. These data comprise embryos described as follows: 8-cell stage (E3: N = 12, n = 78), morula stage (E4: N = 14, n = 187), fifty-eight blastocysts from embryonic day E5 (N = 23, n = 375), E6 (N = 18, n = 415) and E7 (N = 17, n = 465); where N is the number of embryos and n the number of cells. Dataset-2 including scRNA-seq from human oocytes, preimplantation embryos and male human embryonic stem cells were acquired at GEO under the accession number GSE36552 (Gene Expression Omnibus - <https://www.ncbi.nlm.nih.gov/gds>)^[@CR8]^. Specifically: three oocytes, three zygotes, three 2-cell stage embryos (two cells each), three 4-cell stage (four cells each), three 8-cell stage (four cells from embryo \#1 and eight cells from embryos \#2 and \#3), two morula stage (eight cells each), three blastocyst stage (twelve cells from embryo \#1, ten from embryo \#2 and eight from embryo \#3) and one human embryonic stem cell line at passage zero (with seven sequenced cells). These data had also been previously screened by the authors and they found no outlier cells^[@CR8]^. RNA-seq data of two inner cell masses (ICM) and trophectoderm (TE) from one natural monozygotic twin embryo at blastocyst stage were obtained at SRA, under the accession number SRP063754 (Sequence Read Archive - <http://www.ncbi.nlm.nih.gov/sra>)^[@CR10]^. Data from 15 scRNA-seq human fibroblasts (T2N, human fetal primary fibroblast culture derived from female post-mortem skin tissue fetus) were available on the European Genome-phenome Archive (<https://www.ebi.ac.uk/ega/datasets>) under the accession number EGAD00001001084^[@CR9]^. RNA-seq of the female hES cell line H9 were acquired at ArrayExpress -- functional genomics data (EMBL-EBI - <http://www.ebi.ac.uk/>) under the accession number E-MTAB-2857^[@CR11]^. Data processing {#Sec10} --------------- To filter out low-quality reads, we used Trimmomatic v0.33^[@CR25]^ with the default parameters. Briefly, the program was set to remove bases from each read's ends if presenting sequence quality below 3. The average quality score was calculated using a sliding window of 4 bp. If the score was equal or less than 15, the four successive nucleotides were discarded from the read. Our parameters were also set to exclude reads shorter than 36 bp. Reads alignment and counting {#Sec11} ---------------------------- Reads were aligned to human assembly hg19 with the respective UCSC genome annotation (obtained from UCSC, GCA000001405.1 at <http://hgdownload.cse.ucsc.edu>) with TopHat (v2.0.13)^[@CR26]^. Only uniquely aligned reads were considered for further analyses. Aligned reads were then processed with SAMtools^[@CR27]^ and the number of reads mapping to each gene was counted using HTSeq^[@CR28]^. Expression analyses {#Sec12} ------------------- The resulting count files for each cell/sample were used to calculate rpkm (reads per kilobase per million)^[@CR29]^ and tpm (transcripts per million)^[@CR30]^. As defined by Yan *et al*.^[@CR8]^, only genes presenting rpkm ≥0.1 were considered as expressed. As proposed by Wagner *et al*.^[@CR30]^, tpm values were used in order to compare expression levels among different samples. Outlier cells were removed from all expression analyses to increase robustness. Sexing {#Sec13} ------ To determine the sex of each embryo from dataset-2^8^, we considered Y-linked expressed genes (rpkm ≥0.1) outside of the pseudoautosomal region. The total number of Y-expressed genes per embryo was counted and compared to those found in all oocytes and in the male embryonic stem cell line (hESp0) with Fisher's exact test and Bonferroni's correction for 18 tests (corrected *P*-value ≤ 0.00277). As the main embryonic genome activation (EGA) occurs at the 8-cell stage^[@CR8],\ [@CR12],\ [@CR13]^, embryos prior to this phase were not considered for sexing. We expected female embryos to show significantly lower numbers of Y-linked expressed genes than the male stem cell line but equal to oocytes. Analogously, male embryos should present significantly more Y-linked expressed genes than oocytes but not different than the male stem cell line (Supplementary Table [S1](#MOESM2){ref-type="media"}). For dataset-1, we considered sexing as reported by the authors^[@CR7]^. However, we identified one female E3 embryo with low number of X-linked SNPs (embryo E3.1: mean = 2.8; female group mean = 12.44, Wilcoxon non-paired *P*-value = 0.0178), and one male E6 embryo with high number of X-linked SNPs (embryo E6.18: mean = 2.739; male group mean = 0.3885; Wilcoxon non-paired *P*-value = 0.0005), resulting in inconsistent sexing. Those embryos were excluded from our analyses. Ploidy {#Sec14} ------ Post-EGA embryos had their cell ploidy analyzed using methods described by Mayshar *et al*.^[@CR31]^. The male human embryonic stem cell line at passage zero (hESp0, dataset-2), karyotyped as 46,XY, was used as a normal control^[@CR8]^. For each cell from a particular embryo, individual gene expression values (tpm) were normalized by its median value across all embryo cells. In order to obtain a visual profile of each chromosome in different embryos, the normalized gene expression values were then used to build a moving average plot (window size 188 bp). Embryos showing visual abnormalities for a particular chromosome were checked for an excess of over or underexpressed genes using Chi-square test. Genes expressed in one cell by more than 1.5 or less than 0.67 fold in comparison to the embryo median value were considered over/underexpressed, respectively. Variant calling and SNP annotation {#Sec15} ---------------------------------- We used VarScan (v2.3.7, <http://varscan.sourceforge.net>)^[@CR32],\ [@CR33]^ for variant calling with the option mpileup2cns to retrieve reference and alternative allelic read counts covering single nucleotide variants (SNV). SNVs were then annotated using dbSNP Build 138 (GRCh37.p10). Ambiguous dbSNP identification numbers (IDs), *i.e*., SNPs with the same ID annotated in different positions on the genome, were removed prior to annotation. IDs mapped into intergenic regions, pseudogenes, or regions containing overlapping genes were also removed in order to avoid false biallelic detection. Embryo genotyping and detection of monoallelic and biallelic expressed genes {#Sec16} ---------------------------------------------------------------------------- The relative expression ratio of two different alleles was calculated by directly counting the allele-specific reads covering a SNP position mapped to the reference or the alternative allele and then dividing it for all reads covering that position. Only SNPs position covered by at least 20 reads were considered. Biallelic expression was considered when allele relative expression ratio ranged from 0.2 to 0.8; and monoallelic expression otherwise^[@CR9]^. The same SNP should be detected in at least two cells of the same embryo or cell line to be considered in the downstream analyses. We also excluded, from all samples, X-linked SNPs showing biallelic expression in the male hESp0 stem cell line. To consider a gene as heterozygous (informative), it should meet one of the following criteria: (i) to contain SNPs showing biallelic expression or (ii) to have at least one SNP showing monoallelic expression, *i.e*., different cells from the same embryo or cell line expressing different alleles. However, if a gene with two or more SNPs identified in the same cell displayed both (i) and (ii), the inconsistent gene was removed from that cell's analyses. The analyses presented in Petropoulos *et al*.^[@CR7]^ used a minimum of 3 reads for a valid SNP call and considered as biallelic those SNPs with allele relative expression ratio ranging from 0.1 to 0.9. Since the number of reads covering a SNP position influences the rate of false positive and negative biallelic calls, we used more stringent parameters, considering only SNPs position covered by at least 20 reads, and excluding inconsistent genes as described above. Dataset-1 showed low number of reads aligned to genes when compared to other scRNAseq sets (dataset-2 and human fibroblasts^[@CR9]^) (Supplementary Fig. [S2](#MOESM1){ref-type="media"}). Therefore, the number of annotated SNPs and heterozygous genes were also significantly lower (Supplementary Fig. [S2](#MOESM1){ref-type="media"}). We deposited the script used in our analyses in the following link: <https://github.com/gustribeiro/Mello_analysis_pipeline>. Rates of monoallelic and biallelic expressed genes during development {#Sec17} --------------------------------------------------------------------- In female embryos, biallelically expressed genes on the X chromosome indicate that both Xs are active, and therefore an increase in the fraction of heterozygous monoallelically expressed genes is expected when random XCI takes place. In contrast, due to the presence of maternal RNAs, we expected very early male embryos to show biallelic expression of X-linked genes that decreases during development as maternal mRNA resources extinguish^[@CR7],\ [@CR8]^. Later in development, no heterozygous X-linked genes would be expected, as males are hemizygotes (see Supplementary Fig. [S1](#MOESM1){ref-type="media"} for a scheme of the model). In order to test the correlation between developmental stage and the fractions of biallelic and monoallelic expression, we first assessed for each cell the numbers of (i) all expressed genes, (ii) biallelic and (iii) monoallelic heterozygous genes. To calculate the fractions of biallelically and monoallelically expressed genes, the number of biallelic genes as well as the monoallelic genes, respectively, was divided by the total number of expressed genes for each cell at each stage. Genes located in the pseudoautosomal region were excluded from the analyses. Significant increase and decrease in fraction of monoallelically or biallelically expressed genes were assessed by testing respectively positive and negative correlations among those measures and the developmental stages with Pearson correlation coefficient. For comparisons of only two developmental stages, we used non-paired Wilcoxon test. To avoid allelic variance derived from the substantial amount of maternal mRNA^[@CR8]^, we excluded 2- and 4-cell stage embryos from the biallelic correlation analyses in dataset-2. Since two maternal Xs are active at the beginning of embryo development (see X:A expression ratio analyses), pre-EGA embryos should be the starting point for measuring the increase of monoallelic expression. We noted that the number of embryo cells analyzed could increase the chances of detecting monoallelic expression in heterozygous genes. Therefore, we randomly picked four cells of each embryo (4-cell, 8-cell, morula, blastocyst and hESp0) for statistical correlation analyses in Fig. [1](#Fig1){ref-type="fig"}. Males and females were plotted and tested separately for X chromosome analyses, while all embryos were pooled together for analyses of autosomal genes. Similar results were found when considering all cells of each embryo for statistical correlation analyses of monoallelically X-linked expressed genes and using lower numbers of genes, measuring correlations using individual autosomes (Supplementary Fig. [S1](#MOESM1){ref-type="media"} and Supplementary Fig. [S4](#MOESM1){ref-type="media"}). Expression levels of X-linked biallelically expressed genes {#Sec18} ----------------------------------------------------------- Expression levels of X-linked biallelically expressed genes (with rpkm ≥ 0.1) were taken from each cell of female embryos. Transcripts per million (tpm) sum and median were then calculated for each individual cell and grouped by developmental stage (8-cell, morula and blastocyst for each dataset). Differences between the groups were tested with non-paired Wilcoxon test. For the gene-wise analysis of female-to-male expression ratios, we calculated for each X-linked biallelically expressed gene in females the mean expression in female and male cells at each developmental stage ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{{\rm{X}}}$$\end{document}$ ~f~ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{{\rm{X}}}$$\end{document}$ ~m~, respectively). After excluding those with a mean expression \<5 rpkm, we calculated the female-to-male mean expression ratio ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{{\rm{X}}}$$\end{document}$ ~f~/$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{{\rm{X}}}$$\end{document}$ ~m~) for each gene in each developmental stage and then plotted the distribution. The distributions between stages were compared with paired Wilcoxon test. In order to avoid distortions of few very high or very low expressed genes, outliers were removed prior to plots and tests. Allelic frequency on human X chromosome and the *XIST* gene {#Sec19} ----------------------------------------------------------- Allelic frequencies in the X chromosome and the *XIST* gene were obtained from data generated by the 1000 Genomes Project phase 3 (<http://www.1000genomes.org/>)^[@CR34]^. We considered global allelic frequencies in females of all known SNPs on the *XIST* gene and those along the entire X chromosome excluding pseudoautosomal region. The differences were assessed using non-paired Wilcoxon test. X chromosome to autosomes expression ratio {#Sec20} ------------------------------------------ For each individual cell, median gene expressions (tpm) were calculated for all expressed autosomal and X-linked genes outside of pseudoautosomal region. X:A ratios were obtained for individual cells of each embryo by dividing the median tpm values of the X chromosome by those of the autosomes. For dataset-2^8^ non-paired Wilcoxon tests were used to assess differences between pre-EGA samples (oocytes, zygotes, 2-cell and 4-cell stage) and the following stages (8-cell, morula and blastocyst,) male and female hESCs, and female fibroblast cell line; and between male and female embryos at the 8-cell and blastocyst stages. Statistical tests {#Sec21} ----------------- All statistical tests were performed using R version 3.1.3 (http://www.R-project.org/)^[@CR35]^. Electronic supplementary material ================================= {#Sec22} Supplementary Material Supplementary Table S1 Supplementary Table S2 Supplementary Table S3 Supplementary Table S4 Maria D. Vibranovski and Lygia V. Pereira contributed equally to this work. **Electronic supplementary material** **Supplementary information** accompanies this paper at doi:10.1038/s41598-017-11044-z **Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (CEPID 13/08135-2; DR 2009/17481-6; PD 2015/03610-0; JP 2015/20844-4), Conselho Nacional de Desenvolvimento Científico e Tecnológico, Banco Nacional de Desenvolvimento Econômico e Social and Financiadora de Estudos e Projetos. We also would like to thank Drs Noboru Sakabe and Pedro Galante for all the bioinformatics assistance, and Dr Marcelo Nobrega for critical reading of the manuscript. J.C.M.M., conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; G.R.F., collection and assembly of data, data analysis and interpretation, final approval of manuscript; M.D.V., conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript; L.V.P., conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript. Competing Interests {#FPar1} =================== The authors declare that they have no competing interests.

Background {#Sec1} ========== *Typha* is a perennial monocotyledon plant genus that is widely distributed \[[@CR1]\] mainly in wetlands, marshlands and other aquatic habitats. *Typha domingensis* Pers*.* (commonly known as "cattail") is a species of this genus, with an annual growth cycle, that is widely distributed around the world \[[@CR1]\]. It is a warm temperate and pantropical species \[[@CR2]\]. This species is also often found in the Iberian Peninsula and the Balearic Islands \[[@CR3]\]. The reproduction of this species occurs by vegetative spread (rhizomes) and from seed \[[@CR4], [@CR5]\]. Seed production is very high due to a single female spadix that can produce more than 600,000 small single-seeded fruits \[[@CR1]\]. Although considered an invasive plant, cattail provides different raw materials (for weaving) and the rhizomes serve as food for humans and livestock. In recent years, cattail has been used in wastewater treatments and reclamation of industrial sites due to its great capacity to remove particle matter, nutrients and metals from eutrophic waters \[[@CR6]--[@CR8]\]. For wastewater treatment, this species has been used to form green filters that use different types of biological elements (plants and microorganisms). Among the numerous existing green-filter systems, the innovative systems specially designed for small urban agglomerations are called green floating filters (GFFs) \[[@CR9]\]. Different *Typha* species have been used in GFFs, but *T. domingensis* presents an advantage compared to other species: it has the capacity to produce more biomass in deeper water \[[@CR10], [@CR11]\] and to quickly form a floating and filtering plant mat that improves the purification of wastewater in a GFF \[[@CR12]\]. Different studies have demonstrated that managed cattail in constructed wetlands could provide beneficial ecosystem services \[[@CR6], [@CR7]\] and sustainable biomass for biofuel feedstocks \[[@CR13], [@CR14]\] and bioproducts \[[@CR15]\]. Seed germination is an important biological process in plants. The success or failure of introducing a population into a new local habitat is closely related to its seed germination ability. The dynamics of this process are complex and influenced by genetic changes and/or phenotypic variability in the different plant populations \[[@CR16], [@CR17]\]. An example of this complexity is the dormancy mechanism. It is an internal condition of seeds that impedes its germination under otherwise adequate hydric, thermal and gaseous conditions \[[@CR18], [@CR19]\]. This mechanism is very rare in seed crops but common in weed populations and is associated with weed dispersibility \[[@CR20]\]. Dormancy can be ended by one or more environmental factors, such as temperature, water potential, light, and soil pH. However, different dormancy behaviours are related to environmental factors during seed development \[[@CR19]\] and seeds can incorporate the effects of these factors over time \[[@CR21]\]. In the case of cattail, germination studies have focused on avoiding its propensity to invade natural ecosystems and cause negative impacts. Previous studies have demonstrated that environmental requirements, such as temperature, light, depth of water, salinity, pH, and O~2~ concentration could influence the seed germination of different species of *Typha* \[[@CR5], [@CR16], [@CR22]--[@CR24]\]. One of the main factors required for seed germination is the temperature \[[@CR25]\]. This factor has the greatest effect on dormancy, and on the germination rate in the case of seeds that do not exhibit dormancy \[[@CR26]\], and many studies have been implemented using constant temperatures \[[@CR17], [@CR27], [@CR28]\] or/and alternating temperatures \[[@CR29], [@CR30]\] on seed germination. Light is another environmental factor that is important for releasing seeds from dormancy \[[@CR25], [@CR31]\]. Among the environmental requirements related specifically to cattail seed germination, one of the main factors is humidity. Germination of cattail seeds only occurs in wet or flooded environments \[[@CR5], [@CR32], [@CR33]\] at low soil water potentials \< 1 MPa \[[@CR34]\]. Genetic determinants also influence the germination of cattail seeds \[[@CR35], [@CR36]\]. Sometimes, the origins of populations determine the germination conditions of the seeds regardless of whether they are of the same species \[[@CR24], [@CR37]\]. Moreover, the factors that determine the germination of the mother plant (temperature, light, humidity, and others) must be taken into account \[[@CR38]\]. Knowledge of the cattail populations germination capacity can help in the establishment in GFFs or the control of its expansion in different places \[[@CR39]\]. There are various tools for the study of seed germination. In recent years, population-based threshold models have been widely used in germination studies. There are two types of models: empirical and mechanistic. The first is used for a specific objective, but the results are more difficult to apply \[[@CR40], [@CR41]\]. Mechanistic models, on the other hand, are based on known and experimentally quantified the environmental effects on seed dormancy, imbibition, and germination \[[@CR41]\]. These models have been applied, therefore, to explain the most successful seed germination in recent years \[[@CR29], [@CR42], [@CR43]\]. The main models developed are the thermal time model, the hydrotime model, and the hydrothermal model, which describe the effects of temperature and/or water potential on the germination rate by applying a linear relationship \[[@CR42], [@CR44], [@CR45]\]. These models use biological time, which can be quantified by the extent to which water potential and/or temperature of each seed exceeds thresholds (base), below which germination is not completed \[[@CR20]\]. The hydrotime model describes the response germination of seed populations in response to change in water potentials. The thermal time model is based on response germination under variable temperature regimes, and the hydrothermal model is the integration of both temperature and water potential \[[@CR25], [@CR42], [@CR44]\]. The use of these models could provide a way to link ecological observation of germination to laboratory studies \[[@CR20]\]. The thermal time model allows for the estimation of the width of the thermal range over which seeds of a particular species can germinate. This thermal range can be described by the three cardinal temperatures: optimum temperature (T~o~), base temperature (T~b~), and ceiling temperature (T~c~) \[[@CR19]\]. T~o~ is the temperature at which germination is most rapid, while T~b~ and T~c~ are the lowest and highest temperatures at which germination can occur, respectively \[[@CR25]\]. Also, the dormancy status of the seeds can influence the thermal range between T~b~ and T~c~ \[[@CR25]\]. This model is based on mathematical parameters, such as thermal time (*θ*~*T*~, degree-day/degree-hour), the three cardinal temperatures (T~b~, T~o~ and T~c~, °C), the mean temperature of incubation (T~m~,°C), the germination rate (GR) and the time to germination (t) for a specific germination percentile (g). The *θ*~*T*~ is the daily differences between prevailing temperature and T~b~, which are accumulated to complete germination \[[@CR30]\]. GR is considered as the inverse of time to germination for specific germination percentages; moreover, it increases with increasing temperature between T~b~ and T~o~, while it decreases between T~o~ and T~c~ \[[@CR19]\]. The thermal time model has been used successfully to predict the occurrence of seed germination under non-water-limiting conditions, thus explaining approximately 80% of the variation in the cumulative percentile \[[@CR46]\]. The aims of this work, therefore, was to determine the ecophysiological parameters of seeds germination for different populations of *T. domingensis* from Badajoz (Ba), Cuenca (Cu), Madrid (Ma) and Seville (Se) using thermal time model. These parameters allow us to know the germination behaviour of each population and the influence of environmental parameters, such as level and amplitude of temperatures or light exposure, on its germination responses. This analysis can be useful to know which would be the best population for establishing in a new habitat as well as assessing its ability to expand as a weed in a future scenario with warmer temperatures in the Mediterranean zone because of climate change. Results {#Sec2} ======= Final germination responses in the different treatments {#Sec3} ------------------------------------------------------- Table [1](#Tab1){ref-type="table"} shows the germination responses achieved in each treatment. To simplify Table [1](#Tab1){ref-type="table"}, the germination responses to each set of temperatures and darkness treatments within the same population were summarized, and these are shown as lower and upper germination responses for constant (C) and alternating (A) thermal regimes. Table 1Final germination percentage in *T. domingensis* seedsPopulationDarkness treatmentsDT0dDT3dDT5dDT7dDT10dThermal regimenLowerUpperLowerUpperLowerUpperLowerUpperLowerUpperBaC91 ± 1.9 (17.5)99 ± 1.3 (25)59 ± 3.1 (17.5)99 ± 1.6 (30)65 ± 3.8 (17.5)94 ± 2.0 (22.5)69 ± 5.1 (17.5)92 ± 1.8 (25)58 ± 5.3 (17.5)79 ± 4.7 (22.5)A60 ± 4.5 (15/20)99 ± 4.1 (20/25)53 ± 5.2 (15/20)98 ± 1.7 (15/30)53 ± 4.5 (15/20)94 ± 2.6 (15/30)53 ± 6.1 (15/20)93 ± 4.4 (15/30)49 ± 7.6 (15/20)91 ± 4.1 (15/30)CuC92 ± 1.2 (17.5)99 ± 1.7 (22.5)87 ± 2.7 (17.5)99 ± 1.1 (22.5)81 ± 2.9 (17.5)98 ± 1.3 (22.5)74 ± 3.6 (17.5)99 ± 1.8 (22.5)67 ± 4.2 (17.5)94 ± 3.9 (22.5)A61 ± 5.5 (15/20)99 ± 3.2 (15/25)55 ± 6.9 (15/20)97 ± 4.6 (20/30)54 ± 7.7 (15/20)98 ± 1.3 (15/30)49 ± 6.2 (15/20)95 ± 2.7 (20/25)51 ± 7.1 (15/20)94 ± 1.5 (15/30)MaC89 ± 2.2 (17.5)99 ± 2.1 (25)72 ± 2.7 (17.5)98 ± 2.8 (30)73 ± 3.3 (17.5)96 ± 2.0 (25)65 ± 4.5 (17.5)97 ± 2.2 (30)48 ± 6.4 (17.5)86 ± 1.6 (25)A67 ± 6.2 (15/20)97 ± 3.1 (15/30)51 ± 4.3 (15/20)99 ± 2.1 (20/30)48 ± 6.7 (15/20)98 ± 3.6 (20/30)49 ± 7.9 (15/20)94 ± 3.4 (30)49 ± 8.4 (15/20)93 ± 3.4 (15/30)SeC86 ± 3.8 (17.5)99 ± 4.6 (22.5)92 ± 5.8 (17.5)97 ± 4.5 (22.5)75 ± 5.5 (17.5)92 ± 4.2 (22.5)66 ± 6.5 (17.5)91 ± 4.9 (30)53 ± 8.2 (17.5)83 ± 4.4 (22.5)A58 ± 4.5 (15/20)95 ± 3.0 (15/25)52 ± 6.8 (15/20)92 ± 4.3 (15/25)53 ± 6.6 (15/20)84 ± 5.44 (15/30)54 ± 7.2 (15/20)85 ± 5.2 (25/30)50 ± 6.3 (15/20)79 ± 4.9 (15/30)Validation data (To)C74 ± 4.9 (17.5)99 ± 3.1 (22.5)62 ± 8.6 (27.5)92 ± 6.5 (22.5)62 ± 3.9 (17.5)90 ± 4.2 (22.5)56 ± 6.5 (17.5)96 ± 4.9 (30)47 ± 8.2 (17.5)83 ± 4.4 (22.5)A62 ± 5.2 (15/20)96 ± 4.4 (15/25)54 ± 7.9 (15/20)96 ± 3.6 (15/25)49 ± 8.6 (15/20)97 ± 4.3 (15/30)51 ± 9.1 (15/20)97 ± 4.4 (25/30)39 ± 9.8 (15/20)95 ± 3.6 (15/30)Maximum and minimum values ± SD of final germination percentage achieved according to different populations, thermal regimes, and darkness treatments in cattail seeds. The temperature, at which these percentages were reached, is shown in parentheses on the bottom line The results of the multifactor analysis of variance carried out with the data mentioned above are shown in Table [2](#Tab2){ref-type="table"}. There were significant differences among the populations (Po), darkness treatments (DT) and thermal regimes (Tr) when each of these factors was analyzed separately at *p* \< 0.05; however, the interaction between the two factors (PoxDP, DPxTr, PoxTr) and among the three factors (PoxDPxTr) was not significant (Table [2](#Tab2){ref-type="table"}). Table 2Multifactor analysis of variance and multiple range tests for different populations, darkness treatments and thermal regimesMultifactorial ANOVAMultiple Ranges testFactorsFdFPonGR %DTNGR %TrnGR%Po322.66^\*\*\*^Ba6082.2^b^DT0d4891.4^a^17.5_02074.0^d^DT430.77^\*\*\*^Cu6088.2^a^DT3d4887.6^b^17.5_52054.0^e^Tr551.45^\*\*\*^Ma6085.9^a^DT5d4882.3^c^20.0_02083.0^bc^PoxDT120.08Se6076.6^c^DT7d4879.9^c^20.0_102082.0^bc^PoxTr90.68DT10d4875.1^d^22.5_02092.0^a^DTxTr120.7322.5_52089.0^ab^PoxDTxTr360.1522.5_152093.0^a^25.0_02092.0^a^25.0_102087.0^abc^27.5_02088.0^abc^27.5_52082.0^c^30.0_02088.0^abc^*Po* Populations; *DT* Darkness treatments; *Tr* Thermal Regimes (T~m~\_ΔT)Significant codes: \*\*\* 0.001, \*\* 0.01, \* 0.05. Different letters represent statistically significant differences between treatment of each population according to LSD test (p \< 0,05) No germination was obtained in DT20d treatment (20 days with 24 h darkness photoperiod), so the data from these treatments have not been included in the study. These results suggest that the dormancy of cattail seeds was not interrupted independently of thermal treatment or population. In others darkness treatments, different germination responses were reached according to the influence of the factors studied (Table [1](#Tab1){ref-type="table"}), and there was an inverse relationship (Cor. Coeff: − 0.99, p \< 0,05) between the number of dark days and the germination response within the same population and thermal regimes (Table [2](#Tab2){ref-type="table"}). This difference was observed between the DT0d versus DT10d. Regarding the origin of the seeds, there were no significant differences between **Cu** and **Ma**, but differences did exist between **Ba** and **Se**, and those two together were significantly different from **Cu** and **Ma** together. (Table [2](#Tab2){ref-type="table"}). **Cu** and **Ma** had the highest values, while **Se** had the lowest germination responses. Thermal model {#Sec4} ------------- The differences in the thermal regimes depended on the mean temperature (T~m~) of each regime. In Figs. [1](#Fig1){ref-type="fig"} and [2](#Fig2){ref-type="fig"}, the relationships are shown between GR~50~ and cattail seeds of distinct populations, thermal regimes and darkness treatments. The mean value of T~b~ was 16.4 ± 0.2 °C with a minimum of 16.1 °C and a maximum of 16.7 °C. This value explained why no germination occurred in the thermal treatments lower than 17.5 °C and the lowest values of the germination responses occurred in the thermal treatments closest to T~b~ (Table [2](#Tab2){ref-type="table"}). Fig. 1Relationship between GR ~(50)~ and T~m~ of cattail seeds from different populations with ΔT = 0 °C and different darkness treatmentsFig. 2Relationship between GR ~(50)~ and T~m~ of cattail seeds from different populations with ΔT ≥ 0 °C and different darkness treatments In Fig. [3](#Fig3){ref-type="fig"}, the relationships are shown between GR of different percentiles (30, 50 and 70%) and T~m~ of different populations with constant and alternating temperatures and treatments without 24 h dark photoperiod (DT0d). In thermal regimes within constant temperatures (Fig. [3](#Fig3){ref-type="fig"}a), both **Ma** and **Ba** had a T~o~ = 25 °C in the three percentiles of GR, while **Cu** and **Se** had a T~o~ = 22.5 °C. In the regimes with alternating temperatures (Fig. [3](#Fig3){ref-type="fig"}b), only **Ma** had a T~o~ = 25 °C, while the remaining populations had T~o=~22.5 °C. The T~o~ was 22.5 °C, but the optimum temperature difference (ΔT~o~) =2.5 °C was found in the **Ma** population with constant and alternating temperatures and the **Ba** population at constant temperatures (Fig. [3](#Fig3){ref-type="fig"}). Fig. 3Relationship between GR~(g)~ and T~m~ in treatments within constant and alternating temperatures and darkness treatment = DT0d The thermal regimes with T~m~ and the temperature difference (ΔT) =0 °C close to T~b~ achieved the lowest germination responses in all the treatments, while the thermal regimes at temperatures close to T~o~ showed the highest responses. The highest germination responses were observed in treatments with ΔT = 0 °C. The germination responses in the thermal regimes with the same T~m~ but with ΔT = 5 °C/10 °C were lower than ΔT = 0 °C, while the germination in thermal regimes with ΔT = 15 °C was much closer to the germination with ΔT = 0 °C (Table [2](#Tab2){ref-type="table"}). The values of T~o~ with different darkness periods were the same as those at DT0d (Table [3](#Tab3){ref-type="table"}). The highest values of GR~50~ (0.34) were reached within constant regimes close to T~o~ (22.5/25 °C) in all populations (Fig. [3](#Fig3){ref-type="fig"}a). In alternating regimes (Fig. [3](#Fig3){ref-type="fig"}b), these values are all approximately 0.23 in T~o~ = 25 °C. ANOVA of alternating temperatures indicated significant differences between thermal treatments (F ~(5,\ 61)~=9.17, p \< 0,001); therefore, the thermal treatments 15/30 °C and 20/25 °C showed different values of three percentiles of GR (30,50 and 70%). Although they had the same mean temperature (T~m~), this difference could be related to the different ranges utilized in alternating temperatures (15 and 5 °C, respectively). Table 3Parameters of the thermal models for cattail seeds with constant (A) and alternating (B) temperaturesA. Thermal regimes with constant temperaturesB. Thermal regimes with alternating temperaturesCodeT~b~\ °CT~o~\ °CLog(θ~T~(50))\ (log°d)ơθ~T~\ (log°d)θ~T~(50)\ (°d)θ~T~(50)\ (°h)CodeT~b~\ °CT~o~\ °CLog(θ~T~(50))\ (log°d)ơθT\ (log°d)θ~T~(50)\ (°d)θ~T~(50)\ (°h)BaCDT0d16 ± 0.3252.720.515.33368BaADT0d16 ± 0.222.52.810.316.61399BaCDT3d16 ± 0.3252.810.516.61399BaADT3d16 ± 0.222.52.940.318.02454BaCDT5d16 ± 0.3252.870.517.64423BaADT5d16 ± 0.222.53.000.320.09482BaCDT7d16 ± 0.3252.960.519.21461BaADT7d16 ± 0.222.53.060.321.33512BaCDT10d16 ± 0.3253.060.521.27510BaADT10d16 ± 0.222.53.120.322.65544CuCDT0d16 ± 0.222.52.410.411.13267CuADT0d16 ± 0.522.52.700.414.88357CuCDT3d16 ± 0.222.52.500.412.18292CuADT3d16 ± 0.522.52.730.415.45378CuCDT5d16 ± 0.222.52.630.413.87333CuADT5d16 ± 0.522.52.790.416.28391CuCDT7d16 ± 0.222.52.710.415.03361CuADT7d16 ± 0.522.52.870.417.58422CuCDT10d16 ± 0.222.52.840.417.11411CuADT10d16 ± 0.522.52.900.418.23438MaCDT0d16 ± 0.5252.570.513.06314MaADT0d16 ± 0.7252.760.415.80379MaCDT3d16 ± 0.5252.700.514.92358MaADT3d16 ± 0.7252.800.416.44395MaCDT5d16 ± 0.5252.800.516.44395MaADT5d16 ± 0.7252.910.418.30419MaCDT7d16 ± 0.5252.900.518.17436MaADT7d16 ± 0.7253.000.420.09482MaCDT10d16 ± 0.5253.010.520.29487MaADT10d16 ± 0.7253.030.420.78499SeCDT0d16 ± 0.122.52.750.415.70377SeADT0d16 ± 0.522.52.980.419.69473SeCDT3d16 ± 0.122.52.800.416.44395SeADT3d16 ± 0.522.53.020.420.49498SeCDT5d16 ± 0.122.52.890.417.99432SeADT5d16 ± 0.522.53.110.422.42538SeCDT7d16 ± 0.122.53.080.421.76522SeADT7d16 ± 0.522.53.210.424.78595SeCDT10d16 ± 0.122.53.160.423.57566SeADT10d16 ± 0.522.53.240.4.25.53613T~b~ = Base temperature, T~o~ = Optimum temperature; log(θ~T~(50)) = log thermal time 50% germination in °d; σθ~T~ = standard deviation of the log thermal time distribution within the seed population in °d; θ~T~(50)= thermal time 50% germination in °d and °h, respectively In Fig. [4](#Fig4){ref-type="fig"}, the relationship between accumulated germination and log(θ~T~ (50)) in the different thermal regimes (C and A), populations and darkness treatments are shown. The curves of the models within the same population were close but not equal, and slight differences were observed between different darkness treatments and between thermal regimes with constant versus alternating temperatures. The curves of the regimes with alternating temperatures shift to the right when compared with those of constant temperatures. There were similar values of ơθ~T~ (standard deviation of the log thermal time) in all treatments (Table [3](#Tab3){ref-type="table"}) which means that final germination (50%) was reached in all treatments. Fig. 4Relationship between accumulated germination and log(Ɵ~T~(g)) in different populations with temperatures and darkness treatments The treatments of the same population and darkness treatments in thermal regimes with constant temperatures showed lower values of thermal time (Table [3](#Tab3){ref-type="table"}) in comparison with regimes of alternating temperatures. The thermal time was influenced by the darkness treatments because, for all populations, and all temperature levels and temperature amplitudes, it was seen to increase the more days the seeds were kept in darkness(Cor. Coeff: − 0.99, p \< 0,05). According to these results, there was a relationship between log(θ~T~ (50)) and the set of darkness treatments (Table [4](#Tab4){ref-type="table"}) characterized by an R^2^ \> 0.90. Table 4Relationship between log(θ~T~(50)) and darkness treatments (DT) in treatments with the same population and amplitude of temperature regimesCodeAbR^2^BaC2.710.0340.993BaA2.830.0310.965CuC2.390.0440.986CuA2.690.0220.927MaC2.570.0450.996MaA2.750.0300.910SeC2.710.0450.908SeA2.960.0290.938Log(θ~T~(50)) = a + b\* (DT) There were also differences between θ~T~ (50) according to populations (Table [3](#Tab3){ref-type="table"}). The lowest θ~T~ (50) time (Table [3](#Tab3){ref-type="table"}) corresponded to **Cu,** which coincided with the lowest yearly maximum, mean and minimum ambient temperatures (Table [5](#Tab5){ref-type="table"}). **Ma** had θ~T~ (50) higher than that of **Cu** with ambient temperatures slightly higher than those in **Cu**, while **Se** and **Ba** had the highest θ~T~(g) (Table [3](#Tab3){ref-type="table"}) and the highest ambient temperatures (Table [5](#Tab5){ref-type="table"}). Table 5Codes, population name, geographic coordinates and temperatures of five locations where seeds were collectedCodePopulationLatitudeLongitude*MATo*~*ax*~\ *(°C)MATo*\ *(°C)MATo*~*in*~\ *(°C)***Ba**Puebla de Alcocer, Badajoz38°59′N5°15′W23.817.110.3**Cu**Olmedilla del campo, Cuenca40°03′N2°42′W19.313.16.9**Ma**Ciudad Universitaria, Madrid40°26′N3°44′W19.915.010.1**Se**Lantejuela, Seville37°21′N5°13′W25.419.213.0**To**Seseña, Toledo40°04′N3°37′W22.115.89.5MATo~ax~ (yearly mean maximum temperature); MATo (yearly mean temperature) and MATo~in~ (yearly mean minimum temperature). Historical data obtained from The State Meteorological Agency of Spain \[[@CR47]\] The results of the evaluation of the thermal time models are shown in Table [6](#Tab6){ref-type="table"}. The coefficient of determination (R^2^) was the concordance between Log(θ~T~(g)) of each model (expected values in each model) and Log(θ~T~(g))~**To**~ (observed values). Results of R^2^ varied depending on thermal regimes and darkness treatments. A good concordance was found in all treatments with R^2^ values greater than 0.77. The highest values of R^2^ (≥ 0.90) corresponded to treatments without 24 h dark photoperiod (DT0d) in all populations and thermal regimes except **Ma** population (0.87 and 0.85 respectively), which implied a strong coincidence between expected and obtained values. R^2^ values decreased in treatments with 24 h dark photoperiod (DT3d, DT5d, DT7d and DT10d). Table 6Evaluation of the thermal time models. Difference between log(*θ*~*T*~(g)) (expected values) versus log(*θ*~*T*~(g))~To~ (observed values)CODER^**2**^Std ErrCODER^**2**^Std ErrBaCDT0d0.910.13BaADT0d0.910.09BaCDT3d0.840.12BaADT3d0.860.10BaCDT5d0.810.13BaADT5d0.840.11BaCDT7d0.790.13BaADT7d0.830.11BaCDT10d0.800.22BaADT10d0.820.12CuCDT0d0.940.06CuADT0d0.900.14CuCDT3d0.820.11CuADT3d0.820.14CuCDT5d0.810.08CuADT5d0.810.13CuCDT7d0.770.11CuADT7d0.810.13CuCDPT10d0.770.13CuADPT10d0.800.13MaCDT0d0.870.16MaADT0d0.850.12MaCDT3d0.830.14MaADT3d0.820.12MaCDT5d0.810.18MaADT5d0.780.18MaCDT7d0.810.18MaADT7d0.800.27MaCDT10d0.810.15MaADT10d0.800.16SeCDT0d0.940.09SeADT0d0.910.10SeCDT3d0.830.05SeADT3d0.850.08SeCDT5d0.820.11SeADT5d0.790.23SeCDT7d0.780.18SeADT7d0.790.27SeCDT10d0.780.11SeADT10d0.780.23 Discussion {#Sec5} ========== The successful establishment of a plant species in a location is closely related to the rapidity of germination. Different genotype and/or environmental factors can affect this process by increasing or decreasing this rate. Amongst the environmental factors, light is one that does not prevent the germination of seeds, if it acts as a signal \[[@CR25]\] to cause a change in the germination rate and final germination \[[@CR31]\] and, therefore, in thermal time parameters. This factor is one of the main determinants of the accumulation of a persistent seed bank of numerous weeds in the soil \[[@CR48]\], and it is necessary for the germination of many species \[[@CR31]\] mainly of plants with small seeds \[[@CR31], [@CR48]\] because large seeds can emerge from a much greater depth than light can penetrate \[[@CR49]\]. Exposure time to light may be short, less than a minute, or long. Short exposure time is more commonly effective with small weed seeds, such as cattail seeds, than with large weeds \[[@CR31]\]. Light exposure influences the germination of different *Typha* species \[[@CR5], [@CR50]\]. In this work, no germination was obtained in DT20d treatments and a delay in the germination was observed in treatments with a 24 h dark photoperiod (DT3d to DT10d). This effect may be explained by the development of a secondary dormancy related to phytochrome activation/deactivation processes which occur through the stimulus of light on cattail seeds. Phytochromes are the principal mechanism triggering germination of *Typha* because they participate in breaking the dormancy \[[@CR22], [@CR25]\]. These pigments have two mutually photoconvertible forms: Pfr (considered the active form for seed germination) and Pr (considered the inactive form) \[[@CR25], [@CR49]\]. Pfr is established during the formation of the seed in the mother plant; however, this phytochrome form can reconvert to Pr in darkness \[[@CR18], [@CR31]\]. In these circumstances, the secondary dormancy does not break, and a period is needed to reconvert the phytochrome to its active form (Pfr) \[[@CR51], [@CR52]\]. This secondary dormancy can explain the results in darkness treatments. For example, in the darkness treatments (DT3d to DT10d), thermal time increases as the number of days in darkness increases (Table [4](#Tab4){ref-type="table"}). In the case of DT20d treatments, no germination was measured after 20 days in darkness. These cattail seeds, although they absorbed water and began to swell, did not break their coatings to allow germination. This may explain the death of every seed after 20 days in darkness or the delay produced by secondary dormancy. We support this second option but, since no subsequent germination data was collected, a further study would be necessary to determine it. Treatments of the same population had an increase in θ~T~ (50) as the number of days in darkness increased. There was a relationship between log(θ~T~ (50)) and darkness treatments (R^2^ \> 0.90) (Table [4](#Tab4){ref-type="table"}). Initially, a linear increase in thermal time was expected as the number of days in darkness increased. Indeed, there was an increase, but it was not proportional; for example, in the case of the population of **Cu** with constant temperatures, θ~T~ (50) at PT0d was 267 °h, and the value corresponding to DT10d was 411 °h. This means a 50% increase in thermal time, not a 100% increase as expected. This modification would indicate that *T. domingensis* seeds accumulate hours of temperature and that when receiving light, the dormancy is broken by the activation of Prf and the germination response occurs more quickly than expected. Darkness treatments, such as DT3d, DT5d, DT7d and DT10d, had lower seed germination than treatments without 24 h dark photoperiod (DT0d). These results indicate that long days of darkness may decrease the light sensitivity of *T. domingensis*. Dormancy broken in the presence of light and the influence of phytochrome has been studied and is common in small seeds, such as cattails \[[@CR22], [@CR52]\]. A buried environment is associated with darkness and cattail seeds do not germinate in darkness at any temperature; hence, buried seeds of *T. domingensis* could be a control method for the establishment in aquatic ecosystems. Darkness is also related to the depth of water \[[@CR25]\]; so the establishment of cattails in the GFF system may be successful if seeds are sowed above the soil submerged in water or on a floating structure of this system. Although water depth was not a factor in this study, it is another factor that is related to the amount of light and the ease of germination of cattail seeds. The depth used was enough to saturate the paper and seed (\< 0.4 cm) due to the fact that germination in cattail seeds is greater and faster in aquatic conditions \[[@CR2], [@CR24], [@CR33]\]. Some authors have stated that flooded areas increase the germination of *Typha* species, and this increase in germination has a direct relation to depth \[[@CR17], [@CR53]\]. This feature may be caused by a decrease in the level of oxygen, rather than by the lower intensity of light in these situations \[[@CR33]\]. Other studies, however, show no relationship between the germination rate and depth \[[@CR34], [@CR54]\]. The limit of the depth of germination in *Typha* species in clear water is around 40 cm \[[@CR2]\] or 1 cm in sediment \[[@CR55]\]. There is an extreme case where cattail seeds germinated under 80 cm water (and survived 8 weeks) \[[@CR56]\]. The germination response in plants of different origins could also be different \[[@CR49]\]. Differences related to the origin of a population are frequent in numerous species of plants, whether crops \[[@CR57]\] or weeds \[[@CR27], [@CR58]\]. Successfully colonizing a new location is related to the greater adaptive capacity of these populations to harsher environmental conditions compared to other populations \[[@CR59]\], thus allowing these populations to have greater flexibility and adaptability to different locations or future climate change scenarios \[[@CR17]\]. Cattail seeds were grouped into northern (**Cu** and **Ma**) and southern (**Ba** and **Se**) populations (Fig. [5](#Fig5){ref-type="fig"}). Mean temperatures of germination within each group were similar, but there were differences between the groups. The northern populations have lower values than the southern populations (Table [5](#Tab5){ref-type="table"}). The results of the thermal time study also show differences between northern and southern populations. In treatments with the same temperature and darkness periods, the northern population presented lower values of thermal time and a higher germination response than the southern populations (Tables [2](#Tab2){ref-type="table"} and [3](#Tab3){ref-type="table"}). Fig. 5Origin of the populations of *T. domingensis* used in this work. Puebla de Alcocer, Badajoz (**Ba**); Olmedilla del Campo, Cuenca (**Cu**); Lantejuela, Seville (**Se**), the macrophytes nursery of GA, Madrid (**Ma**) and Seseña (**To**). **To** population was used to validate the thermal time parameters These differences among populations are consistent with the results of other studies carried out with *Typha latifolia* L. in fifteen European populations \[[@CR17]\] and USA populations \[[@CR60]\]; in both studies, in comparison to northern populations, southern populations germinated at a lower temperature. However, in our study, the opposite scenario occurred. Before providing conclusions, some points concerning these studies must be clarified. For example, *T. domingensis* is a species more adapted to warmer areas compared to *T. latifolia*. In the European study, only two Mediterranean populations were used, and both populations germinated more rapidly than northern populations; the distances between the origins of the populations were greater than those in our study. Some authors mention that other factors, such as temperature or nutrient supply, are more important than the origin of the seeds in the case of neighbouring populations \[[@CR17]\]. In this study, the estimated mean T~b~ was 16.4 °C and no differences greater than 0.6 °C were observed regardless of origin, darkness treatments, or level or amplitude of temperatures. We could have considered that T~b~ was constant; however, other studies with crops \[[@CR46]\] or weeds \[[@CR61]\] estimated different T~b~ values for the different amplitudes of temperatures. There were significant differences in the germination responses both in terms of the level and amplitude of temperatures (Table [2](#Tab2){ref-type="table"}). In comparison to treatments with other T~m~, treatments with T~m~ close to T~b~ achieved a lower germination response in all treatments (Table [2](#Tab2){ref-type="table"}). No data were found for the calculated T~b~ for *Typha* species, but the estimated values of T~b~ for cattail seeds in this study were very similar to those obtained in other studies with summer weeds \[[@CR29], [@CR62]\]. Steinmaus (2000) established a relation between the slope of the line used to estimate T~b~ and germination rate; this rate will be greater with a higher slope. In our study, higher slopes occurred in **Cu** in thermal regimes with both constant and alternating temperatures and coincided with the lower θ~T~ (50) of all populations studied (Figs. [1](#Fig1){ref-type="fig"} and [2](#Fig2){ref-type="fig"}). Differences in T~o~ were obtained in the results of the multifactor analysis, mainly between the northern (**Cu** and **Ma**) and southern populations (**Ba** and **Se**) (Table [4](#Tab4){ref-type="table"}). This difference in T~o~ is comparable with the results of other studies with different populations of weeds or with *T. latifolia* \[[@CR17], [@CR22], [@CR23]\]. The T~o~ for the Swedish populations of *T. latifolia* was approximately 20 °C \[[@CR23]\] or 10/30 °C with alternating temperatures in Italian populations \[[@CR22]\]. Australian populations of the *Typha* genus germinate readily at high temperatures and decline when the T~m~ is lower than 20 °C \[[@CR63]\]. Table [7](#Tab7){ref-type="table"} shows the results from different studies of the seed germination of *T. latifolia* and *T. domingensis*. There are few studies on the seed germination of *T. domingensis*. Lorenzen et al. (2000) stated that a T~o~ of 30 °C and 25/10 °C occurred in south-eastern American populations of *T. domingensis* at constant and alternating temperatures, respectively. These T~o~ values are distinct from those obtained in our study (22.5--25 °C), but there are other studies with T~o~ values very similar to those obtained in this work (Table [7](#Tab7){ref-type="table"}). These results showed different T~o~ values according to the places of origin of the seed and were closely related to climatic conditions at each location \[[@CR17]\]. Some conditions, such as the temperature of the mother plants \[[@CR38], [@CR65]\], may determine the germination of populations, regardless of whether the seeds were of the same species \[[@CR24], [@CR37]\]. Table 7Optimal temperature in *T. domingensis* and *T. latifolia* in different populations from various studiesPlant speciesReferenceCASeed location*Typha domingensis*This study22.5; 25 °CSpainLorenzen et al. (2000) \[[@CR5]\]30 °C25/10 °CFlorida, U.S.Royal Botanic Gardens (2002) \[[@CR64]\]20 °CWakehurst, England*Typha latifolia*Sifton H.B (1959) \[[@CR50]\]30 °C20/30 °COntario, CanadaBonnewell, V. et al. (1983) \[[@CR33]\]35 °CMinnesota, U.S.Lombardi, T et al. (1997) \[[@CR22]\]20/30 °CPisa, ItalyEkstam and Forseby (1999) \[[@CR23]\]20 °CLinköping, SwedenHeinz, S (2011)25 °C10/25 °CGermanyMeng, H. et al. (2016) \[[@CR24]\]25/15 °CNortheast of China.*C* constant temperature. *A* alternating temperature In the *Typha* genus, temperature and amplitude were shown to be factors related to germination \[[@CR23]\]. The favourable effect of alternating temperatures on the germination response is well known in different weeds \[[@CR22]\] because the effect enables a seed to understand when it is buried and to inhibit germination. In nature, seeds of the cattail are usually submerged. In this situation, fluctuations in the ambient temperature are rare; therefore, an increase in this fluctuation could indicate that seeds have reached land and germination could be stimulated. In this study, both thermal factors (level and fluctuation in temperatures) influenced the final germination of cattail seeds. In the treatments within the same population and in the darkness treatment, there was a greater germination response as the temperature approached T~o~ from values close to T~b~, causing the existence of significant differences depending on the temperature level (Tables [1](#Tab1){ref-type="table"} and [2](#Tab2){ref-type="table"}). An increase in the germination response is obtained with higher temperatures up to T~o~; above this value, germination begins to decrease. The same results occur in other studies with *Typha* \[[@CR17], [@CR22], [@CR23], [@CR33]\] and weeds \[[@CR27], [@CR29]\]. The use of different amplitudes of temperature is related to the loss of dormancy in weeds \[[@CR29], [@CR66]\] or crops such as lentil \[[@CR30]\]. In the case of cattail seeds, the loss of dormancy is related to changes in germination responses. Treatments with ΔT = 0 °C and 15 °C had a higher germination response than those with ΔT = 5 °C and 10 °C (Table [1](#Tab1){ref-type="table"}), so these last two amplitudes of temperature negatively affect germination. However, in studies with *T. latifolia*, treatments with constant temperature regimes (ΔT = 0 °C) achieved a lower germination response than alternating regimes (ΔT \> 0 °C) \[[@CR17]\]. On the other hand, θ~T~ (50) corresponds to treatments of the same population, and ΔT = 0 °C is lower than treatments with ΔT ≥ 0 °C (Fig. [4](#Fig4){ref-type="fig"}), in contrast to *Solanum physalifolium* \[[@CR29]\] whose thermal time is considerably reduced in an alternating regime (Table [3](#Tab3){ref-type="table"}). These data are consistent with the germination rate (Fig. [3](#Fig3){ref-type="fig"}), in which treatments with alternating temperatures reach lower values than those corresponding to constant temperatures. According to these results, the best season to germinate *T. domingensis* would be late spring because these seasons have a temperature regime of approximately ΔT = 15 °C under natural conditions in the five locations where seeds were collected \[[@CR47]\]. The thermal time value of different populations of cattail seeds (Table [3](#Tab3){ref-type="table"}) was substantially lower than that of other weeds such as different species of *Solanum* \[[@CR20], [@CR22]\] or tropical species such as *Pennisetum typhoydes* \[[@CR45], [@CR67]\]. This indicates a rapid germination response compared with those of other plant species. There were also differences between populations, with **Cu** being the one with the lowest thermal time, both in ΔT = 0 and ΔT \> 0 treatments. Although **Cu** and **Ma** obtained similar germination values (Table [2](#Tab2){ref-type="table"}), θ~T~(50) was the highest in **Ma**. Therefore, **Cu** could be the population that presents the most vigour during this process because this population had the fastest germination under the conditions tested. The final germination percentages were very similar in all populations. It would be necessary to carry out new tests to determine whether the development in other stages of plant growth would also be fast in this population. In comparison to other species of the genus, such as *T. angustifolia*, *T. domingensis* is a plant species more adapted to warm temperatures. In Spain, it has been observed that *T. domingensis* has been colonizing places where *T. angustifolia* once stood \[[@CR3]\]. If this capacity occurs with an increase in temperatures due to climate change, then it is possible to consider that *T. domingensis* might increase its expansion to the detriment of other *Typha* species such as *T. angustifolia.* According to the evaluation of the models developed in this work (Table [6](#Tab6){ref-type="table"}), there were some differences between the results that were related to thermal regimes and darkness treatments. The greatest coincidences are in the models developed with constant and alternating thermal regimes and DT0d treatments with R^2^ mean values ≥0.9, except for the population of **Ma** that present values slightly below 0.9 in both thermal regimes. The best coincidence was between expected and observed values in constant regimes and treatments without 24 h dark photoperiod (DT0d). Darkness treatments affect the coincidence between expected and observed values, the R^2^ decreased to mean values between 0.84 to 0.78. In the DT3d treatments, R^2^ values were greater than the other darkness treatments. In the remaining darkness treatments (DT5d, DT7d and DT10d), R^2^ values were similar (Table [6](#Tab6){ref-type="table"}). **Ba** and **Ma** populations show both the most and least coincidental values in all treatments, respectively, but the differences were small among all populations. This work shows that environmental factors and the origin of populations affect the germination responses of cattail seed; moreover, how these parameters could be used to develop models that predicting seed behaviour in a new habitat. Conclusions {#Sec6} =========== The thermal time model for the different populations of *T. domingensis* allows an understanding of the germination response of each population established in a new habitat, such as a GFF system. The germination response of *T. domingensis* was affected by thermal regimes, darkness treatments, and populations. Among the different populations of *T. domingensis*, T~o~ in **Ma** and **Ba** were 25 °C, and those in **Cu** and **Se** were 22.5 °C. However, the T~b~ was the same in the four populations (mean 16.4 ± 0.2 °C). Therefore, the best population can tolerate a vast range of temperatures in a new habitat was **Cu** due to this population had the highest germination rates and the highest germination percentage. If growth chambers are used to proceed with the germination of *T. domingensis* seeds, then the most appropriate temperature treatment will be a constant temperature of 22.5 °C. Under natural conditions, the best time for seed germination occurs when there is a temperature regime of approximately 15 °C in the Mediterranean zone such as Spain, which mainly occurs in the late spring. Values of T~m~ near T~o~ and ΔT values approximately to 15 °C are common in the four populations; hence, these values show that *T. domingensis* could readily germinate in a new habitat and expand as a weed if an increase in the mean temperature occurs. Methods {#Sec7} ======= Plant material {#Sec8} -------------- The plant material used for this study was obtained and subsequently identified by experts of the Botany Unit of the Department of Agrarian Production (UPM). The Botanical key used was: Flora Iberica, Vol. XVIII, Gen. Typha \[[@CR3]\]. *T. domingensis* is a species widely distributed throughout Spain. For this reason, no specimens were taken to be included in any Herbarium. According to the International Union for Conservation of Nature and Natural Resources (IUCN) Red List Categories, *T. domingensis* does not qualify as critically endangered, endangered, vulnerable or near threatened \[[@CR68]\] so permissions were not necessary to collect samples. The seed material for this study came from natural *T. domingensis* stands at five different locations in Spain (Fig. [5](#Fig5){ref-type="fig"}). For the thermal study, mature spadices were collected from Puebla de Alcocer (**Ba**), Olmedilla del Campo (**Cu**), and Lantejuela (**Se**). The plants of these populations were located in naturally flooded areas (ponds, lagoons, and marshes) and in this study they are represented by **Ba**, **Cu,** and **Se,** for plant populations from Badajoz, Cuenca and Seville, respectively (Table [5](#Tab5){ref-type="table"}). **Ma** location was the fourth population. The seeds of this population were obtained from a macrophyte nursery in the experimental fields of GA, Madrid, whose initial source was the Manzanares River, which is very close to these facilities. The fifth population was collected in Seseña, Toledo (**To**). This population was used to evaluate the results of the thermal models obtained for the other populations. According to the classification of Köppen-Geiger, the five locations are classified as having temperate climates with dry and hot summers. The geographic coordinates and temperatures of the different locations are shown in Table [5](#Tab5){ref-type="table"} \[[@CR47]\]. Mature spadices of these populations were collected from plants grouped into the pure mass of cattails between late summer and early autumn of 2017. These spadices were obtained from various *T. domingensis* plants (7--12 plants per population) that grew in the same physical location. The seeds of each spadix were mixed. In the laboratory, the seeds were removed from these spadices by agitating the fruits in water. Only seeds settling to the bottom of the container were selected as viable seeds for the germination test. Then, the selected seeds were dried on filter paper and stored in a refrigerator (at 5 °C) until they were used in the germination test in the following year. Previous experiments had been carried out to verify that most of the seeds were viable. Shortly before the experiment, the seeds were treated with 1% sodium hypochlorite to prevent infection during the assay \[[@CR69]\], washed with sterile distilled water to eliminate any residue and dried rapidly at room temperature. Germination tests {#Sec9} ----------------- Germination tests were carried out in three identical growth chambers with two photoperiodic regimes: (i) 24 h darkness, the dishes were covered with aluminium foil and placed in black plastic bags to prevent the passage of light, and (ii) 12 h light /dark under continuous irradiation illuminated by 8 fluorescent sources of white light (Sylvania Grolux 35 W). PAR was measured in different positions of the growth chamber (PAR sensor LI-COR-DATALOGGER Inc.USA) at the beginning and the end of the experiments; the mean value was 85 ± 9 μmol m^− 2^ s^− 1^. Different darkness treatments and thermal regimes on five cattail seed populations (**Ba**, **Cu**, **Ma**, **Se** and **To**) were studied as factors that could alter the final germination and the result of the germination model. The relationship between thermal time, temperature and darkness for the cattail seeds was studied. For this reason, different darkness treatments (0, 3, 5, 7, 10 and 20 dark days) were included in this study. The longest number of days was the same (20 days) for the seeds, so the number of days with 12 h light /dark photoperiod was reduced successively (Table [8](#Tab8){ref-type="table"}). In the treatment DT20d, the seeds were incubated in total darkness for the entire time. Table 8Thermal regimes and darkness period treatments used in the experimentThermal regimes (°C)Darkness treatmentsTreatmentsT~m~ΔTTreatmentsDescriptionC 17.517.50DT0d20 days with 12 h light /darkC 20.0200DT3d3 days (24 h darkness) and 17 days (12 h light /dark)C 22.522.50DT5d5 days (24 h darkness) and 15 days (12 h light /dark)C 25.0250DT7d7 days (24 h darkness) and 13 days (12 h light /dark)C 27.527.50DT10d10 days (24 h darkness) and 20 days (12 h light /dark)C 30.0300DT20d20 days with 24 h darknessA 15/2017.55A 15/252010A 15/3022.515A 20/2522.55A 20/302510A 25/3027.55 Different thermal regimes were included in this study. These regimes include different levels of constant or alternating temperatures, as explained below. The constant temperatures used (C) were: 17.5, 20.0, 22.5, 25.0, 27.5 and 30.0 °C; the alternating temperatures used (A) were 15/20, 15/25, 15/30, 20/25, 20/30, and 25/30 °C. In the alternating regimes, higher temperatures coincide with light periods and lower temperatures with the dark period of each photoperiodic regime used, and the temperature difference (ΔT) between the dark and light periods was ΔT = 5, 10 and 15 °C (Table [8](#Tab8){ref-type="table"}). The mean value of both temperatures (T~m~) was used to calculate the models. Temperatures above 30 °C were not used because *T. domingensis* is a plant species whose germination season coincides with middle spring, so it would be very odd if the mean temperature above 30 °C was reached at that time in the study area. The treatment name was composed of the name of the population, mean temperature value, letter of the temperature regime C/A with a number that indicated the ΔT and darkness treatment, such as Ba25C0DT5d and Ba25A10DT3d. The experimental design was completely randomized. A total of 360 treatments were carried out and three replicates of 33 seeds each were used for each treatment. The germination test was conducted in a filter paper-lined Petri dish filled with 15 ml of distilled water. To prevent evaporation losses, the edges of the Petri dishes were closed using laboratory film. HOBO U12 (Onset Computer Corporation, Pocasset, MA, USA) data loggers were used to monitor the temperature inside the growth chambers. Data from the chamber were accepted if the temperature registered showed a difference of less than ±0.5 °C. All treatments were set up at 9:00 h. Germinated seeds were counted daily for 20 days. A seed was considered to have germinated when the coleoptile broke the pericarp \[[@CR22]\]. Thermal time model {#Sec10} ------------------ Seed germination of different populations of *T. domingensis* was described as a function of the thermal time model. According to the model presented by Garcia-Huidrobo et al. (1982), the parameter thermal time *θ*~*T*~ (degree-day/ degree-hour) for the percentile g is: $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\uptheta}_T(g)=\left(T\hbox{-} {T}_b\right){t}_g $$\end{document}$$ Another parameter defined in this model is the germination rate (GR~g~), which is the inverse of the time to radicle emergence of a specific percentile of the population defined by eq. [1](#Equ1){ref-type=""}. $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {GR}_g=\frac{T\hbox{-} {T}_b}{\theta_T(g)}=\frac{1}{t_g} $$\end{document}$$ There is a linear regression line between GR~g~ and T when the temperature is between T~b~ and T~o~. Under these circumstances, the slope of this linear regression is equal to the reciprocal of thermal time *θ*~*T*~(g). If the change in *θ*~*T*~(g) within a seed population is a log-normal distribution, then the relation between GR~g~ and *θ*~*T*~(g) can be described using the probit function \[[@CR46]\]. $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\mathrm{prob}}_{\mathrm{g}}=\left(\frac{1}{\sigma_{\uptheta_{\mathrm{T}}}}\right)\log \left(\mathrm{T}\hbox{-} {\mathrm{T}}_{\mathrm{b}}\right){\mathrm{t}}_{\mathrm{g}}\hbox{-} \frac{\log \left({\uptheta}_{\mathrm{T}}(50)\right)}{{}^{\sigma_{\uptheta_{\mathrm{T}}}}} $$\end{document}$$ In this function, prob~g~ is the probit transformation of the cumulative germination percentile g, *θ*~*T*~(50) is the thermal time to 50% germination, and σ*θ*~*T*~ is the standard deviation of *θ*~*T*~ for individual seeds in the population \[[@CR70]\]. Alternative use of probit transformation is logistic transformation when the sample size is not very large. In this case, the midpoint of the logit regression (logit = 0) is the same as that obtained with probit transformation, and the slope (α) is related to the standard deviation of the normal distribution (σ) as defined \[[@CR71]\] as: $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \upsigma =\frac{\uppi}{\sqrt{3}}\ast \frac{1}{\upalpha} $$\end{document}$$and the log *θ*~*T*~(50) is related to the intercept of the logit regression (β) \[[@CR27]\] as: $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \log \left({\uptheta}_{\mathrm{T}}(50)\right)=-\frac{\beta }{\alpha } $$\end{document}$$ Data from the different temperature regimes were normalized following the concept of thermal time basis (Covell et al., 1986) where *θ*~T~(50) is the mean thermal time to 50% germination used for the log thermal time distribution that was estimated from the equation: $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \theta {T}_{(50)}=\frac{\left({T}_m-{T}_b\right)}{GR_{50}} $$\end{document}$$ The different variables to solve this equation were obtained as follows: Equation 2 (1/t ~(50)~) was used to calculate GR~50~ for different treatments. In each treatment, the results of GR~50~ were used to calculate a linear regression whose x-intercept represented the estimated value of T~b~ \[[@CR46], [@CR61]\]. The T~o~ was obtained from the relationship between different percentiles of GR (30, 50 and 70%) and the T~m~ of each thermal regime (C or A) with the same darkness treatments where T~o~ was the point on the x-axis that coincided with the maximum GR~30~, GR~50,~ and GR~70~ of the above relationship. The estimated θ~T~(50) was used to obtain the σ of the log thermal time in the different treatments and using the logit model \[[@CR29]\], the median germination time was estimated using the values to logit = 0 as was mentioned above. Only GR% values less than 95% from T~b~ to T~o~ were included in the logit regression \[[@CR28]\] Validation of thermal time model {#Sec11} -------------------------------- The evaluation process is an important part of the design of a model because it allows checking the concordance between expected and observed results. The method of evaluation used in this work is based on the coefficient of determination (R^2^) and the root-mean-square error (RMSE). This method provides an estimation of the difference between log(*θ*~*T*~(g)) obtained in each model (expected values) and log(*θ*~*T*~(g))~To~ (observed values), where log(θT(g)~To~ was calculated applying thermal parameters of each model (Table [4](#Tab4){ref-type="table"}) to data germination of **To** population. Statistical analyses {#Sec12} -------------------- The multifactor analysis of variance with final germination response as a percentage was carried out with the software package Statgraphics Centurion XVI (Starpoint Technologies, 2011) to determine the relationships among origin, thermal conditions and darkness period of cattail seeds. Germination responses were transformed to meet the assumption of the ANOVA. In this case, the transformation used was arcsine (√final germination %). A multiple range test was also performed to determine which variables were significantly different from the others. The method to make the comparisons was LSD (Least Significance Difference). Statistical differences were defined as *p* \< 0.05. Availability of data and materials {#Sec13} ---------------------------------- All data generated or analysed and its [supplementary information files](#MOESM1){ref-type="media"} during this study are included in this published article. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Supplementary information ========================= {#Sec14} **Additional file 1.** ΔT : The temperature difference *θ*~*T*~(g) : Thermal time σ*θ*~*T*~ : Standard deviation of *θ*~*T*~ A : Alternating ANOVA : Analysis of variance Ba : Badajoz C : Constante Cu : Cuenca DT : Darkness treatments g : the percentile of germination GA : Agroenergy Group GFF : Green floating filter GR : Germination rate IUCN : International Union for Conservation of Nature and Natural Resources LD : light/dark LSD : Least Significance Difference Ma : Madrid PAR : Photosynthetic Active Radiation prob~g~ : Probit transformation Se : Seville T~b~ : Base temperature T~m~ : Mean temperature t(~g~) : time of germination T~o~ : Optimum temperature To : Toledo USA : United States of America **Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information ========================= **Supplementary information** accompanies this paper at 10.1186/s12870-020-02573-3. FMC is supported by a grant from the National Program of Scholarships and Educational Loan (PRONABEC) -- Ministry of Education Peru. FMC performed and analyzed the seed germination test. PLA interpreted and proceeded with the data analyzed. MCM and MTC review and improved the thermal time model. FMC and PLA conceived the study, planned experiments, and draft the manuscript. All authors read and approved the final manuscript. Not applicable. All data generated or analyzed and its supplementary information files during this study are included in this published article. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Not applicable. Not applicable. The authors declare that they have no competing interests.

Introduction {#s1} ============ Hexachlorocyclohexane (HCH), a highly recalcitrant organochlorine pesticide (OCP), has been widely used in agriculture, public health, and ectoparasite controls since 1940 s [@pone.0051043-Li1]. Technical HCH production between 1945 and 1992 was estimated to be 1400 kt, with approximately 400 kt of that were contributed by US producers [@pone.0051043-Barrie1]. Although HCH usage has been banned for decades, the production of HCH is still allowed in the EU (Stockholm Convention in 2004). Besides, a large amount is still used in developing countries because of its low cost [@pone.0051043-Feng1]. Therefore, the residues of HCH may still be present in soil and other environmental matrices, and may accumulate in food crops and pose potential health risk to humans [@pone.0051043-Babu1]. The common characteristic of OCPs is the persistence in the environment and the bioconcentration in organism lipid compartments [@pone.0051043-Waliszewski1]. For example, HCH residues in a contaminated point-source in German were analyzed by Ricking and Schwarzbauer [@pone.0051043-Ricking1] and they found that groundwater and a riverine sediment core was heavily affected by the HCH and its degradation products in the same industrial point source. Because of the high bioconcentration factor and its longer half-life, HCH had been detected in human milk from different countries [@pone.0051043-Willett1]. Technical grade HCH is a mixture of eight isomers \[α (contains a pair of enantiomers), β, γ, δ, ε, η, θ\], the toxic effect caused by HCH isomers is mainly focused on animals, despite that plants are providing vital ecosystem service. For instance, researchers have reported the central nervous system, reproductive and endocrine damage induced by HCH isomers in rats [@pone.0051043-Pomes1]--[@pone.0051043-Bigsby1]. Besides, comparing with other HCH isomers, β-isomer may currently be the most toxicologically significant HCH due to the reports of its estrogenic effects in mammalian cells, laboratory mammals, and fish [@pone.0051043-Willett1]. Growing vegetation can absorb OCPs through their roots or leaves [@pone.0051043-Paterson1], so vegetation has been used in pesticide monitoring [@pone.0051043-Safe1], [@pone.0051043-MahugijaMarco1] on the one hand, and on the other hand they has also been used to restore the contaminated sites [@pone.0051043-Rugh1], [@pone.0051043-VanAken1]. In fact, plants, as the sink for lipophilic pollutants due to their large surface area and associated lipids in plants [@pone.0051043-Simonich1], are also seriously affected with deleterious pollutants such as HCH. However, to our knowledge, although the residues difference of HCH isomers in different parts of plants was determined [@pone.0051043-Waliszewski1], [@pone.0051043-Pereira1], the phytotoxicity and toxic difference of HCH isomers in high plant is seldom considered. For the purpose of giving a comprehensive risk assessment on OCPs, studies of vegetation contamination caused by OCPs such as HCH and the mechanism of phytotoxicity are expected. In the previous studies, the evaluation of phytotoxicity induced by organic chemicals focused on the basic physical indexes such as plant biomass, dry matter, and the rate of germination [@pone.0051043-Wieczoreka1], [@pone.0051043-Pereira2]. Nevertheless, little was known about the phytotoxicity in plant photosynthetic and antioxidant defense systems. Although the changes of plant photosynthesis induced by OCPs were hardly referred to, many articles have reported that photosynthesis could be influenced dramatically when plants are subjected to a variety of adverse environmental conditions, such as chilling, desiccation and drought [@pone.0051043-Golding1]--[@pone.0051043-Huang1]. Meanwhile, excess generation of reactive oxygen species (ROS) can also be induced by those abiotic stresses; pesticide is also one of the adverse environmental factors that can induce oxidative stress [@pone.0051043-Bowler1], [@pone.0051043-RomeroPuertas1]. Considering ROS excess accumulation can lead to plant cell death, plant has evolved a whole antioxidant defense systems that can be divided into two categories: one that reacts with ROS and keeps them at low levels (peroxidase, superoxide dismutase and catalase), and one that regenerates the oxidized antioxidants (ascorbate peroxidase and glutathione reductase) [@pone.0051043-Smirnoff1]. Moreover, plant photosynthetic system and antioxidant defense system not only act independently of each other but also link intimately to perform signal function of ROS and response to adverse conditions. First, the sources of ROS are intimately correlated with photosystems in plants. It is generally accepted that PSI is the major site of superoxide generation in the photosynthetic electron transport chain, while superoxide (one kind of ROS) production by autoxidation of PSII components has been discussed. If superoxide forms in a short time and the antioxidant enzymes are unable to keep pace, superoxide as a signal molecule can trigger programmed cell death (PCD) in plants [@pone.0051043-Foyer1]. In addition, given that ROS scavenge by a number of enzymatic processes demands high energy, e. g. ascorbate peroxidase (APX) can detoxify H~2~O~2~ in the participation of NADPH [@pone.0051043-Asada1]. Ott et al. [@pone.0051043-Ott1] suggested that ROS would also be avoided in the first place by regulating photosynthetic electron transport. In this study, the stereoselective toxicity of four HCH isomers absorbed from roots of *Arabidopsis thaliana* was studied. *Arabidopsis thaliana* is a common model plant widely used in studying plant physiology and toxicology. The changes of phenotype and substructure in *A. thaliana* treated by HCH isomers were observed. Parameters of photosynthetic system and enzymatic antioxidant defense system were first involved to investigate the stereoselective toxicity and oxidative stress when induced by HCH in model plant *A. thaliana.* Results {#s2} ======= The Effects of HCH Isomers on Morphology of *Arabidopsis thaliana* {#s2a} ------------------------------------------------------------------ As compared to controls, after treated by HCH isomers for four weeks, the pesticide affected the root dramatically, accessory roots numbers and the root length decreased. The root length of all isomers treated plants were inhibited obviously, and the order from long to short was con \>β\>α\>γ\>δ. At the macroscopic level, the leaf shape and size were not affected dramatically except for δ-HCH treatment, of which the leaves turned yellow. The fresh weight of the α-, β-, γ-HCH treatments were not significantly different when compared to controls, however, the fresh weight was dramatically decreased after four weeks exposure to δ-HCH isomer **(** [**Figure 1**](#pone-0051043-g001){ref-type="fig"} **,** [**Table 1**](#pone-0051043-t001){ref-type="table"} **)**. Besides, the differences of root length and fresh weight among the four HCH isomers were also analyzed especially and shown in [Table 1](#pone-0051043-t001){ref-type="table"}. {#pone-0051043-g001} 10.1371/journal.pone.0051043.t001 ###### Changes of root length and fresh weight in *Arabidopsis thaliana* exposed to α-, β-, γ- and δ-HCH for four weeks. {#pone-0051043-t001-1} con α β γ δ -------------------------------- ------------------------------------------- ---------------- ---------------- ---------------- ----------------------------------------------- **Root length (cm)** 10.2±1.0[\#](#nt101){ref-type="table-fn"} 7.5±0.5^\*\*a^ 9.7±0.8^\*\*b^ 6.5±0.4^\*\*c^ 1.8±0.6^\*\*d^ **Fresh weight (mg)** 26.0±3.2 25.0±3.6^a^ 26.1±0.7^a^ 30.3±2.9^a^ 14.6±2.5[\*](#nt102){ref-type="table-fn"} ^b^ **Root length inhibition (%)** -- 25.9 4.7 36.0 82.8 mean value ± standard deviation; or \*\* indicate the values were significantly different as compared with controls (*p*\<0.05 or *p*\<0.01, respectively); Different letters show significant differences between values of the four HCH isomers (*p* = 0.05). Photos of Transmission Electron Microscopy of *A. thaliana* Leaves {#s2b} ------------------------------------------------------------------ As shown in [**Figure 2**](#pone-0051043-g002){ref-type="fig"}, the substructure of *A. thaliana* leaves was employed to acquire the structure changes of chloroplast (cp) in where the photosynthesis takes place. Mesophyll cell was infected seriously by HCH ([**Figure 2A**](#pone-0051043-g002){ref-type="fig"}), the shape was not longer rounded or oval but irregular, especially for δ-HCH treatment. The number of chloroplasts in each cell was decreased. After exposing to HCH isomers, see [**Figure 2B**](#pone-0051043-g002){ref-type="fig"}, the morphology of chloroplast was also altered obviously. Expansion could be observed in β-, γ- and δ-HCH treatments, and the size of chloroplast was hardly affected in α-HCH treatment. The number and size of starch granules (sg) was increased in HCH treatments as compared with controls, δ-HCH treatment was the most affected one among the four isomers. For the structure changes of thylakoid, as shown in [**Figure 2C**](#pone-0051043-g002){ref-type="fig"}, the appressed regions of granal thylakoid (gt) were thinner since the extrusion by swelled starch granules and the stromal thylakoid links between the granal thylakoids were vague in HCH treatments. Furthermore, α-HCH treatment was the least affected one among the four isomers again. {#pone-0051043-g002} The Effects of HCH Isomers on Photosynthetic System of *Arabidopsis thaliana* {#s2c} ----------------------------------------------------------------------------- In *A. thaliana*, the effective quantum yield was inhibited in both photosystem I and photosystem II ([**Figure 3**](#pone-0051043-g003){ref-type="fig"}). Compared to controls, statistical significance was shown almost in every treatment except for α-HCH treatment in photosystem I. The dramatic difference among the four isomers could also be observed and the highest and the least inhibition rate came from δ-HCH and α-HCH treatment, respectively. ![The effective quantum yield of photosystems \[Y (I) and Y (II)\] of *Arabidopsis thaliana* treated by HCH isomers.\ \* and \*\* depict a statistically significant difference as compared to controls (*p*\<0.05 or 0.01 respectively, ANOVA's test). Different letters above the columns of HCH isomer treatments show statistically significant differences between them (*p* = 0.05).](pone.0051043.g003){#pone-0051043-g003} There were similar trend between photosynthetic electron transport rates (ETR) and the effective quantum yield of photosystems in *A. thaliana.* The inhibition effect of HCH isomers on ETR from serious to small was δ\>γ\>β\>α in turn (See [**Figure 4**](#pone-0051043-g004){ref-type="fig"}). As shown in [**Figure 5**](#pone-0051043-g005){ref-type="fig"}, non-photochemical quantum yield of PSI caused by donor-side limitation \[Y (ND)\] was dramatically stimulated in all HCH treatments, especially in γ- and δ-HCH treatments. However, Non-photochemical quantum yield of PSI caused by acceptor-side limitation \[Y (NA)\] was inhibited in α-HCH treatment but stimulated in δ-HCH treatment. Quantum yields of non-light-induced non-photochemical fluorescence quenching \[Y (NO)\] were inhibited only in α- and β-HCH treatments, and Quantum yields of light-induced non-photochemical fluorescence quenching \[Y (NPQ)\] were stimulated in γ- and δ-HCH treatments. Moreover, the significant differences of all the photosynthetic parameters between the treatments of HCH isomers were also analyzed ([Figure **3**](#pone-0051043-g003){ref-type="fig"}-[**Figure 5**](#pone-0051043-g005){ref-type="fig"}). ![Changes of photosynthetic electron transport rates \[ETR (I) and ETR (II)\] in *Arabidopsis thaliana* treated by HCH isomers.\ Different letters above the columns of HCH isomer treatments show statistically significant differences between them (*p* = 0.05).](pone.0051043.g004){#pone-0051043-g004} {#pone-0051043-g005} Activities of Anti-oxidative Enzymes and Malondialdehyde Content in *A. thaliana* Exposed to HCH Isomers {#s2d} -------------------------------------------------------------------------------------------------------- The activity of the anti-oxidative enzyme was shown in [**Figure 6A--6C**](#pone-0051043-g006){ref-type="fig"}. Compared to controls, CAT activity was inhibited in the first two weeks after treatment with α-, β-, or γ-HCH treatments and was stimulated later on. However, the activity was always dramatically stimulated by δ-HCH. For the activity of SOD, inhibition was obviously observed in all HCH treatments at three weeks; however, after exposing for four weeks, the significant difference was only shown in δ-HCH treatment comparing with controls. The activity of POD was same as the CAT that a phenomenon of inhibition first and then stimulation was presented. POD activity was the most dramatically stimulated by δ-HCH and the least by α-HCH. The MDA level affected by the four HCH isomers was also investigated at different time. MDA content increased in all HCH treatments at different exposure time when compared to controls. Within the four isomers, δ-HCH treatment was also the most affected one and the β-HCH was the least affected one ([**Figure 6D**](#pone-0051043-g006){ref-type="fig"}). Similarly, the significant differences of all the anti-oxidative enzymes and MDA between the treatments of HCH isomers were specially analyzed ([**Figure 6**](#pone-0051043-g006){ref-type="fig"}). {#pone-0051043-g006} Discussion {#s3} ========== Recently, phytotoxicity of HCH on nine plant species was conducted by Pereira et al. [@pone.0051043-Pereira2]. All of the species displayed signs under stress in response to the presence of HCH, although to different degrees. However, the reports about the toxic differences of HCH isomers in plant were rare despite there are lots of reports on stereospecific toxicity of HCH isomers in other organisms. For instance, Srivastavaa and Shivanandappa [@pone.0051043-Srivastava1] investigated the toxicity of HCH isomers and its mechanism in Ehrlich Ascites tumor (EAT) cells. Their studies show differential cytotoxicity of α, β, γ, δ-HCH isomers, δ isomer being the most toxic and β the least. Nagata et al. [@pone.0051043-Nagata1] reported the differential effects of α, β, γ, and δ-HCH isomers on the GABA-induced chloride currents in three combinations subunits of GABA~A~ receptor expressed in human embryonic kidney cells. They found that differential actions of HCH isomers might produce variable effects on different regions of nervous systems and in different species of animals. Willett et al. [@pone.0051043-Willett1] summarized the different toxicity of HCH isomers on animals: α-HCH resulted in the highest incidence of hepatic nodules and hepatocellular carcinomas in male mice orally exposed; γ-HCH has the lowest oral cancer potency factor as compared to other isomers in mice; Of the four isomers, β-HCH has the greatest physical and metabolic stability that reflected in the environmental and biological persistence of this isomer [@pone.0051043-Bidlan1], [@pone.0051043-Kumar1]. Moreover, β-isomer could produce estrogen-like effects through non-classical estrogen-dependent mechanisms of action and therefore had the most important potential risks. After observing the phenotype and substructure of *Arabidopsis thaliana* exposed to HCH isomers, we concluded that the stereoselective toxic difference of HCH isomers was obvious, δ-HCH was the most toxic one while α-HCH the least. Therefore, the stereoselective toxic difference and action mode of HCH isomers in different organisms or even in different systems of same species was disparate. In our experiment, photosynthetic system and antioxidant defense system were first chosen to investigate the plant responses to HCH isomers. Photosynthesis is the basic course of organic chemicals synthesis by which plants and algae using sun energy. When plant treated with HCH, its photosynthetic system was down-regulated inevitably as we had discovered in results. In fact, although HCH toxicity mechanism on plants is unknown, over half the herbicides in current use act primarily on the light reaction of photosynthesis [@pone.0051043-Delorenzo1]. Oxidative stress is the situation by which many kinds of ROS were induced and the plant cells were damaged or killed because of the excessive accumulation of ROS. The antioxidant enzymes in plants would response to oxidative stress by up-regulating or down-regulating their activities. Pesticides such as paraquat could be one of the abiotic stresses that induced oxidative stress [@pone.0051043-Bchanan1]. Both photosystems were affected dramatically by the HCH isomers in our study when we carried out our experiments on these two systems separately. Besides, as the main photosynthetic organelle, oxygen content of chloroplasts is higher, and the roles of ROS in plant responses to stress were undoubtedly focused on photosynthetic tissues such as cells with chloroplasts [@pone.0051043-Asada2]. Moreover, as researchers have reviewed, photosynthetic cells indisputably have several unique ROS producing pathways and a multiplicity of ROS metabolizing systems under stress conditions [@pone.0051043-Foyer1]. And antioxidant enzymes in plants can scavenge the excessive accumulation of ROS that threaten the normal cell, therefore the antioxidant defense and photosynthetic systems are tightly interacted with each other when plant responses to stress. There are a number of useful photosynthetic parameters that can be derived from saturation pulse-induced fluorescence analysis. The dual-PAM-100 measuring system has been used as an effective tool in determining these parameters that showed the effects of environmental stress on the plant photosynthesis [@pone.0051043-Gao1], [@pone.0051043-Huang1]. In this study, the effective quantum yield \[Y (I) and Y (II)\]) and photosynthetic electron transport rates \[ETR (I) and ETR (II)\] of PSI and PSII in *Arabidopsis thaliana* were profoundly decreased in all HCHs treatments especially in γ- and δ-HCH treatments. The energy absorbed by PSII is divided between the fraction used in photochemistry and that lost non-photochemically [@pone.0051043-Kramer1]. An increase in non-photochemical fluorescence quenching (NPQ) played as a protective process when Y (II) was decreased. [**Figure 5B**](#pone-0051043-g005){ref-type="fig"} depicted the changes of Y (NPQ) and Y (NO) that were protective indicator and injury index respectively in PSII. The increase of Y (NPQ) in γ- and δ-HCH treatments indicated that plant treated with HCHs could use its regulation mechanism such as a dissipation of energy in heat form to protect its photosynthetic system [@pone.0051043-Golding1]. Similarly, Y (ND) is an important protective indicator in PSI when plants face to high light induced by environmental stress, if the value of Y (ND) is high, it shows that plant is suffered from excess light for one thing, and for another plant can protect itself by increasing heat dissipation. Because Calvin-Benson cycle is blocked, the electron in acceptor-side of PSI is accumulated. Then it leads to the increase of Y (NA), which is the mark of light injury [@pone.0051043-Kim1], [@pone.0051043-Niewiadomska1]. In current experiment, Y (ND) was higher in all the HCH treatments; however, the remarkable injury was only turned on in δ-HCH treatment, as shown in [**Figure 5A**](#pone-0051043-g005){ref-type="fig"}. Besides, some of the reactive oxygen species (ROS) such as triplet oxygen and superoxide generate in photosynthetic course [@pone.0051043-Foyer1]. Ott et al. [@pone.0051043-Ott1] observed a decrease of electron transport rate between PSII and PSI and suggested that this regulation functions could limit superoxide formation under stress conditions. This is good news for plant how to avoid ROS toxicity but also restrict the efficiency of photosynthetic system as shown in [**Figure 3**](#pone-0051043-g003){ref-type="fig"}. Growing evidence suggests a model for redox homeostasis in which antioxidant interaction acts as a metabolic interface for signals derived from metabolism and from the environment [@pone.0051043-Foyer2]. However, excessive ROS accumulation was harmful to plants [@pone.0051043-delRo1]. Oxidative stress is a state of imbalance between generation of ROS like hydroxyl and superoxide radicals and the level of antioxidant defense system [@pone.0051043-Anilakumar1]. Since plant could not totally control the production and accumulation of ROS by electron transport course, oxidative stress occurred. The changes of activity of antioxidant enzymes and the increase of lipid peroxidation in our study confirmed that *Arabidopsis thaliana* plant, when exposed to HCHs isomers tried to eliminate the ROS by their enzymatic detoxification systems. Although little is known about the changes of antioxidant enzymes activity induced by HCHs in plants, there are also many reports about oxidative stress induced by HCHs in other organisms. For example, HCH induced oxidative stress in Ehrlich Ascites tumor cells was studied by Srivastava and Shivanandappa [@pone.0051043-Srivastava2], their results could be characterized by glutathione depletion, lipid peroxidation (LPO), reactive oxygen species (ROS) production and inhibition of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). Moreover, effect of repeated oral administration of HCH on antioxidant defense system and lipid peroxidation in the rat testis was conducted by Samanta et al. [@pone.0051043-Samanta1], they also found that the pesticide elicited a significant decrease in the activities of cytosolic SOD and CAT. Although the use of HCH has been inhibited, considering the long retention time of HCHs in environmental matrices and plant bioconcentration of HCHs [@pone.0051043-Lu1], the pollution by HCHs in some special places was arresting, e. g. the surrounding areas of facilities that once used for HCH production. The environmental pollution by HCHs of high concentration [@pone.0051043-Ricking1] indicated that large residue of HCH isomers in special area was possible and deserved more concern. Plants, as one important part of ecosystem, an integral phytotoxicity study and residues detection are essential for understanding the global cycle and fate of OCPs, as well for assessing the risk of transfer to the trophic chain and for the development of phytoremediation techniques. Moreover, if we try to use vegetation to monitor or restore the environmental matrices contaminated by OCPs, more knowledge should be known, such as the tolerances of vegetation, transference and the metabolism of these pesticides after being absorbed by plant. Materials and Methods {#s4} ===================== Regents and Plant Materials {#s4a} --------------------------- The HCH isomers were purchased from Iprochem Co., Ltd (Shenzhen, Guangdong Province, China). *Arabidopsis thaliana* (ecotype Columbia \[Col\]) seeds were kindly provided by Prof. Jirong Wang (National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences). All the organic reagents used in this experiment were analytically pure. Cultivation and Phenotype Analysis of *A. thaliana* {#s4b} --------------------------------------------------- The *A. thaliana* seeds were sterilized with hypochlorite (4%) for 15 min first and with 75% ethanol for 1 min, repeated three times. After that, seeds were washed several times with distilled water and were vernalized at 4°C for 2--3 d. The MS medium was sterilized at 121°C for 20 min and 5 mg/L of α-, β-, γ- and δ-HCH was separately added in different conical beakers. The medium with different treatments was sufficiently blended and was removed into a 24-orifice plate respectively. All of the operations above were executed when the medium was still liquid. When the medium solidified, the vernalized seeds were sowed on. The plants used to taken morphological photos were sowed in vertical plates and all plates were sealed to avoid contamination. The plates were put into a constant temperature (25±0.5°C) culture room, equipped with cool-white fluorescence lights of 300 µmol/m^2^/s fluorescence intensity and a 12 h light/12 h dark cycle. After four weeks exposure, the plants were harvested for taking photos, meanwhile, the root length and fresh weight of the plants were measured. Substructure Detection by Transmission Electron Microscopy (TEM) {#s4c} ---------------------------------------------------------------- Leaf samples of controls and HCH-treated plantlets were sliced into crumbs of about 1 cm^2^square and fixed for over 2 h in cacodylate buffer solution containing 2.5% glutaraldehyde. Samples were then treated with 1.0% OsO~4~ for 1.5 h and dehydratedin acetone several times. After that, samples were embedded in epoxy resin. Ultra-thin sections (70--90 nm) were obtained using a Reichert Ultracutsultramicrotome, stained with uranyl acetate then by lead citrate. Finally, the samples of leaves were observed with a JEM-1230microscope (JEOL Ltd., Tokyo, Japan). Determination of Photosynthetic System Parameters in *Arabidopsis thaliana* {#s4d} --------------------------------------------------------------------------- The photosynthetic system parameters in *Arabidopsis thaliana* Chloro fluorescence of PSII and PSI were measured concomitantly by using a Dual-PAM-100 fluorometer (Walz) connected to a computer. The automated induction and recovery curve routine in the Dual-PAM soft ware was used, with repetitive application of saturation pulses for the assessment of fluorescence and P700 parameters from which the quantum yields of PSI and PSII were derived by the software [@pone.0051043-Klughammer1], [@pone.0051043-Schreiber1]. Y (NO), Y (NPQ), Y (ND) and Y (NA) were calculated automatically by Dual-PAM soft ware and saved in a report file [@pone.0051043-Pfndel1]. The ETR (I) and ETR (II) were also calculated by Dual-PAM software. Antioxidation Enzyme Extraction and Analysis {#s4e} -------------------------------------------- *A. thaliana* plantlets were grounded using a mortar with 2 ml PBS buffer (pH 7.4) on an ice bath. Every treatment was done with four replicates. After centrifuging at 2500 r/min for 10 min, the supernatant was collected to assay the antioxidant enzyme activity and malondialdehyde (MDA) level. The activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and lipid peroxidation level reflected by MDA level was determined by using the kit came from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The related mechanisms of determination could refer to these articles [@pone.0051043-Sunderman1]--[@pone.0051043-Pattichis1]. Statistical Analysis {#s4f} -------------------- Data were presented as mean±standard error of the mean (SD) and statistical significance was analyzed by Origin 6.0 (Microcal Software, Northampton, MA, USA). If the probability (*p*) was less than 0.05, the values of HCH treatments were considered significantly difference when compared to controls. For a statistical evaluation of difference between the four HCH isomers, the PASW Statistics 18.0 software (SPSS, Inc.) was used. Means of at least four repetitions of each parameter were assessed. The significance of the differences between every two treatments was evaluated by the analysis of the variance based on the method of Tukey contrasts (one-way ANOVA' test) with a *p* value of 0.05. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: Qiong Zhang MZ WL. Performed the experiments: Qiong Zhang Quan Zhang CZ HQ. Analyzed the data: Qiong Zhang MZ. Contributed reagents/materials/analysis tools: WL Quan Zhang CZ. Wrote the paper: Qiong Zhang MZ WL.

Introduction ============ The human immunodeficiency virus-1 (HIV-1) enters target cells by binding its envelope glycoprotein gp120 to the CD4 receptor and/or coreceptors such as the C-C chemokine receptor type 5 (CCR5; R5) and C-X-C chemokine receptor type 4 (CXCR4; X4).[@b1-dddt-9-5447] R5-tropic and X4-tropic viral strains use CCR5 and CXCR4, respectively, as coreceptor to enter and infect target cells. Some HIV-1 strains are dual tropic and can use CCR5 and/or CXCR4.[@b2-dddt-9-5447] CCR5 is expressed on several cell types, including T-cells, dendritic cells, and leukocytes.[@b2-dddt-9-5447],[@b3-dddt-9-5447] In HIV-infected humans, R5-tropic viruses predominate during the early stages of infection, whereas X4-tropic viruses usually emerge during the later stages.[@b1-dddt-9-5447],[@b2-dddt-9-5447] The importance of CCR5 in HIV/AIDS was demonstrated by studies showing that a 32-base-pair deletion in the CCR5 gene results in resistance to HIV-1 infection or slower progression to AIDS.[@b4-dddt-9-5447],[@b5-dddt-9-5447] Given CCR5's importance in HIV-1 transmission, infection, and AIDS progression, drugs targeting CCR5 have been an important area of research. In a short-term trial with HIV-infected patients, aplaviroc, the first CCR5 antagonist to enter clinical trials, demonstrated significant antiretroviral activity.[@b6-dddt-9-5447] However, in subsequent Phase II trials, testing was discontinued due to increased cases of idiosyncratic hepatotoxicity.[@b7-dddt-9-5447] Vicriviroc (VCV) significantly reduced viral loads (VL).[@b8-dddt-9-5447] Two subsequent Phase II trials confirmed VCV antiretroviral activity and safety.[@b9-dddt-9-5447],[@b10-dddt-9-5447] However, in a third Phase II and two Phase III trials, VCV showed higher rates of virological failure than other antiretroviral drugs,[@b11-dddt-9-5447],[@b12-dddt-9-5447] and its further development was terminated. Cenicriviroc, a CCR5 and CCR2 inhibitor, has completed Phase IIb trials and showed potent antiretroviral activity in vitro and in vivo.[@b13-dddt-9-5447],[@b14-dddt-9-5447] Maraviroc (MVC, Pfizer) is a small molecule, reversible CCR5 antagonist,[@b15-dddt-9-5447] currently approved for treatment of patients infected with R5-tropic HIV-1.[@b15-dddt-9-5447] This paper will review MVC discovery and development, its efficacy against HIV-1/AIDS, pharmacokinetics, pharmacodynamics and drug resistance, and its use in other diseases. MVC discovery ============= MVC, originally called UK-427,857 (empirical formula: C~29~H~41~F~2~N~5~O), was developed by Pfizer during CCR5 ligand studies.[@b16-dddt-9-5447] High-throughput screening to identify small molecules that could inhibit the binding of macrophage inflammatory protein-1-beta to CCR5 stably expressed in HEK-293 cells lead to the discovery of imidazopyridine, UK-107,543.[@b17-dddt-9-5447] UK-107,543 displayed efficient and potent inhibition of macrophage inflammatory protein-1-beta binding to CCR5, with a half-maximal inhibitory concentration of 650 nM. However, UK-107,543 had no antiretroviral activity. MVC was the result of UK-107,543 optimization for binding potency against CCR5, antiretroviral activity, absorption, pharmacokinetics, and selectivity for the human ERG channel.[@b16-dddt-9-5447] This optimization is summarized in [Figure 1](#f1-dddt-9-5447){ref-type="fig"}. Modifications of UK-107,543 to UK-372,673 resulted in increased binding to CCR5 and antiretroviral activity, with 90% inhibitory concentration (IC~90~) of 75 nM.[@b18-dddt-9-5447] Further modifications to make UK-382,055 increased its antiretroviral activity (IC~90~: 3 nM), but blocked potassium channels.[@b18-dddt-9-5447] Modifications to make UK-396,794 further increased anti-retroviral activity (IC~90~: 0.6 nM) and increased absorption, but UK-396,794 was rapidly metabolized.[@b18-dddt-9-5447] In total, 956 analogues were screened before finally getting MVC, which displayed good antiretroviral activity (IC~90~ below 2 nM), did not block potassium channels, was not rapidly metabolized, and had good absorption.[@b18-dddt-9-5447] MVC clinical trials =================== In Phase I trial, MVC reached steady-state plasma concentrations after 7 days' treatment and was well tolerated at clinically relevant doses (\<900 mg/kg).[@b19-dddt-9-5447] A phase II trial in asymptomatic HIV-1-infected humans receiving MVC, 300 mg/kg twice daily showed that VL decreased by 1.6 log~10~ copies/mL compared to 0.02 log~10~ copies/mL in infected humans receiving placebo.[@b20-dddt-9-5447] These results validated MVC as a non-viral target antiretroviral therapy (ART). The MOTIVATE trials were two Phase III trials to determine the efficacy and safety of MVC in patients infected with R5-tropic HIV-1.[@b21-dddt-9-5447] Patients received oral MVC or placebo once or twice daily, with doses adjusted based on other drugs in the patient's ART regimen. MVC decreased VL by −1.84 log~10~ copies/mL compared to −0.79 log~10~ copies/mL in placebo-treated patients.[@b21-dddt-9-5447] MVC showed efficacy throughout the 96 weeks of treatment; 86.7% and 81.4% of patients receiving MVC twice daily and once daily, respectively, who had VL below 50 copies/mL at week 48, maintained virological suppression up to week 96.[@b22-dddt-9-5447] From week 48 to week 96, the median CD4 count increased by 89 and 113 cells/mm^3^ in patients receiving MVC once daily and twice daily, respectively.[@b22-dddt-9-5447] The incidences of adverse events were similar for patients on ART regimens containing MVC twice daily, MVC once daily, or placebo, at week 96 and year 5.[@b23-dddt-9-5447] Subgroup analyses of pooled data from week 48 of the MOTIVATE trials were performed based on parameters such as ethnicity, baseline VL, CD4 count, tropism, genetic polymorphisms, and ART regimens. Results showed that MVC was effective in multiple patients infected with R5-tropic HIV-1 who had previously been treated with other ART drugs, including patients with low baseline CD4 count or high VL.[@b24-dddt-9-5447] Furthermore, more patients receiving MVC who failed treatment had X4-tropic HIV-1 at treatment failure compared to those receiving placebo, but MVC-treated patients showed no decrease in CD4 count.[@b24-dddt-9-5447] The MERIT study was a Phase IIb/III trial to determine the efficacy of MVC versus efavirenz (EFV) in treatment-naïve patients infected with R5-tropic HIV-1.[@b25-dddt-9-5447] MVC treatment did not initially demonstrate noninferiority for patients with VL below 50 copies/mL, with 65.3% and 69.3% of patients receiving EFV and MVC, respectively, having VL below 50 copies/mL. However, in post hoc reanalysis after removal of patients with previously undetectable X4-tropic viruses, MVC was noninferior to EFV, with 68.3% and 68.5% of patients in the EFV and MVC groups, respectively, having VL below 50 copies/mL.[@b25-dddt-9-5447] Analysis at 5 years showed that 50.8% and 45.9% of patients receiving MVC and EFV, respectively, had maintained VL below 50 copies/mL.[@b26-dddt-9-5447] The mean increased in CD4 count was 293 cells/µL in MVC-treated patients compared to 271 cells/µL in EFV-treated patients;[@b26-dddt-9-5447] compared to the EFV group, fewer MVC-treated patients discontinued treatment due to adverse events.[@b26-dddt-9-5447] Following these studies, MVC was approved by United States Food and Drug Administration and the European Commission for treatment-experienced HIV-1-infected humans, and was subsequently also approved for treatment-naïve HIV-1-infected humans.[@b15-dddt-9-5447] MVC pharmacokinetics ==================== Dosage, absorption, and bioavailability --------------------------------------- Standard MVC dosage is 300 mg/kg twice daily;[@b15-dddt-9-5447] for patients receiving a cytochrome P450-3A4 (CYP3A4) inhibitor or inducer, dosage is adjusted to 150 or 600 mg/kg twice daily, respectively.[@b15-dddt-9-5447] In an effort to reduce possible toxicity associated with high drug concentrations, simplify drug regimens, and reduce costs, the clinical outcomes of 150 or 300 mg/kg MVC once daily have been examined. In the MOTIVATE trials, once daily MVC showed virological suppression (mean change in plasma HIV-1 RNA levels of −1.68 log~10~ copies/mL), although this was not as robust as the levels of virological suppression in subjects receiving MVC twice daily.[@b21-dddt-9-5447] The Maraviroc Once Daily with Darunavir Enhanced by Ritonavir in a New Regimen study, comparing the efficacy of 150 mg/kg MVC plus darunavir/ritonavir (DRV/RTV) once daily versus tenofovir (TDF) plus DRV/RTV once daily, was terminated due to inferiority of the MVC-containing regimen.[@b27-dddt-9-5447] However, other studies have shown that 150 mg/kg MVC plus DRV/RTV once daily can effectively suppress HIV-1 infection.[@b28-dddt-9-5447] Virological suppression was also demonstrated with nucleoside reverse-transcriptase inhibitor (NRTI)-sparing regimens of 150 mg/kg MVC once daily plus lopinavir (LPV)/RTV and atazanavir (ATV)/RTV.[@b29-dddt-9-5447],[@b30-dddt-9-5447] Another study examined the effect of MVC-containing regimens with TDF-containing regimens on bone loss; the authors used a dose of 150 mg/kg MVC once daily in combination with DRV/RTV and emtricitabine (FTC), and showed that, although bone mineral density decreased in both groups, the magnitude of decrease was less in patients receiving MVC.[@b31-dddt-9-5447] However, this latter study did not analyze the effects of treatment regimens on viremia. MVC (molecular weight: 513.67 g/mol) is moderately lipophilic, with a distribution constant at pH 7.4 (log *D*~7.4~) of 2.1.[@b32-dddt-9-5447] Drug lipophilicity can increase its ability to penetrate lipid membranes such as the blood--brain barrier;[@b33-dddt-9-5447] and the optimal log *D*~7.4~ for diffusion from plasma into cerebrospinal fluid (CSF) is 1--10.[@b34-dddt-9-5447] MVC is a weak base with a p*K*~a~ of 7.3,[@b35-dddt-9-5447] is highly soluble at pH 1--7.5,[@b15-dddt-9-5447] and 76% of MVC binds to plasma proteins.[@b35-dddt-9-5447] At a single MVC dose of 300 mg/kg, time to maximum concentration (Tmax) occurred by 2 hours post-treatment in humans;[@b32-dddt-9-5447] with higher MVC levels in plasma (median maximum concentration \[Cmax: 800 ng/g\]) compared to whole blood (Cmax: 489 ng/g).[@b32-dddt-9-5447] MVC exposure (area under the curve; AUC) was 4,497 ng h/L for plasma and 2,251 ng h/L for blood.[@b32-dddt-9-5447] MVC AUC decreased by 33%--37% after a high fat meal,[@b15-dddt-9-5447],[@b36-dddt-9-5447] but this did not affect MVC antiviral activity.[@b15-dddt-9-5447] At 100 and 300 mg/kg, the absolute MVC oral bioavailability is 23.1% and 33%, respectively.[@b32-dddt-9-5447] Distribution ------------ MVC is distributed throughout the body and can be detected in the seminal plasma (SP), vaginal tissues (VT), cervicovaginal fluids (CF), rectum, and CSF.[@b15-dddt-9-5447],[@b37-dddt-9-5447]--[@b47-dddt-9-5447] Pharmacokinetic data are summarized in [Table 1](#t1-dddt-9-5447){ref-type="table"}. MVC concentrations and AUC in the SP are lower than in the blood plasma (plasma).[@b37-dddt-9-5447] However, the median protein binding in the SP is 9% compared to 76% in the plasma; more pharmacologically available MVC are found in the SP,[@b37-dddt-9-5447] and most patients on MVC had undetectable VL in SP,[@b38-dddt-9-5447] suggesting that MVC might reduce sexual HIV-1 transmission. MVC has higher concentrations in vaginal tissue and CF than in plasma, and also has lower protein binding (7.6%) in CF than in plasma.[@b40-dddt-9-5447] Compared to plasma, MVC concentrations in rectal tissues are 7.5-fold higher after a single dose and 26-fold higher after multiple doses.[@b37-dddt-9-5447] These results were corroborated by animal studies showing that MVC concentrations in macaques' rectal tissues were 23% higher than in plasma;[@b39-dddt-9-5447] however, despite high concentrations in rectal tissues, MVC could not prevent simian-HIV (SHIV) rectal transmission.[@b39-dddt-9-5447] MVC CSF concentrations are often low but vary; studies of asymptomatic HIV-1-infected humans[@b42-dddt-9-5447],[@b43-dddt-9-5447],[@b45-dddt-9-5447],[@b46-dddt-9-5447] showed lower concentrations than a study of HIV-1-infected patients with central nervous system (CNS) impairment.[@b44-dddt-9-5447] These discrepancies could be due to differences in drug regimens or increased drug entry into the brain due to blood--brain barrier inflammation in patients with CNS impairment. Despite low CSF concentrations, MVC suppressed CSF VL.[@b42-dddt-9-5447],[@b43-dddt-9-5447] MVC is detectable in humans' saliva and correlates with its plasma concentrations;[@b37-dddt-9-5447] thus, measuring MVC saliva concentrations could be a less invasive option of monitoring adherence. Quantification of MVC in humans' intestines has not been done, however, in MVC-treated humanized mice, the highest drug concentrations were in the intestine.[@b47-dddt-9-5447] This is likely due to MVC excretion through feces.[@b35-dddt-9-5447] Metabolism ---------- Unmetabolized MVC constitutes 42% of circulating drug.[@b32-dddt-9-5447] The most abundant metabolite in the plasma is a secondary amine product of N-dealkylation (UK-408,027).[@b32-dddt-9-5447] MVC is metabolized by CYP3A4 and CYP3A5;[@b35-dddt-9-5447],[@b48-dddt-9-5447] CYP3A4 is its major enzyme,[@b35-dddt-9-5447] while CYP3A5 is involved in the formation of mono-oxygenated metabolites.[@b48-dddt-9-5447],[@b49-dddt-9-5447] In fact, ketoconazole, a potent CYP3A4 inhibitor, reduced MVC metabolism and UK-408,207 formation by over 80%,[@b50-dddt-9-5447] while specific inhibitors of CYP1A2, CYP2C9, CYP2C19, and CYP2D6 had no significant effect.[@b50-dddt-9-5447] MVC is a P-glycoprotein (Pgp) substrate; in MVC-treated Pgp-knockout-friend-leukemia-virus-B mice Cmax and AUC increased by 108% and 183%, respectively, compared to MVC-treated wild-type mice.[@b35-dddt-9-5447] Genetic polymorphisms --------------------- CYP3A5 is involved in the oxidative metabolism of MVC, and it has been suggested that CYP3A5 genetic polymorphisms may influence MVC pharmacokinetics.[@b49-dddt-9-5447] The wild-type homozygous allele, CYP3A5\*1, is associated with the highest CYP3A5 protein expression, whereas the CYP3A5\*3, CYP3A5\*6, and CYP3A5\*7 alleles are associated with nonfunctional or dysfunctional CYP3A5 expression.[@b49-dddt-9-5447],[@b51-dddt-9-5447]--[@b54-dddt-9-5447] CYP3A5 expression varies by ethnicity, 80%--90% of Caucasians are non-expressers due to a high prevalence of CYP3A5\*3, while the majority of African--Americans are expressers, with 45% being homozygous for CYP3A5\*1.[@b51-dddt-9-5447],[@b55-dddt-9-5447] Individuals with no CYP3A5\*1 had similar MVC plasma concentrations as those with heterozygous CYP3A5\*1.[@b49-dddt-9-5447] However, compared to humans with no CYP3A5\*1 and heterozygous subjects, individuals with homozygous CYP3A5\*1 allele had lower MVC exposures and lower MVC plasma concentrations, with half of the individuals in the homozygous group having average MVC concentrations below 100 ng/mL, the threshold value below which there is increased risk of virological failure.[@b49-dddt-9-5447] Compared to individuals with no CYP3A5\*1 and heterozygous individuals, MVC Cmax in homozygous individuals decreased by 33% and 22%, respectively; and MVC exposure also decreased in homozygous individuals by 41% and 30%.[@b49-dddt-9-5447] Individuals with no CYP3A5\*1 showed decreased exposure to the predominant MVC oxidative metabolite compared to the homozygous group.[@b49-dddt-9-5447] MVC is a substrate for the organic anion transporter polypeptide-1B1, an influx transporter, also known as solute carrier organic anion transporter family member-1B1.[@b56-dddt-9-5447] The solute carrier organic anion transporter family member-1B1 gene has two functional single nucleotide polymorphisms (SNPs), c.521 T\>C and c.388 A\>G.[@b56-dddt-9-5447]--[@b58-dddt-9-5447] The c.521 T\>C SNP contains two haplotypes, \*5 (388A-521 C) and \*15 (388G-521 C), that have been associated with reduced activity and increased plasma concentrations of several drugs.[@b58-dddt-9-5447]--[@b60-dddt-9-5447] HIV-1-infected patients who were heterozygous for the c.521 T\>C SNP had a 55% increase in MVC concentrations compared to patients who were homozygous for the wild-type allele.[@b56-dddt-9-5447] Excretion --------- MVC is primarily excreted through feces, with 76% excreted through this route, and 19%--20% excreted through urine at 36--96 hours post-dose.[@b32-dddt-9-5447],[@b35-dddt-9-5447] Unmetabolized MVC accounts for 33% of excreted drug,[@b35-dddt-9-5447] major excreted metabolites include a product of hydroxylation of the methyl group of the triazole moiety, four products of mono-oxidation in the difluorocyclohexyl ring, and N-dealkylation adjacent to the tropane ring, resulting in a secondary amine.[@b32-dddt-9-5447],[@b35-dddt-9-5447] MVC pharmacodynamics ==================== In radioligand-binding assays, MVC inhibited the binding of chemokine (C-C motif) ligand (CCL)-3, CCL4, and CCL5 to CCR5 with a half-maximal inhibitory concentration of 3.3, 7.2, and 5.2 nM, respectively.[@b35-dddt-9-5447] Furthermore, MVC potently inhibited downstream CCR5 signaling and did not induce CCR5 internalization, suggesting that MVC is a functional CCR5 antagonist.[@b35-dddt-9-5447] MVC showed no significant affinity for other receptors, including CCR2, which has sequence similarity to CCR5 and is susceptible to other CCR5 antagonists.[@b61-dddt-9-5447],[@b62-dddt-9-5447] MVC inhibits HIV-1 binding to peripheral blood mononuclear cells (PBMC) (IC~90~: 3.1 nM), and up to 10 µM MVC had no effect on cell proliferation.[@b35-dddt-9-5447] MVC effectively inhibited infection of several R5-tropic HIV-1 strains, but not X4-tropic HIV-1.[@b35-dddt-9-5447] MVC concentrations increase by 11%--32% in humans with mild-to-moderate hepatic impairment, but with proper monitoring, no dose adjustment is necessary.[@b63-dddt-9-5447] In individuals with severe renal impairment, MVC renal clearance was fourfold lower than in patients with normal renal function (27 versus 110 mg/min, respectively).[@b64-dddt-9-5447] In a patient with end-stage liver disease and renal impairment receiving 300 mg/kg MVC, raltegravir (RAL), and enfuvirtide (ENF) twice daily, MVC serum concentrations were 25-fold higher than expected, and remained high after doses were adjusted to every 48 hours.[@b65-dddt-9-5447] No dose adjustment is necessary in patients with mild-to-moderate renal impairment, but in patients with severe renal impairment or end-stage renal disease, MVC should not be given if the patient is also taking CYP3A4 inducers or inhibitors.[@b15-dddt-9-5447] Compared to untreated patients, MVC treatment of humans infected with R5-tropic HIV-1 resulted in improved lipid profiles, increased CD4 count, and decreased levels of soluble endothelial protein-C receptor.[@b66-dddt-9-5447],[@b67-dddt-9-5447] MVC and immune cell restoration =============================== MVC improves T-cell counts, and higher baseline CD4 counts correlate with better response to MVC treatment.[@b68-dddt-9-5447] In the MOTIVATE trials, patients receiving MVC had larger CD4^+^ T-cell increases than those receiving placebo, even after adjusting for the greater virological potency of MVC-containing regimens.[@b69-dddt-9-5447] Short-term MVC treatment of patients with persistent virological failure resulted in increased CD4^+^ and CD8^+^ T-cells and positively correlated with improved antiviral activity.[@b70-dddt-9-5447] Following virological failure, infected patients treated for 96 weeks with RAL, etravirine (ETR), and MVC showed a greater increase in CD4^+^ T-cells than patients receiving RAL, ETR, and DRV/RTV.[@b71-dddt-9-5447] Patients on MVC intensification showed increased CD4 count slope,[@b72-dddt-9-5447] maintained higher CD4 counts after MVC discontinuation,[@b73-dddt-9-5447] and showed decreased VL in memory T-cells.[@b74-dddt-9-5447] In vitro studies also showed that MVC blocks CCR5 and CCR2 internalization and inhibits T-cell chemotaxis,[@b74-dddt-9-5447] increases CCR5 expression on activated T-cells, decreases the expression of CD25, CD38, and HLA-DR, and increases the expression of CD69 in CD4^+^ and CD8^+^ T-lymphocytes.[@b75-dddt-9-5447] In patients with suppressed viremia, MVC treatment modestly increased CD4 and CD8 counts and reduced the percentages of regulatory T-cells (Tregs).[@b76-dddt-9-5447] ART regimens containing MVC or MVC monotherapy decreased the frequency of Tregs in treatment-naïve HIV-1-infected patients from day 8 post-treatment initiation, and dose-dependently reduced Tregs frequency in PBMC.[@b77-dddt-9-5447] Treatment of virologically suppressed patients with ART regimens containing MVC decreased the expression of natural killer cell activation markers, decreased the frequency of inflammatory monocytes, including CD16^+^ monocytes, and this was associated with improved neuropsychological performance.[@b78-dddt-9-5447],[@b79-dddt-9-5447] However, in some HIV-1-infected patients with low baseline T-cells counts, MVC intensification had limited-to-no effect on CD4 counts, compared to patients on ART regimens without MVC;[@b80-dddt-9-5447]--[@b82-dddt-9-5447] although patients on MVC-containing ART regimens showed increased CD8 counts.[@b80-dddt-9-5447] MVC and HIV-1 mother-to-child transmission ========================================== Mother-to-child transmission (MTCT) is the most common route of pediatric HIV-1 transmission and occurs in utero, intrapartum, or during breastfeeding.[@b83-dddt-9-5447] The mother's plasma VL can predict the likelihood of HIV-1 transmission to the child; in developed countries where ART is readily available, MTCT is below 2%.[@b84-dddt-9-5447] A study of MVC placental transfer in macaques showed that after a single MVC dose 2 hours pre-delivery, maximal plasma MVC concentrations in infants occurred immediately after birth, Cmax: 22 ng/mL.[@b85-dddt-9-5447] Infants' Cmax were 44-fold lower than mothers' Cmax, and MVC was not detectable in infants 3.5 hours after birth.[@b85-dddt-9-5447] This suggests that a single MVC dose may not protect babies during vaginal delivery. Previous World Health Organization (WHO) recommendation for reducing MTCT, when access to ART is insufficient for a complete antepartum, intrapartum, and postpartum regimen, was a single-dose nevirapine (NVP) during labor.[@b85-dddt-9-5447] This single NVP dose reduced MTCT by approximately 50%,[@b86-dddt-9-5447] but increased drug resistance.[@b87-dddt-9-5447] The macaque study[@b85-dddt-9-5447] sought to determine whether MVC could replace NVP as a single intrapartum dose option. However, while WHO guidelines have since been revised, many low-income countries continue to use single-dose NVP.[@b88-dddt-9-5447] There have been no other major studies on MVC placental transfer after multiple doses. The low MVC placental transfer was confirmed using an ex vivo human perfused cotyledon model, which reproduces the third-trimester pregnancy conditions, and is considered the gold standard method for measuring fetal exposure risk during maternal treatment.[@b83-dddt-9-5447] The MVC fetal transfer rate was 8% and its clearance index was 0.26;[@b83-dddt-9-5447] low MVC transfer was associated with transporters-mediated drug efflux across placental membranes.[@b83-dddt-9-5447] However, an ART regimen consisting of 600/100 mg DRV/RTV, 150 mg MVC, and 200 mg ETR twice daily, with TDF/FTC prevented HIV-1 MTCT.[@b89-dddt-9-5447] In this case, MVC concentration in the umbilical cord blood was 69 ng/mL and the umbilical cord-to-plasma ratio was 0.37.[@b89-dddt-9-5447] Studies are needed to determine MVC levels in breast milk, quantify MVC transfer after multiple/prolonged dosing, and determine the long-term efficacy of MVC in preventing HIV-1 MTCT. MVC drug--drug interactions =========================== MVC interactions with protease inhibitors ----------------------------------------- Protease inhibitors (PIs) bind to HIV-1 protease, preventing its cleavage of Gag and GagPol precursors into their mature/infective forms.[@b90-dddt-9-5447] PIs, including saquinavir (SQV), RTV, LPV, ATV, tipranavir (TPV), amprenavir (APV), and DRV, are common CYP3A4 inhibitors[@b91-dddt-9-5447] and would increase MVC concentrations. Co-administration of MVC with SQV, SQV/RTV, LPV/RTV, RTV, ATV, ATV/RTV, or DRV/RTV increased MVC plasma Cmax by 332%, 423%, 161%, 128%, 209%, 267%, and 229%, respectively.[@b92-dddt-9-5447],[@b93-dddt-9-5447] However, TPV/RTV had no effect on MVC plasma concentrations.[@b92-dddt-9-5447] Fosamprenavir (FPV), an APV pro-drug, is used as a slow-release version of APV. In a treatment regimen including FPV, RTV, and MVC, MVC AUC and Cmax increased by 249% and 152%, respectively;[@b94-dddt-9-5447] APV AUC and Cmax decreased by 35% and 34%, respectively;[@b94-dddt-9-5447] RTV AUC and Cmax decreased by 34% and 39%, respectively.[@b94-dddt-9-5447] While FPV/RTV increasing MVC concentrations is not surprising, since both are CYP3A4 inhibitors, the mechanisms of MVC reducing RTV and FPV concentrations are not known. This may involve protein-binding displacement, which is associated with increase in unbound FPV and RTV ready to be metabolized. Overall, adjustments of MVC doses to 150 mg/kg twice daily are recommended when co-administered with PIs, except for TPV/RTV[@b15-dddt-9-5447] ([Table 2](#t2-dddt-9-5447){ref-type="table"}). MVC interactions with nucleoside/nucleotide analog reverse-transcriptase inhibitors ----------------------------------------------------------------------------------- NRTIs and nucleotide analog reverse-transcriptase inhibitors (NtRTIs) are nucleoside or nucleotide analogs that block the reverse transcriptase activity by incorporating themselves into viral RNA. TDF, a NtRTI, does not affect MVC concentration;[@b95-dddt-9-5447] MVC treatment had no effect on lamivudine/zidovudine (3TC/ZDV) concentrations, both NRTIs.[@b96-dddt-9-5447] NRTIs are mostly cleared renally and are not metabolized by CYPs enzymes;[@b97-dddt-9-5447] therefore, no dose adjustments are necessary when NRTIs are co-administered with MVC[@b15-dddt-9-5447] ([Table 2](#t2-dddt-9-5447){ref-type="table"}). MVC interactions with non-nucleoside reverse-transcriptase inhibitors --------------------------------------------------------------------- Non-nucleoside reverse-transcriptase inhibitors (NNRTIs) bind to the reverse transcriptase to prevent RNA conversion to cDNA. EFV, a CYP3A4 inducer, reduced MVC concentrations by over 50% in healthy subjects[@b98-dddt-9-5447] and by 25%--40% in HIV-1-infected patients.[@b99-dddt-9-5447] ETR, another CYP3A4 inducer[@b100-dddt-9-5447],[@b101-dddt-9-5447] decreased MVC Cmax by 60%, but co-administration of MVC with ETR/DRV/RTV increased MVC Cmax by 176%.[@b93-dddt-9-5447] MVC dosage should be increased to 600 mg/kg twice daily when co-administered with EFV or ETR.[@b15-dddt-9-5447] Lersivirine is a weak CYP3A4 inducer, although its further development was stopped in 2013.[@b102-dddt-9-5447] In healthy subjects given 300 mg MVC and 500 mg Lersivirine twice daily, MVC Cmax and AUC increased by 3.4% and 6.2%, respectively, compared to subjects given MVC plus placebo.[@b102-dddt-9-5447] NVP increased MVC Cmax and AUC by 101% and 154%, respectively[@b99-dddt-9-5447] ([Table 2](#t2-dddt-9-5447){ref-type="table"}). MVC interactions with integrase inhibitors ------------------------------------------ Integrase inhibitors are a newer class of ART drugs that inhibit integrase to prevent insertion of the HIV-1 genome into cellular DNA. Co-administration of RAL with MVC decreased RAL and MVC plasma concentrations, respectively, by 33% and 20%.[@b103-dddt-9-5447] However, another study showed no MVC effects on RAL concentrations.[@b104-dddt-9-5447] Elvitegravir (EVG) is often given in combination with RTV, and is metabolized by CYP3A4.[@b105-dddt-9-5447],[@b106-dddt-9-5447] Co-administration of EVG/RTV with MVC resulted in a 215% increase in MVC plasma concentration, while EVG and RTV concentrations remained unchanged.[@b105-dddt-9-5447] No dose adjustments are necessary when integrase inhibitors are co-administered with MVC.[@b15-dddt-9-5447] MVC interactions with non-HIV-1 drugs ------------------------------------- MVC does not affect the pharmacokinetics of midazolam, a benzodiazepine metabolized by CYP3A4, and had no effect on oral contraceptives ethinyloestradiol and levonorgestrel.[@b96-dddt-9-5447] When used with alcohol, MVC increased alcohol concentrations by 12%, but alcohol use did not affect MVC plasma concentrations.[@b107-dddt-9-5447] Ketoconazole, an antifungal commonly used for the treatment of opportunistic infections in HIV-1/AIDS patients, and a CYP3A4 inhibitor, increased MVC plasma Cmax by 338%; thus, MVC dosage should be decreased to 150 mg/kg twice daily when given with ketoconazole.[@b92-dddt-9-5447] Cotrimoxazole, an antibiotic commonly used to treat HIV-1-associated pneumocystis jiroveci infections, had no effect on MVC plasma concentrations or its clearance.[@b95-dddt-9-5447] Boceprevir, an antiviral used for hepatitis-C treatment, increased MVC plasma concentrations by 300%, thus, MVC dosage should be decreased to 150 mg/kg twice daily when given with boceprevir.[@b108-dddt-9-5447] Telaprevir is a hepatitis-C PI and inhibitor of CYP3A and Pgp.[@b108-dddt-9-5447] Co-administration of telaprevir and MVC to healthy subjects increased MVC AUC and Cmax, respectively, by ninefold and tenfold, compared to subjects treated only with MVC.[@b108-dddt-9-5447] Data on MVC interactions with other drugs are summarized in [Table 2](#t2-dddt-9-5447){ref-type="table"}. MVC drug resistance =================== Resistance due to the outgrowth of X4-tropic HIV-1 strains ---------------------------------------------------------- Resistance to MVC often occurs when previously undetectable X4-tropic HIV-1 are selected under pressure from drug treatment.[@b25-dddt-9-5447] This occurs when R5-tropic viruses, which previously constitute the majority of viral species, are sufficiently repressed. Viruses capable of using X4, which previously constituted a small minority, then multiply and become the dominant viral species.[@b109-dddt-9-5447] In the MOTIVATE trials, patients who were unresponsive to MVC treatment often had detectable X4-tropic viruses at treatment failure;[@b24-dddt-9-5447] 76 (57%) of MVC-treated subjects that failed treatment had X4-tropic or dual-R5X4-tropic HIV-1 at the time of treatment failure, whereas only 6 (6%) of subjects receiving optimized background therapy plus placebo had detectable X4-tropic or dual-R5X4-tropic viral strains.[@b24-dddt-9-5447] In deep-sequencing analysis of plasma samples from subjects who experienced treatment failure, subjects who had a tropism shift had multiple amino acid mutations in the gp120 third variable (V3) region; the most common mutations included substitutions of glycine (G) to arginine (R) at position 11 (G11R), proline (P) to R at position 13 (P13R), and alanine to lysine (K) at position 25 (A25K).[@b110-dddt-9-5447] Deep sequencing further showed that most of these patients had some non-R5-tropic viruses at the time of initial screening.[@b110-dddt-9-5447] In a study of 62 individuals infected with R5-tropic HIV-1 who had been receiving MVC for 10 days, only two showed emergence of dual-X4R5-tropic HIV-1 strains.[@b20-dddt-9-5447],[@b109-dddt-9-5447] Phenotype and genotype screening showed that one patient had dual-tropic strains at baseline, and HIV-1 variants using X4 were genetically distinct from variants using R5.[@b109-dddt-9-5447] In one patient on MVC-containing regimen who had failed treatment, X4-tropic HIV-1 strains were rescued from previously stored PBMC samples, and became the main circulating strain under selective pressure from MVC.[@b20-dddt-9-5447] Mutations associated with resistance to MVC ------------------------------------------- ### Mutations found in vitro Variable mutations in the V3 loop of gp120 can occur but may not confer complete resistance to MVC,[@b111-dddt-9-5447] and there has been no signature pattern of mutations described that can help predict resistance. Substitutions from alanine to threonine at position 316 (A316T), isoleucine to valine at position 323 (I323V), and alanine to serine at position 319 (A319S) were shown by genetic sequencing of clades-B and -G primary HIV-1 isolates following sequential passage.[@b112-dddt-9-5447] Deletions of isoleucine and serine at position 315 and position 317, respectively, also occurred in some viral isolates.[@b112-dddt-9-5447] Viral site-directed mutagenesis of the substitutions at position 316 and position 323 back to their original sequence, alanine to threonine and isoleucine to valine for position 316 and position 323, respectively, had full MVC response.[@b112-dddt-9-5447] Site-directed mutagenesis of either substitution alone only partially restored MVC response.[@b112-dddt-9-5447] That study also showed mutations outside the V3 loop: in the V1; V2; V4; and constant domains (C)-3, C4, and C5 regions of gp120, and in gp41.[@b112-dddt-9-5447] The authors suggested that mutations outside the V3 loop could help accommodate the resistance mutations occurring inside the V3 loop. Mutations due to serial passage of CC1/85 and Bal HIV-1 strains in vitro include valine to methionine (M) at position 169 (V169M) and asparagine (N) to K at position 192 (N192K) in the V2 region, leucine (L) to tryptophan (W) at position 317 (L317W) in the V3 region, I408A in the V4 region, aspartate (D) to N at position 462 (D462N), N463T, S464T, and N465aD in the V5 region, and L820I, I829V, and tyrosine (Y) to cysteine (C) at position 837 (Y837C) in gp41.[@b113-dddt-9-5447] Other mutations include T199K and T275M in the C2 region and I304V/F312W/T314A/glutamic acid (E) to D at position 317 (E317D)/I318V in the V3 loop.[@b114-dddt-9-5447] The T199K mutation has been shown to increase viral fitness;[@b114-dddt-9-5447] combination of F312W/T314A/E317D mutations with either I304V or I318V mutation was necessary for V3 loop binding to CCR5 in the presence of MVC.[@b114-dddt-9-5447] These mutations resulted in changes in the V3 loop configuration and secondary structure that enabled gp120 binding to drug-bound CCR5.[@b114-dddt-9-5447] In vitro selected mutations of a subtype-A HIV-1 showed mutations in the C4 but not the V3 region.[@b115-dddt-9-5447] Common mutations associated with resistance to MVC are summarized in [Table 3](#t3-dddt-9-5447){ref-type="table"}. ### Mutations found in clinical specimens Mutations seen in patients enrolled in the MOTIVATE trials include G11S + I26V, S18G + A22T, A19S + I26V, I20F + A25D + I26V, and I20F + Y21I in the V3 region.[@b110-dddt-9-5447] In a patient resistant to MVC, the mutations present included P/T to histidine (H) at position308 (P/T308H), T320H, and I322aV in the V3 loop; D407G and loss of a glycosylation site at residue-386 in the V4 region; and V489I in the C5 region.[@b116-dddt-9-5447] The P/T308H mutation seemed to be the most important, resulting in complete resistance to MVC treatment, while the H320T and V322aI mutations resulted in lesser resistance.[@b116-dddt-9-5447] Insertion of the resistant V3 loop into a MVC-sensitive ENV clone resulted in partial resistance to MVC, while addition of mutations from the V4 region showed complete resistance.[@b116-dddt-9-5447] However, when V4 mutations were added without V3 mutations, there was no resistance to MVC.[@b116-dddt-9-5447] Studies of MVC-naïve patients harboring commonly seen resistance mutations showed that 9.8% (93/951 subjects) had V3 mutations associated with MVC resistance.[@b117-dddt-9-5447] Common mutations included G11S/I26V (6.4%) and I20F/A25D/I26V (2.2%); 14 isolates with these mutation patterns were selected for MVC sensitivity tests, 13 were fully sensitive to MVC; the MVC-resistant isolate had I20F/Y21I mutations and reversing this mutation restored the sensitivity to MVC.[@b117-dddt-9-5447] However, when V3 MVC-resistant mutations from one isolate were cloned into other isolates, there was lesser or no resistance to MVC, suggesting that resistance due to V3 mutations may be dependent on which env sequence they occur in.[@b117-dddt-9-5447] Common mutations associated with resistance to MVC are summarized in [Table 3](#t3-dddt-9-5447){ref-type="table"}. ### Resistance-associated mutations and viral subtype It has been suggested that naturally occurring mutations to MVC might be more common in subtype-C than subtype-B HIV-1. Analysis of 65 samples showed that 52.3% (75% of subtype-C and 18.2% of subtype-B) had at least one mutation associated with MVC resistance.[@b118-dddt-9-5447] A commonly seen mutation was A316T in the gp120 region, occurring in 67.8% of subtype-C samples and 18.2% of subtype-B samples.[@b118-dddt-9-5447] The I323V mutation in the gp120 region occurred in 7.14% of subtype-C and was not seen in subtype-B samples.[@b118-dddt-9-5447] This observation was further confirmed in treatment-naïve HIV-1 infected patients in Zambia, all with subtype-C HIV-1.[@b119-dddt-9-5447] In an analysis of patients' PBMC from three different study populations, the A316T mutation was found in 68%, 80.7%, and 64.5% of the 28, 32, and 90 samples analyzed, respectively.[@b119-dddt-9-5447] The I323V mutation in the gp120 was rare but was only present with the A316T mutation.[@b119-dddt-9-5447] A high prevalence of the A316T mutation, which results in partial resistance to MVC, was found in Zambian mother--infant pairs infected with HIV-1 subtype-C.[@b120-dddt-9-5447] A study of 80 HIV-infected patients in Brazil who had failed treatment showed that 27.5% harbored the the A316T, I323V, and/or S405A mutations in the gp120.[@b121-dddt-9-5447] However, in another study of 498 individuals infected with R5-tropic, subtype-B HIV-1, mutation patterns associated with MVC resistance were less than 5%.[@b122-dddt-9-5447] Single mutations were more commonly observed, but their significance on MVC resistance was not examined.[@b122-dddt-9-5447] Mechanisms involved in resistance to MVC ---------------------------------------- Resistance to MVC is typically non-competitive, characterized by a decrease in maximal percentage inhibition (MPI), with most resistant viruses having 80%--95% MPI.[@b111-dddt-9-5447],[@b123-dddt-9-5447] The mechanisms through which resistance to MVC evolves have not been fully elucidated, but involve mutations that result in increased affinity of gp120 to MVC-bound CCR5, enabling gp120 binding to CCR5 despite conformational changes from MVC binding.[@b124-dddt-9-5447] Resistant viruses could interact with CCR5 in the presence of MVC through increased binding to the CCR5 N-terminal domain.[@b111-dddt-9-5447] In fact, CCR5 antagonists work by binding to a hydrophobic pocket in the CCR5 transmembrane region;[@b111-dddt-9-5447] this results in conformational change in the extracellular loop (ECL) region, but little-to-no conformational change in the N-terminal domain.[@b111-dddt-9-5447] Therefore, mutations resulting in increased binding to the N-terminal domain would enable binding to MVC-bound CCR5. This could cause broad cross-resistance among several CCR5 antagonists, since most do not affect N-terminal conformation.[@b111-dddt-9-5447] In fact, three HIV-1 isolates resistant to VCV also displayed resistance to MVC, as well as resistance to TAK-779, another small-molecule CCR5 inhibitor.[@b125-dddt-9-5447] Another model for resistance to MVC is the requirement of both the N-terminal and ECL domains for viral binding; mutations in the V3 loop would thus allow its binding to the ECL in the presence of MVC, due to increased affinity for the binding site.[@b124-dddt-9-5447] Conformational change to the ECL region differs among CCR5 antagonists, thus, mutations resulting in the V3 loop binding to MVC-bound CCR5 may not result in binding in the presence of other CCR5 antagonists.[@b116-dddt-9-5447] The I322a residue is in contact with D11, Y10, and Y14, three key amino acid residues of the CCR5 N-terminal domain that are predicted to interact with the V3 loop.[@b126-dddt-9-5447] The hydrophobic side chain of I322a intercalates between all three residues, making hydrophobic contacts with the aromatic rings of both Y residues and the backbone of D11.[@b126-dddt-9-5447] Replacement of I with V likely alters the hydrophobic packing of these interactions and thereby modulates how V3 interacts with the CCR5 N-terminal.[@b126-dddt-9-5447] Use of MVC in HIV-1 infection ============================= Routine treatment ----------------- Several studies have examined MVC use for treatment of HIV-1 infected humans. In a study of 32 patients, the most common reasons for MVC initiation were treatment failure, intolerance to previous ART regimens, and treatment intensification.[@b127-dddt-9-5447] After 3 and 6 months MVC treatment, 75% and 78% of patients, respectively, had fully suppressed viremia;[@b127-dddt-9-5447] the median increase in CD4^+^ count was 141 and 124 cells/µL at 3 and 6 months, respectively.[@b127-dddt-9-5447] In a study of 27 patients (20 with R5-tropic HIV-1, 1 with dual-R5X4-tropic HIV-1, and 6 with undetermined viral tropism) receiving MVC-containing ART regimens, 59% (10/17) of patients with detectable viremia before MVC initiation achieved viral suppression; HIV-1 remained undetectable in 60% (6/10) of patients who had undetectable virus before MVC initiation.[@b128-dddt-9-5447] CD4 count increased in 78%, remained unchanged in 11%, and decreased in 11% of patients.[@b128-dddt-9-5447] Another study of 25 patients infected with R5-tropic HIV-1 who had failed treatment showed that 12 months' MVC intensification resulted in increased CD4 count and undetectable VL in 21 patients, and two patients without suppressed viremia showed a switch to X4-using HIV-1 by 24 months.[@b129-dddt-9-5447] Another study of 27 patients receiving MVC who had experienced treatment failure showed that at the time of treatment failure, 12 patients had X4-using viruses and 15 had R5-using viruses.[@b130-dddt-9-5447] Of the 12 patients with X4-tropic HIV-1 at treatment failure, four were infected with X4-tropic and dual-R5X4-tropic HIV-1 before MVC initiation.[@b130-dddt-9-5447] Resistance profiles of four patients with R5-using viruses at treatment failure showed that two patients had MVC-resistant HIV-1.[@b130-dddt-9-5447] Drug monitoring --------------- The United States Department of Health and Human Services guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents suggest a minimum MVC plasma concentrations of 50 ng/mL.[@b131-dddt-9-5447] Intent-to-treat analysis of the MERIT study found that the probability of virological suppression decreased when average plasma concentrations were below 75 ng/mL.[@b132-dddt-9-5447] Additional studies are needed to determine which concentrations are optimal for accurate drug monitoring. Since several drugs, including other ART drugs, can affect MVC concentrations, monitoring is important to ensure that patients maintain sufficient plasma drug concentrations.[@b92-dddt-9-5447],[@b95-dddt-9-5447],[@b96-dddt-9-5447],[@b98-dddt-9-5447] Several groups have developed high-performance liquid chromatography (HPLC) with ultra violet (UV) detection or with tandem mass spectrometry (MS/MS) for drug quantification.[@b133-dddt-9-5447]--[@b139-dddt-9-5447] There are several drawbacks with many of these methods, including not being able to quantify low amounts of drug, the high cost of tests and equipment, and complicated procedures that may not be suitable for routine clinical practice, especially clinical practices in resource-limited settings. In the past few years, newer methods have been developed to address some of these concerns.[@b140-dddt-9-5447] Simiele et al developed a method that used less sample volume (could quantify MVC in 100 µL, compared to 500 µL of sample required with similar HPLC methods), could be completed in a shorter time (20 minutes), and used a less expensive HPLC-MS instrument that had a UV detector instead of a more costly HPLC-MS/MS instrument.[@b140-dddt-9-5447] This method had a similar lower limit of MVC quantification as other methods (4.9 ng/mL) and did not show any significant interference with other drugs likely to be used concomitantly with MVC.[@b140-dddt-9-5447] Emory et al developed a LC-MS/MS method that could quantify MVC levels from 0.5 ng/mL.[@b138-dddt-9-5447] This method can be performed using a 96-well plate, making it suitable for high-throughput screening.[@b138-dddt-9-5447] Overall, progress is being made in developing methods that will make drug monitoring easier in the future. Pre-exposure and post-exposure prophylaxis ------------------------------------------ Pre-exposure prophylaxis (PrEP) can reduce the risk of HIV-1 infection by over 90% when taken consistently.[@b141-dddt-9-5447] The only drug currently approved for use as HIV-1 PrEP is tenofovir/emtricitabine (Truvada).[@b142-dddt-9-5447] Investigation of MVC for PrEP has been mostly limited to animal studies. MVC treatment, 62 mg/kg by oral gavage, protected humanized Rag22/2cc2/2(RAG-hu) mice against vaginal HIV-1 challenge;[@b143-dddt-9-5447] MVC-treated mice had undetectable HIV-1 RNA and DNA, while all placebo-treated mice became infected.[@b143-dddt-9-5447] MVC protective effects were further confirmed by 10 weeks monitoring data showing stable CD4^+^ T-cells in MVC-treated mice while placebo-treated mice had decreased CD4^+^ T-cells.[@b39-dddt-9-5447],[@b143-dddt-9-5447] However, MVC treatment, 44 mg/kg by oral gavage, did not protect macaques against rectal SHIV transmission, despite high MVC concentrations in rectal tissues.[@b39-dddt-9-5447] This could be due to the high density of CCR5^+^ cells and activated memory CD4^+^ T-cells in macaques' gastrointestinal mucosa, which would suggest that higher MVC concentrations are needed to prevent rectal SHIV transmission.[@b39-dddt-9-5447] These macaques received one dose 24 hours pre- and post-SHIV rectal challenge,[@b39-dddt-9-5447] and accumulation of MVC in tissues from daily or twice daily use may be more effective at preventing rectal viral transmission. ART drugs can also be used as microbicides in PrEP, often as gels or time-released rings applied vaginally or rectally. A MVC gel protected against SHIV and HIV-1 infection in macaques and RAG-hu mice, respectively.[@b144-dddt-9-5447],[@b145-dddt-9-5447] In macaques, vaginal application of MVC gel prevented SHIV infection in a time-dependent and dose-dependent manner, with half-maximal protection at 4 hours and 0.5 mM.[@b144-dddt-9-5447] MVC gels were also able to prevent HIV-1 via rectal transmission in macaques.[@b146-dddt-9-5447] Vaginal application of MVC gel to RAG-hu mice (5 mM 1 hours before HIV-1 vaginal challenge) protected all mice against infection.[@b145-dddt-9-5447] An ex vivo model sought to determine whether MVC oral administration could block HIV-1 infection in Langerhans cells (LCs) and viral transmission to CD4^+^ T-cells.[@b147-dddt-9-5447] Twenty healthy volunteers were given 300 mg/kg MVC twice daily for 1, 2, 3, and 14 days and epithelial tissue explants infected with HIV-1.[@b147-dddt-9-5447] One or 2 days MVC pretreatment partially inhibited infection of LCs within epithelial tissues and 3 or 14 days pretreatment completely inhibited LCs infection.[@b147-dddt-9-5447] MVC treatment also prevented HIV-1 transmission from LCs to co-cultured CD4^+^ T-cells.[@b147-dddt-9-5447] A Phase I clinical trial (MTN-013/IPM 026) evaluated the pharmacokinetics and pharmacodynamics of vaginal rings containing MVC.[@b148-dddt-9-5447] The trial enrolled 48 HIV-1 negative individuals using vaginal rings containing 100 mg MVC and 25 mg dapivirine (DPV) or 100 mg MVC for 28 days. MVC concentrations in the CF peaked at day 2 of ring use (Cmax: 22×10^6^ pg/mL and AUC: 3.7×10^9^ pg h/mL with the MVC-only ring).[@b148-dddt-9-5447] With the MVC/DPV ring, MVC concentrations in the CF peaked at day 1 of ring use and were higher than in subjects with MVC ring alone (Cmax: 97×10^6^ pg/mL and AUC: 6.2×10^9^ pg h/mL).[@b148-dddt-9-5447] MVC was only detectable in the cervical tissues (CT) in 4 out of 12 individuals with the MVC-only ring and undetectable in all individuals with the MVC/DPV ring.[@b148-dddt-9-5447] MVC plasma concentrations were below the limit of detection in both MVC and MVC/DPV rings subject groups.[@b148-dddt-9-5447] Ex vivo HIV-1 challenge of CT from MVC ring users did not show any drug-associated viral inhibition.[@b148-dddt-9-5447] Overall, although data are limited to animal and ex vivo models, MVC (oral or microbicides) could be useful in HIV-1 PrEP. The only reported case of MVC use in post-exposure prophylaxis was a student exposed to multidrug-resistant HIV-1 using a needlestick.[@b149-dddt-9-5447] The student received LPV/RTV, FPV, TDF, and 3TC 15 minutes after injury, and 3 days later LPV/RTV was replaced with MVC.[@b149-dddt-9-5447] The student remained HIV-1-negative after a 6-month follow-up and the drug regimen was well tolerated.[@b149-dddt-9-5447] Dual therapy ------------ The MVC + DRV/RTV (MIDAS) study assessed the efficacy of MVC (150 mg) + DRV/RTV (800/100 mg) once daily, in 24 ART-naïve R5-tropic HIV-1-infected subjects.[@b150-dddt-9-5447] At week 48, 92% had VL below 50 copies/mL; their median CD4 count increased by 216 cells/mm^3^ at week 96.[@b150-dddt-9-5447] A retrospective cohort study of 60 treatment-experienced R5-tropic HIV-1-infected patients[@b28-dddt-9-5447] corroborated these findings; in this second study patients received MVC (150 mg) + DRV/RTV (800/100 mg) once daily. At week 48, 78% of patients had VL below 50 copies/mL and median CD4 count increased by 70 cells/µL.[@b28-dddt-9-5447] This suggests that MVC + DRV/RTV may be a viable NRTI-sparing regimen,[@b28-dddt-9-5447] however, additional studies with larger sample sizes and appropriate controls are needed to confirm this hypothesis. Combination of MVC (600 mg), RAL (400 mg), and ETR (200 mg) twice daily is effective in treatment-experienced R5-tropic HIV-1-infected patients.[@b151-dddt-9-5447] At week 48, all 28 patients had VL below 400 copies/mL; 93% had VL below 50 copies/mL and the median CD4 count increased by 267 cells/µL.[@b151-dddt-9-5447] The long-term efficacy of this regimen was confirmed at week 204, where 96% of patients had VL below 50 copies/mL and median CD4 count increased by 267 cells/mm^3^.[@b152-dddt-9-5447] In both studies, the treatment regimen was well tolerated.[@b151-dddt-9-5447],[@b152-dddt-9-5447] A 24-week study of 26 treatment-experienced patients given RAL (400 mg) + MVC (300 mg) twice daily showed higher than expected levels of viral rebound.[@b153-dddt-9-5447] In the ROCnRAL study, RAL + MVC treatment of HIV-1-infected patients with lipoatrophy did not suppress viremia, despite improvements in lipid profile and bone density.[@b154-dddt-9-5447] Another study assessed the efficacy of LPV/RTV + MVC (150 mg, once daily) or TDF/FTC, in HIV-1-infected patients with VL\>1,000 copies/mL.[@b155-dddt-9-5447] At week 48, VL was below 50 copies/mL in all patients receiving MVC + LPV/RTV, and in 96% of patients receiving TDF/FTC + LPV/RTV.[@b155-dddt-9-5447] Furthermore, patients receiving MVC + LPV/RTV showed a larger median increase in CD4 counts (MVC group: 286 cells/µL versus TDF/FTC group: 199 cells/µL).[@b155-dddt-9-5447] MVC (150 mg) + ATV/RTV (300/100 mg) once daily had beneficial antiviral activity comparable to TDF/FTC (300/200 mg) + ATV/RTV; at week 48 74.6% (44/60) of patients had VL below 50 copies/mL, median CD4 count increased by 173 cells/mm^3^ in the MVC-treated group and 187 cells/mm^3^ in the TDC/FTC-treated group.[@b156-dddt-9-5447] NRTIs are associated with endothelial dysfunction and increased inflammation.[@b157-dddt-9-5447] Previously treated R5-HIV-1-infected patients with undetectable VL receiving a NTRI-sparing dual regimen (MVC (150 mg once daily) + RTV-boosted PI) showed improved endothelial function.[@b157-dddt-9-5447] Treatment of human PBMC and polymorphonuclear neutrophils with MVC and DRV decreased cell apoptosis and migration.[@b158-dddt-9-5447] The OPTIPRIM study[@b159-dddt-9-5447] compared the standard three-drug regimen versus a five-drug regimen in reducing viral DNA load in HIV-1-infected patients. Patients in the five drugs group were given RAL (400 mg) + MVC (150 mg) twice daily with a fixed-dose combination of TDF (300 g) + FTC (200 g) + DRV (800 g) + RTV (100 g) once daily; patients in the three drugs group were given TDF (300 g) + FTC (200 g) + DRV (800 g) + RTV (100 g) once daily.[@b159-dddt-9-5447] After 24 weeks treatment, there was no significant difference in HIV-1 DNA loads of patients in the three or five drugs regimen (2.25 log~10~ per 10^6^ PBMC versus 2.35 log~10~ per 10^6^ PBMC), suggesting that there was no virological benefit to the intensive five-drug regimen.[@b159-dddt-9-5447] MVC and dual-tropic HIV-1 infection ----------------------------------- MVC efficacy in dual/mixed-R5X4-tropic HIV-1 infection was examined in a randomized, placebo-controlled, double-blind, Phase II study of treatment-experienced patients (A4001029 study).[@b160-dddt-9-5447] Patients received MVC (150 or 300 mg) or placebo plus optimized background therapy once or twice daily.[@b160-dddt-9-5447] By week 24, 61% (38/62), 60% (38/63), and 52% (32/61) in the placebo, MVC once daily, and MVC twice daily groups, respectively, had discontinued treatment because of inefficacy.[@b160-dddt-9-5447] The mean decrease in VL was 0.97 log~10~, 0.91 log~10~, and 1.2 log~10~ copies/mL in patients receiving placebo, once daily MVC, and twice daily MVC, respectively;[@b160-dddt-9-5447] the mean CD4 count increased by 36, 60, and 62 cells/µL in patients receiving placebo, once daily MVC, and twice daily MVC, respectively.[@b160-dddt-9-5447] At treatment failure more patients receiving MVC had X4-tropic HIV-1 compared to patients receiving placebo.[@b160-dddt-9-5447] These results showed that MVC treatment of humans infected with dual/mixed-R5X4-tropic HIV-1 have little-to-no virological benefit. Deep sequencing showed that patients receiving MVC twice daily had greater virological response when X4-using virus constituted less than 10% of the total viral population compared to patients with higher percentages of X4-using virus.[@b161-dddt-9-5447] In vitro studies showed that dual-R5X4-tropic HIV-1 strains that preferably use R5 are genetically and phenotypically similar to R5-tropic strains and can be inhibited by MVC.[@b162-dddt-9-5447],[@b163-dddt-9-5447] Human studies correlating HIV-1 tropism to virological response to short-term MVC exposure (8 days treatment, 300 mg/kg twice daily) showed that patients in the control group and patients with dual/mixed-tropic HIV-1 had no significant change in VL, but in patients with R5-tropic HIV-1, VL decreased by 1.41 log~10~ copies/mL.[@b164-dddt-9-5447] There was 93.5% concordance between virological response to MVC and viral tropism, with positive virological response in 95% (19/20) of patients with R5-tropic HIV-1 and negative virological response in 90.9% (10/11) of patients with dual/mixed virus.[@b164-dddt-9-5447] These results suggested that short-term MVC exposure could help determine/confirm the genotypic or phenotypic HIV-1 tropism, particularly in patients with non-reportable results by a Trofile assay.[@b164-dddt-9-5447] However, subsequent studies found no concordance between standard V3-based genotypic tropism assays and virological response to MVC monotherapy,[@b165-dddt-9-5447] and determined that short-term MVC treatment of HIV-1-infected, treatment-naïve patients could not predict viral tropism.[@b166-dddt-9-5447] In this later study, following 10 days MVC treatment (300 mg/kg twice daily) of 30 patients infected with R5-tropic and 10 patients infected with dual/mixed-tropic HIV-1, VL decreased by 1.52 log~10~ and 1.62 log~10~ copies/mL in patients with R5-tropic and dual/mixed-tropic virus, respectively;[@b166-dddt-9-5447] thus, short-term MVC exposure cannot predict viral tropism in treatment-naïve patients. The discrepancies between findings in these different studies could be due to differences in the study population.[@b166-dddt-9-5447],[@b164-dddt-9-5447] It is also possible that short-term MVC treatment could help predict viral tropism in treatment-experienced patients, but not in treatment-naïve patients. MVC and HIV-1/hepatitis-C co-infection -------------------------------------- After 6-months MVC treatment of HIV-1/hepatitis-C virus (HCV) co-infected patients, there was no significant increase in serum mediators of fibrogenesis and fibrosis, such as transforming growth factor beta-1, tissue inhibitors of metalloproteinases-1, and matrix metalloproteinase-2.[@b167-dddt-9-5447] The GUSTA study examined the effect of MVC/DRV/RTV dual therapy on hepatic injury in HIV-1/HCV co-infected patients;[@b168-dddt-9-5447] patients on dual therapy did not show any increases in the incidence of adverse events or severe liver abnormalities.[@b168-dddt-9-5447] MVC also reduced the progression of hepatic fibrosis in HIV-1/HCV co-infected patients.[@b169-dddt-9-5447] MVC and immune reconstitution inflammatory syndrome --------------------------------------------------- Immune reconstitution inflammatory syndrome (IRIS) occurs when a suppressed immune system begins to recover, and produces a massive inflammatory response to previously acquired pathogens. ART initiation in a treatment-naïve HIV-1 and polyomavirus-JC (JCV) co-infected patient with high VL and low CD4 count resulted in IRIS and decreased cognitive impairment, and this was markedly improved after ENF/MVC treatment.[@b170-dddt-9-5447] MVC treatment of HIV-1/JCV co-infected patients with IRIS, cognitive impairments, and progressive multifocal leukoencephalopathy (PML) resulted in decreased levels of CCR5^+^ immune cells in the CSF and improved patients' conditions;[@b171-dddt-9-5447] this was reversed by treatment interruption, and restarting MVC treatment again improved the patients' conditions.[@b171-dddt-9-5447] However, another HIV-1/JCV + patient with virological failure, low CD4 count and PML-associated IRIS, treated with MVC displayed rapid clinical deterioration and died 21 days after MVC initiation.[@b172-dddt-9-5447] This difference in MVC treatment outcomes could be due to advanced AIDS and PML in this latter patient. Synergistic antiviral effects of MVC and antibodies or peptides --------------------------------------------------------------- Antibodies directed against the second CCR5 ECL such as HGS004 and HGS101 had greater antiviral activity against MVC-bound than MVC-free CCR5, and inhibited infection by MVC-resistant HIV-1 more potently with MVC-bound than with free CCR5.[@b173-dddt-9-5447],[@b174-dddt-9-5447] HGS004 and MVC have potent antiviral synergy against R5-tropic HIV-1,[@b173-dddt-9-5447] and the IgG-CD4-gp120(Bal) fusion protein synergizes with MVC.[@b175-dddt-9-5447] The CCL5-derived R4.0 peptide, CCL5, and MVC exhibited concomitant interactions with CCR5 and promoted synergic inhibition of HIV-1 in acute-infection assays.[@b176-dddt-9-5447] Another CCR5 antibody, PRO-140, also showed a synergistic antiviral effect with MVC.[@b177-dddt-9-5447] MVC and HIV-2 infection ======================= Of the 34 million HIV-infected individuals worldwide, one-to-two million (mostly in West Africa) are infected with HIV-2.[@b178-dddt-9-5447] Although HIV-2 can progress to AIDS, VL are often lower and disease progression is slower compared to HIV-1 infection.[@b179-dddt-9-5447] Treatment is complex due to limited clinical trials with HIV-2-infected patients; in fact, studies on current ART drugs were done using mostly HIV-1 isolates and HIV-1-infected patients. HIV-2 is resistant to NNRTIs and has reduced sensitivity to PIs.[@b180-dddt-9-5447] Like HIV-1, the major HIV-2 coreceptors are CCR5 and CXCR4.[@b181-dddt-9-5447],[@b182-dddt-9-5447] In vitro studies showed that similar concentrations of MVC and other CCR5 antagonists that inhibit HIV-1 could also inhibit infection by primary CCR5-tropic HIV-2, with comparable MPI.[@b183-dddt-9-5447],[@b184-dddt-9-5447] Treatment of an HIV-2-infected AIDS patient with cognitive impairment and resistant to NRTIs, NNRTIs, and PIs, with a salvage therapy consisting of TDF + FTC + TPV/RTV + RAL + MVC, decreased blood VL to undetectable levels, but CSF VL remained high, neurological impairments continued, and blood VL later increased.[@b185-dddt-9-5447] Genotyping analyses suggested that treatment failure was not due to viral escape, but to poor CNS penetration of ART drugs.[@b185-dddt-9-5447] However, salvage therapy containing MVC successfully repressed blood VL in other HIV-2-infected patients resistant to other ART drugs.[@b186-dddt-9-5447],[@b187-dddt-9-5447] The presence of neurocognitive impairment and high CSF VL before initiation of MVC salvage therapy may have contributed to treatment failure in the first study,[@b185-dddt-9-5447] likely because of a large CNS/CSF viral reservoir, but it is not known whether this factor alone could explain the failure of MVC salvage therapy in that patient. The latter two studies[@b186-dddt-9-5447],[@b187-dddt-9-5447] did not indicate the patients' CSF VL or their neurocognitive status. Furthermore, none of these studies tested the tropism of HIV-2 strains circulating in the patients, therefore, it is possible that the patient who failed MVC salvage therapy[@b185-dddt-9-5447] had mixed-/dual-R5X4-tropic HIV-2 strains, or HIV-2 strains using coreceptors other than CCR5. In fact, in addition to CCR5 and CXCR4, HIV-2 can use CCR3, G-protein-coupled receptor-15, or CXCR6 to enter and infect target cells.[@b181-dddt-9-5447],[@b182-dddt-9-5447] Overall, in vitro and in vivo evidence suggests that MVC can be effective against R5-tropic HIV-2. Summary of MVC use in HIV-1 therapy ----------------------------------- MVC is a small-molecule CCR5 antagonist used for the treatment of R5-tropic HIV-1 infection in both treatment-naïve and treatment-experienced patients.[@b15-dddt-9-5447] MVC has favorable safety, pharmacokinetic and pharmacodynamic profiles. MVC is a substrate for CYP3A4, CYP3A5, Pgp, and organic anion transporter polypeptide-1B1, but is primarily metabolized by CYP3A4.[@b35-dddt-9-5447],[@b48-dddt-9-5447],[@b56-dddt-9-5447] As a substrate for CYP3A4, MVC's pharmacokinetics is affected by the concurrent use of CYP3A4 inhibitors and inducers.[@b49-dddt-9-5447] Typical dosage is 300 mg/kg twice daily.[@b15-dddt-9-5447] However, when combined with a CYP3A4 inducer the dosage is increased to 600 mg/kg twice daily; and when combined with a CYP3A4 inhibitor the dosage in decreased to 150 mg/kg twice daily.[@b15-dddt-9-5447] MVC has been used successfully for routine treatment of HIV-1-infected patients, with both decreases in VL and increases in CD4^+^ cell levels observed in treated individuals.[@b127-dddt-9-5447],[@b128-dddt-9-5447] MVC has shown promise in pre- and post-exposure prophylaxis, although current data are conflicting. Microbicide gels containing MVC reduced vaginal and rectal SHIV transmission in macaques;[@b144-dddt-9-5447],[@b146-dddt-9-5447] but women using vaginal rings containing MVC had low drug concentrations in their CF, and their CT was not resistant to ex vivo HIV-1 infection.[@b148-dddt-9-5447] NRTIs are often associated with harsh side effects and because MVC has a favorable safety profile, MVC has been used in NRTI- and PI-sparing dual regimens.[@b28-dddt-9-5447],[@b150-dddt-9-5447],[@b156-dddt-9-5447] MVC could reduce hepatic fibrosis in HIV-1/HCV co-infected patients, reduce the severity of IRIS, and suppress VL in HIV-2-infected humans.[@b169-dddt-9-5447]--[@b171-dddt-9-5447],[@b186-dddt-9-5447],[@b187-dddt-9-5447] In addition to its virological benefits, MVC has been shown to increase the levels of immune cells, including CD4^+^ and CD8^+^ cells, suggesting an additional immunological benefit.[@b70-dddt-9-5447],[@b76-dddt-9-5447] Resistance to MVC is often associated with the outgrowth of previously undetectable X4-tropic viral strains.[@b109-dddt-9-5447] Overall, current studies showed that MVC is safe and efficacious against infection with R5-tropic virus in vivo, ex vivo, and in vitro. Use of MVC in other diseases ============================ CCR5 has been implicated in other diseases besides HIV-1/AIDS, including cancer, inflammatory and graft-versus-host diseases (GVHDs).[@b188-dddt-9-5447]--[@b206-dddt-9-5447] Therefore, it was proposed that blocking CCR5 can attenuate the severity or progression of these diseases. Cancer ------ CCL5 and CCR5 expression are increased in breast cancer cells, correlate with poor prognosis,[@b188-dddt-9-5447] and breast cancer patients with CCR5-delta32 have longer metastasis-free survival.[@b207-dddt-9-5447] MVC blocked CCR5 on breast cancer cells and this was associated with decreased CCL5-induced calcium signaling and cell invasion.[@b188-dddt-9-5447] In vivo studies also showed that MVC significantly reduces the number and size of breast cancer metastasis in mice,[@b188-dddt-9-5447] increases survival, reduces weight loss, attenuates liver damage, and reduces the number and size of hepatic tumors in a mouse model of hepatocellular carcinoma.[@b189-dddt-9-5447] CCR5 expression negatively correlates with gastric cancer progression, and MVC reduced the number of peritoneal and mesenteric nodules, and decreased tumor burden in mice.[@b190-dddt-9-5447] CCR5 is also involved in prostate cancer metastasis; the proto-oncogene tyrosine-protein kinase induces CCR5 signaling in prostate epithelial cells and there is increased CCR5 expression in humans with prostate cancer, with higher CCR5 expression in metastases, compared to primary tumors.[@b191-dddt-9-5447] MVC treatment of mice injected with a prostate cancer cell line reduced the bone and brain metastasis burden by 80% and 60%, respectively.[@b191-dddt-9-5447] Perineural invasion in salivary adenoid cystic carcinoma (SACC) is also associated with increased CCR5 and CCL5 expression,[@b192-dddt-9-5447] and MVC blocked CCL5-induced increase in migration and perineural invasion of salivary adenoid cystic carcinoma.[@b192-dddt-9-5447] Graft-versus-host disease ------------------------- GVHD often occurs following an allogenic transplant, and its incidence is 30%--70% among transplant patients.[@b193-dddt-9-5447] During GVHD activated donor leukocytes recognize the recipient's antigens as foreign, resulting in tissue damage. CCR5 is involved in GVHD pathology as it binds CCL5 and recruits leukocytes to tissue sites;[@b193-dddt-9-5447],[@b194-dddt-9-5447] humans without genetically functional CCR5 have longer survival rates after renal transplantation.[@b193-dddt-9-5447] MVC blocks T-lymphocyte chemotaxis in vitro, and adding MVC to the standard therapy of patients undergoing hematopoietic stem-cell transplantation resulted in lower GVHD incidence.[@b194-dddt-9-5447] The use of MVC for GVHD prevention is currently in Phase II trials.[@b193-dddt-9-5447] Heart and lung diseases ----------------------- Pulmonary arterial hypertension (PH) is partly caused by chronic lung inflammation, and chemokines, including CCL5, play a major role in pulmonary vascular remodeling, a PH hallmark pathological feature.[@b195-dddt-9-5447],[@b196-dddt-9-5447] CCR5 expression in the lungs of humans with PH is increased compared to healthy donors.[@b197-dddt-9-5447] MVC treatment decreased PH development, or reversed PH in CCR5-knockout mice that had murine CCR5 replaced by human CCR5, and in animals subjected to PH-inducing conditions.[@b197-dddt-9-5447] Cardiac dysfunction is prevalent among HIV-1-infected patients,[@b198-dddt-9-5447] resulting from damage to cardiomyocytes by proinflammatory mediators and viral proteins.[@b199-dddt-9-5447] In MVC-treated Simian Immunodeficiency Virus (SIV)-infected macaques, diastolic function was similar to uninfected animals, whereas untreated and infected animals displayed diastolic dysfunction.[@b199-dddt-9-5447] Diastolic dysfunction was associated with myocardial macrophages activation and MVC decreased the expression of the macrophage marker CD163 compared to untreated animals, but CD68 expression remained unchanged.[@b199-dddt-9-5447] CCR5 antagonists also reduced the atherosclerotic burden and secretion of proinflammatory Th1-cytokines in dyslipidemia mouse models.[@b208-dddt-9-5447],[@b209-dddt-9-5447] RTV induces inflammation in adipose tissues, and this is associated with increased expression of proinflammatory cytokines and accelerated formation of atherosclerotic aortic plaques.[@b210-dddt-9-5447] C57BL6/J ApoE−/− mice treated with RTV + MVC were protected against aortic plaque progression; and showed less macrophage infiltration into the aortic wall, decreased levels of intercellular adhesion molecule-1, vascular cell-adhesion protein-1, CCL2, interleukin-17A, CCL5, and tumor necrosis factor-alpha, compared to mice treated with RTV alone.[@b211-dddt-9-5447] These data suggest that MVC has protective and anti-inflammatory properties on the vasculature. Hemorrhage ---------- Trauma-induced hemorrhage often results in increased inflammation and liver damage.[@b202-dddt-9-5447] This likely involves CCR5, as MVC treatment attenuates liver injury in rats subjected to trauma-induced hemorrhage by increasing peroxisome proliferator-activated receptor-gamma activity and decreasing proinflammatory factors.[@b202-dddt-9-5447] Peroxisome proliferator-activated receptor-gamma activation also improved liver function during trauma-induced hemorrhage;[@b201-dddt-9-5447] and CCR5-deficient mice have lower inflammatory pain under chemical or inflammatory stimuli.[@b212-dddt-9-5447] Rheumatoid arthritis -------------------- CCR5 ligands and other chemokines are increased in synovial fluids during rheumatoid arthritis (RA), resulting in tissue and joint damage.[@b204-dddt-9-5447],[@b205-dddt-9-5447] Therefore, blocking CCR5 could reduce inflammation at synovial joints and reduce RA symptoms. In support of this hypothesis, previous work demonstrated that CCR5-delta32 mutation had protective effects in patients with RA.[@b206-dddt-9-5447] However, in a Phase IIa study, MVC showed no efficacy in the treatment of RA.[@b203-dddt-9-5447] Liver disease ------------- CCL5 has been implicated in the pathogenesis of different hepatic disorders: HCV-infected patients with advanced stage liver injury have high levels of CCL5 mRNA,[@b213-dddt-9-5447] and hepatic CCL5 expression is increased in a mouse model of hepatic steatosis.[@b214-dddt-9-5447] Furthermore, CCL5 promotes hepatic inflammation and fibrosis in experimental fibrogenesis models.[@b215-dddt-9-5447] Mice on high fat diet treated with MVC showed less weight gain or liver damage compared to untreated mice on a similar diet.[@b215-dddt-9-5447] Furthermore, MVC-treated mice had lower hepatic triglycerides, lower degrees of steatosis, and decreased CCL5 expression compared to untreated mice on high fat diet.[@b216-dddt-9-5447] Summary ======= MVC, a CCR5 antagonist, is effective against infection with R5-tropic HIV-1 in vivo and in vitro;[@b15-dddt-9-5447],[@b20-dddt-9-5447],[@b25-dddt-9-5447] it is well tolerated and safe for most individuals. MVC has a good pharmacokinetic profile, with relatively low protein binding and high bioavailability.[@b32-dddt-9-5447] It also has a wide distribution throughout the body, with high concentrations found in the vaginal and rectal tissues.15,37--41,43--47,143,145 MVC appears to be safe in patients with mild-to-moderate hepatic and renal disorders.[@b39-dddt-9-5447],[@b63-dddt-9-5447] Limited studies have examined MVC effects on HIV-1 MTCT; however, MVC placental MVC is low.[@b83-dddt-9-5447],[@b85-dddt-9-5447] MVC is metabolized by CYP3A4, and dosing must be adjusted when given with CYP3A4 inducers or inhibitors.[@b15-dddt-9-5447] True resistance to MVC is rare, as most cases of virological failure seen are associated with outgrowth of previously undetectable X4-tropic HIV-1 strains.[@b25-dddt-9-5447],[@b109-dddt-9-5447] Current evidence suggests that MVC is not effective against dual-/mixed-R5X4-tropic HIV-1 infections. Although there have been mutations associated with resistance to MVC, mutation patterns observed vary with HIV-1 strains and no specific mutation patterns identified can predict resistance to MVC. In addition to its role against HIV-1 infection, clinical trial data and animal studies suggest that MVC may help in the treatment of other diseases, including cancer, GVHD, and inflammatory diseases.[@b188-dddt-9-5447]--[@b206-dddt-9-5447] Additional studies are needed to further our knowledge about the safety of long-term MVC use, its effectiveness against HIV-1 MTCT, its antiviral efficacy in HIV-1 reservoirs such as the CNS and lymphoid tissues, and its potential role in post-exposure prophylaxis. This work was partly supported by grants from the National Institute of Health, National Institute of Mental Health, to GDK (MH081780 and MH094160). **Disclosure** The authors report no conflicts of interest in this work. ![Development of maraviroc.\ **Notes:** Panels show the sequential optimization from the initial compound UK-107,543 to UK-372,673; UK-382,055; UK-396,794; UK-408,030; and finally maraviroc. Reprinted from *Prog Med Chem*, 43. Wood A, Armour D. The discovery of the CCR5 receptor antagonist, UK-427,857, a new agent for the treatment of HIV infection and AIDS., 239--271, Copyright © 2015, with permission from Elsevier.[@b18-dddt-9-5447]](dddt-9-5447Fig1){#f1-dddt-9-5447} ###### Maraviroc tissue distribution Tissue/sample Host Dose Cmax (range) Tmax (range) AUC (range) ---------------------------------------- ----------------- ---------------------------- ----------------------------------- ---------------------------- ------------------------------------ Plasma[@b32-dddt-9-5447] Human 300 mg/kg twice daily 800 ng/mL 2 hours 4,497 ng h/mL Semen[@b37-dddt-9-5447] Human 300 mg/kg twice daily 190 ng/mL (90.2--664 ng/mL) 2.2 hours (2.4--6.3 hours) 1,123 ng h/mL (633--2,087 ng h/mL) Semen[@b38-dddt-9-5447] Human 150--600 mg/kg twice daily 197 ng/mL (15.8--1,650 ng/mL) N/A N/A Rectal tissue[@b37-dddt-9-5447] Human 300 mg/kg twice daily 7,119 ng/mL 1.3 hours 57,326 ng h/g Rectal tissue[@b39-dddt-9-5447] Rhesus macaques Single dose of 44 mg/kg 10,242 ng/g N/A 131,164 ng h/mL Vaginal tissue[@b40-dddt-9-5447] Human 300 mg/kg twice daily 848 ng/mL 4 hours 4,857 ng h/mL Cervicovaginal fluid[@b40-dddt-9-5447] Human 300 mg/kg twice daily 1,230 ng/mL (409--1,810 ng/mL) 6 hours (0--12 hours) 750 ng h/mL (3,078--9,090 ng h/mL) Cerebrospinal fluid[@b42-dddt-9-5447] Human 150--600 mg/kg twice daily 3.63 ng/mL (1.83--12.2 ng/mL) N/A N/A Cerebrospinal fluid[@b43-dddt-9-5447] Human 150--600 mg/kg twice daily 2.58 ng/mL (\>0.5--7.22 ng/mL) N/A N/A Cerebrospinal fluid[@b44-dddt-9-5447] Human 150--600 mg/kg twice daily 102 ng/mL (35--173 ng/mL) N/A N/A Cerebrospinal fluid[@b45-dddt-9-5447] Human 150 mg/kg twice daily 6.32 ng/mL (3.41--8.67 ng/mL) N/A N/A Cerebrospinal fluid[@b46-dddt-9-5447] Human 150--300 mg/kg twice daily 2.4 ng/mL (1.5--4.0 ng/mL) N/A N/A Saliva[@b37-dddt-9-5447] Human 300 mg/kg twice daily 186 ng/mL (58.6--325 ng/mL) 3.0 hours (1.0--6.1 hours) 827 ng h/mL (252--1,298 ng h/mL) Intestines[@b43-dddt-9-5447] Humanized mice 62 mg/kg once daily 33,462 ng/g (31,958--36,819 ng/g) 4 hours 140,159 ng g/mL **Abbreviations:** Cmax, maximum concentration; Tmax, time at Cmax; AUC, area under the curve; N/A, Not available. ###### Interaction of MVC with other drugs Drugs Net effect on CYP3A4 Effect on MVC concentrations Recommended MVC dosage --------------------------------------------------------------- ---------------------- ------------------------------ ------------------------ Protease inhibitors  Saquinavir[@b92-dddt-9-5447] Inhibits Increase by 332% 150 mg/kg twice daily  Lopinavir/ritonavir[@b92-dddt-9-5447] Inhibits Increase by 128% 150 mg/kg twice daily  Atazanavir[@b92-dddt-9-5447] Inhibits Increase by 267% 150 mg/kg twice daily  Ritonavir[@b92-dddt-9-5447] Inhibits Increase by 209% 150 mg/kg twice daily  Darunavir/ritonavir[@b93-dddt-9-5447] Inhibits Increase by 229% 150 mg/kg twice daily  Tipranavir/ritonavir[@b92-dddt-9-5447] Inhibits No effect 300 mg/kg twice daily  Fosamprenavir/ritonavir[@b94-dddt-9-5447] Inhibits Increased by 152% 150 mg/kg twice daily Nucleoside/nucleotide analog reverse-transcriptase inhibitors  Lamivudine/zidovudine[@b96-dddt-9-5447] N/A No effect 300 mg/kg twice daily  Tenofovir[@b95-dddt-9-5447] N/A No effect 300 mg/kg twice daily Non-nucleoside reverse-transcriptase inhibitors  Efavirenz[@b98-dddt-9-5447],[@b99-dddt-9-5447] Induces Decrease by 25%--40% 600 mg/kg twice daily  Etravirine[@b93-dddt-9-5447] Induces Decrease by 60% 600 mg/kg twice daily  Lersivirine[@b102-dddt-9-5447] Induces Increase by 3.4% 300 mg/kg twice daily  Nevirapine[@b99-dddt-9-5447] Induces Increase by 101% 150 mg/kg twice daily Integrase inhibitors  Raltegravir[@b103-dddt-9-5447] Induces Decrease by 20% 300 mg/kg twice daily  Elvitegravir/ritonavir[@b105-dddt-9-5447] Inhibits Increase by 215% 150 mg/kg twice daily Other (non-HIV) drugs  Ethinyloestradiol/leveonorgestrel[@b96-dddt-9-5447] N/A No effect 300 mg/kg twice daily  Ethanol[@b107-dddt-9-5447] N/A No effect 300 mg/kg twice daily  Ketaconazole[@b92-dddt-9-5447] Inhibits Increase by 338% 150 mg/kg twice daily  Cotrimoxazole[@b95-dddt-9-5447] Inhibits No effect 300 mg/kg twice daily  Boceprevir[@b108-dddt-9-5447] Induces Increase by 300% 150 mg/kg twice daily  Telaprevir[@b108-dddt-9-5447] Inhibits Increase by 9% 300 mg/kg twice daily **Abbreviations:** MVC, maraviroc; CYP3A4, cytochrome P450-3A4; N/A, not available. ###### Mutations associated with resistance to MVC Mutations HIV region Specimens ----------------------------------------------- ------------- -------------------------------------- Mutations found in clinical specimens  G11R[@b110-dddt-9-5447] V3 of gp120 Plasma  P13R[@b110-dddt-9-5447] V3 of gp120 Plasma  A25K[@b110-dddt-9-5447] V3 of gp120 Plasma  A316T[@b112-dddt-9-5447],[@b118-dddt-9-5447] V3 of gp120 Primary clinical isolates and plasma  P/T308H[@b116-dddt-9-5447] V3 of gp120 Plasma  T320H[@b116-dddt-9-5447] V3 of gp120 Plasma  I322aV[@b116-dddt-9-5447] V3 of gp120 Plasma  D407G[@b116-dddt-9-5447] V4 of gp120 Plasma  V489I[@b116-dddt-9-5447] C5 of gp120 Plasma  I20F + Y21I[@b110-dddt-9-5447] V3 of gp120 Plasma Mutations found in vitro  A319S[@b112-dddt-9-5447] V3 of gp120 Primary clinical isolates  V169M[@b113-dddt-9-5447] V2 of gp120 HIV strain CC1/85  N192K[@b113-dddt-9-5447] V2 of gp120 HIV strain CC1/85  L317W[@b113-dddt-9-5447] V3 of gp120 HIV strain CC1/85  I408A[@b113-dddt-9-5447] V4 of gp120 HIV strain CC1/85  D462N[@b113-dddt-9-5447] V5 of gp120 HIV strain CC1/85  N463T[@b113-dddt-9-5447] V5 of gp120 HIV strain CC1/85  S464T[@b113-dddt-9-5447] V5 of gp120 HIV strain CC1/85  N465aD[@b113-dddt-9-5447] V5 of gp120 HIV strain CC1/85  L820I[@b113-dddt-9-5447] gp41 HIV strain CC1/85  I829V[@b113-dddt-9-5447] gp41 HIV strain CC1/85  Y837C[@b113-dddt-9-5447] gp41 HIV strain CC1/85  T199K/T275M[@b114-dddt-9-5447] C2 of gp120 HIV-1JR-FL-P17 and/or HIV-1V3Lib-P17  T275M[@b114-dddt-9-5447] C2 of gp120 HIV-1JR-FL-P17 and/or HIV-1V3Lib-P17  I304V[@b114-dddt-9-5447] V3 of gp120 HIV-1JR-FL-P17 and/or HIV-1V3Lib-P17  F312W[@b114-dddt-9-5447] V3 of gp120 HIV-1JR-FL-P17 and/or HIV-1V3Lib-P17  T314A[@b114-dddt-9-5447] V3 of gp120 HIV-1JR-FL-P17 and/or HIV-1V3Lib-P17  E317D[@b114-dddt-9-5447] V3 of gp120 HIV-1JR-FL-P17 and/or HIV-1V3Lib-P17 **Abbreviation:** MVC, maraviroc.

Abbreviations: RGCs, retinal ganglion cells; NMDA, N-methyl-D-aspartate; AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid INTRODUCTION {#sec1-1} ============ Loss of vision in acute optic nerve injury results from optic nerve axonal injury and subsequent death of retinal ganglion cells (RGCs)\[[@ref1][@ref2][@ref3][@ref4]\]. Slowing or inhibiting secondary degeneration of RGCs following optic nerve injury can provide the basis for effective treatment of optic nerve injury, and can promote the recovery of visual function limited by the low regenerative capability of RGCs\[[@ref5]\]. Recent reports have demonstrated that following optic nerve injury, altered microenvironment greatly affects the repair and regeneration of the optic nerve\[[@ref6][@ref7][@ref8][@ref9]\]. Additional studies have shown that following acute optic nerve injury and death of RGCs, the resulting elevated intravitreal glutamate levels are potentially neurotoxic in the microenvironment of RGCs\[[@ref10][@ref11]\]. Glutamate acts directly on N-methyl-D-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, indirectly stimulating the combined conduction of photoreceptor, bipolar cell, and retinal cell processes\[[@ref12]\]. Wang *et al*\[[@ref13]\] reported that the NMDA receptor antagonist dizocilpine can effectively reduce apoptosis of RGCs in a rabbit model of optic nerve injury. To investigate whether an AMPA receptor antagonist also exhibits anti-apoptotic effects on RGCs, we established a rabbit model of traumatic optic nerve injury, treated it with AMPA antagonist GYKI 52466, and observed changes in the number of RGCs following optic nerve injury. RESULTS {#sec1-2} ======= Quantitative analysis of experimental animals {#sec2-1} --------------------------------------------- Eighty New Zealand rabbits were used as models of traumatic optic nerve injury. They were randomly divided into a treatment group and a control group, with 40 rabbits in each group. In the treatment group, GYKI 52466 was administered intraperitoneally, and in the control group, physiological saline was administered intraperitoneally. The right eyes served as model controls and the left eyes served as blank controls. At 1, 3, 7, 14, and 21 days after optic nerve injury, eight rabbits from each group were used for experimentation. All 80 rabbits were included in the final analysis. Gross changes in operated eyes of rabbits with optic nerve injury {#sec2-2} ----------------------------------------------------------------- After induction of traumatic optic nerve injury, incisions healed well, without inflammatory reaction, and corneas were transparent. No traumatic cataract, intravitreal inflammatory reaction, or hematocele was observed. Retinal blood supply was normal. There was no hemorrhage or retinal detachment. The pupils of the operated eyes dilated 2--4 mm and relative afferent pupillary defect occurred. All symptoms indicated success in induction of traumatic optic nerve injury, and traumatic optic nerve injury was achieved in all 80 rabbits. GYKI 52466 reduced loss of RGCs in rabbits with traumatic optic nerve injury {#sec2-3} ---------------------------------------------------------------------------- Hematoxylin-eosin staining showed that at the early stage of traumatic optic nerve injury (1, 3 days), cells in the retinal cell layer and inner nuclear layer were arranged in a disorderly manner, chromatin was decentralized towards the periphery, nuclei were shrunken, cellular interspace was increased, cells were swollen, and RGCs decreased in number. At 7, 14, and 21 days after traumatic optic nerve injury induction, the above-mentioned changes progressed and the number of RGCs were further reduced. At 1, 3, 7, 14, and 21 days after injury, RGCs in each tissue area (25 μm × 25 μm) stained by hematoxylin-eosin were reduced by 28.32%, 37.96%, 39.27%, 56.72%, and 64.51%, respectively. These findings suggested that following acute optic nerve injury, apoptosis of RGCs is a continually programmed process. After GYKI 52466 administration, cell morphology in the retinal cell layer and inner nuclear layer were relatively normal, the degree of injury decreased, and the loss of RGCs was reduced ([Figure 1](#F1){ref-type="fig"}, [Table 1](#T1){ref-type="table"}). {#F1} ###### Quantitation of rabbit retinal ganglion cells in each group (number of surviving retinal ganglion cells/25 μm × 25 μm)  GYKI 52466 inhibited apoptosis of RGCs in rabbits with traumatic optic nerve injury {#sec2-4} ----------------------------------------------------------------------------------- TUNEL staining showed that following optic nerve injury, most RGCs were stained, and at 3 days after injury, loss of RGCs and DNA fragmentation occurred. At 14 days after optic nerve injury, TUNEL-positive cells could be observed in the retinal cell layer. At 3 and 14 days after optic nerve injury, there were significantly fewer TUNEL-positive cells in the treatment group than in the model control group ([Figure 2](#F2){ref-type="fig"}). {#F2} DISCUSSION {#sec1-3} ========== Following acute optic nerve injury, retinal ischemia causes injury of the retinal cell layer and inner nuclear layer, and cells in the retinal cell layer are greatly reduced\[[@ref14]\]. We successfully established a rabbit model of traumatic optic nerve injury as confirmed by gross observation and pupillary reflex. Using this model, loss of RGCs began at the early stage of optic nerve injury and was characterized by *in situ* DNA fragments, nuclear chromatin marginalization, and morphological condensation. At 1, 3, 7, 14, and 21 days after acute optic nerve injury, RGCs were greatly reduced. Understanding of persistent apoptosis of RGCs would help further explain the mechanism of retinal ischemia\[[@ref15]\]. Glutamate, which is a major excitatory neurotransmitter in retina, increases rapidly following acute optic nerve injury\[[@ref12]\]. Elevated levels of intracellular glutamate lead to programmed death of RGCs by overstimulation of NMDA and AMPA receptors, eventually resulting in a significant decrease in RGC number\[[@ref14]\]. During development of the visual system, glutamate is involved in neural plasticity\[[@ref16]\]. Subcutaneous injection of glutamate can destroy the retinal cell layer and inner nuclear layer\[[@ref17][@ref18][@ref19]\]. Therefore, inhibition of glutamate neurotoxicity could potentially provide therapeutic value for acute optic nerve injury. AMPA receptors can mediate rapid excitatory synaptic transmission, which greatly impacts neuronal integrity and synaptic plasticity. AMPA receptor activation can also regulate NMDA receptor activation\[[@ref20][@ref21]\]. Following optic nerve injury, AMPA receptor components altered followed by a rapid influx of calcium ions and increase in intracellular calcium ion concentration, resulting in neuronal apoptosis\[[@ref22][@ref23]\]. Glutamate receptor antagonists can therefore potentially reverse the excitatory toxicity of glutamate. Systematic application of glutamate receptor antagonists for the NMDA receptor and AMPA receptor could effectively treat tissue injury caused by high concentrations of glutamate after optic nerve injury, and reduce apoptosis of RGCs\[[@ref24][@ref25]\]. Nevertheless, there have been no reports describing use of AMPA receptor antagonists to treat acute optic nerve injury. Wang *et al*\[[@ref13]\] reported that the AMPA/kainate (KA) receptor of adult neurons is not permeable to calcium ions, but upregulated expression of AMPA/KA or KA receptors was detected in injured RGCs\[[@ref26]\]. The results of the present study showed that at an early stage of treatment, there were significantly more surviving RGCs in the treatment group than in the model control group. Furthermore, at each time point after injury, there were significantly fewer apoptotic cells in the treatment group than in the model control group. This suggests that at an early stage of acute optic nerve injury, AMPA receptor antagonist GYKI 52466 interacts with glutamate AMPA receptor to inhibit accumulation and inflow of calcium ion, reduce the release of related enzymes, inhibit apoptosis of RGCs\[[@ref27][@ref28]\], and exert effects during the later programmed apoptosis of RGCs. This could possibly occur because the AMPA receptor antagonist reduces early apoptosis of RGCs in the injury site and then reduces secondary apoptosis of RGCs in the periphery. Taken together, our results demonstrate that following acute optic nerve injury, apoptosis of retinal ganglion cells is a programmed process that can be inhibited by an AMPA receptor antagonist. MATERIALS AND METHODS {#sec1-4} ===================== Design {#sec2-5} ------ A randomized controlled animal experiment Time and setting {#sec2-6} ---------------- This study was performed at The Laboratory Animal Center of the Second Affiliated Hospital of Xinjiang Medical University in China between January 2011 and November 2011. Materials {#sec2-7} --------- Eighty male New Zealand rabbits, 4--5 months of age and weighing 2.0--2.5 kg, were provided by the Laboratory Animal Center, Xinjiang Medical University in China (license No. 16-003). All rabbits were raised in a 12-hour light-dark cycle. No eye disorders were detected. All procedures were performed in accordance with the *Guidance Suggestions for the Care and Use of Laboratory Animals*, formulated by the Ministry of Science and Technology of China\[[@ref29]\]. Methods {#sec2-8} ------- ### Model of traumatic optic nerve injury {#sec3-1} Traumatic optic nerve injury was induced in the right eyes of rabbits using the following protocol. Following anesthesia by intraperitoneal injection of 3% sodium pentobarbital, rabbits were placed in a lateral position with four limbs fixed and head towards one side. Physiological saline (250 mL) supplemented with 8 × 10^5^ U penicillin was intravenously transfused. The scalp skin was dissected using a 3--4 cm incision made on a line between tragus and lateral canthus. The temporal muscle was then bluntly dissected to expose the periosteum. After pulling away the temporal muscle using a retractor, the periosteum was dissected. The cranial bone was stripped and a bone window (2--3 cm × 2--3 cm) was made. Using a microscope, dura mater was dissected up to the bone margin, then cerebrospinal fluid was extracted. Brain tissue was slowly lifted until the white optic nerve entering into brain tissue *via* the optic foramen was exposed. The optic nerve at an approximate length of 3 mm was dissociated and occluded for five seconds using a non-traumatic vascular clamp. The resulting nerve was compressed but not fragmented. After washing with physiological saline, the skull was closed if no hemorrhage was detected. All rabbits survived during this traumatic optic nerve injury induction. After surgery, pupillary size was determined and the relative afferent pupillary defect was detected. After nerve injury induction, the animals were fed chow containing cephalexin, and erythromycin ophthalmic ointment (Beijing Shuangji Pharmaceutical Co., Ltd., China) was applied to the right eye to prevent infection. ### Intervention and specimen preparation {#sec3-2} After traumatic optic nerve injury induction, rabbits from the treatment group were intraperitoneally administered 15 mg/kg GYKI 52466 (Sigma, St. Louis, MO, USA), once a day. The control group rabbits received equal amounts of physiological saline. At 1, 3, 7, 14, and 21 days after injury induction, rabbits were sacrificed using 3% sodium pentobarbital, and eyeballs were excised and marked. After fixation for 72 hours with 4% paraformaldehyde, and dehydration and paraffin embedding, the retina was cut into 7 μm sections along the meridian of eyeballs, and the sections were stained with hematoxylin-eosin and TUNEL. ### Hematoxylin-eosin staining for RGC morphology and quantitation {#sec3-3} The sections were deparaffinized, cleared, dehydrated in a gradient ethanol series, stained with hematoxylin for 5 minutes, washed with tap water, treated in hydrogen ethanol for several seconds, washed with tap water for 1 minute, stained with eosin for 5 minutes, dehydrated, cleared, and mounted with neutral gum. Six tissues were randomly selected from each specimen. Using an optical microscope (Olympus, Tokyo, Japan) at 400 × magnification, RGCs in six areas (each area, 25 μm × 25 μm), 300 μm above and below the optic papilla were counted, and the average number of RGCs across six areas was calculated. All cell counting was done in a "single blind" manner, by a person who did not know the identity of the samples. ### TUNEL detection of RGC apoptosis {#sec3-4} The DeadEnd™ Fluorometric TUNEL System (Promega Biotechnology Co., Ltd., Beijing, China) was used for detection of apoptosis. Following deparaffinization, sections were incubated with 100 μL of 20 μg/mL proteinase K for 8--10 minutes at room temperature, washed, post-fixed with 4% formaldehyde, treated with 100 μL balanced buffer solution (200 nM postassium cacodylate, 25 nM Tris-HCl, 0.2 nM dithiothreitol, 0.25 mg/mL bovine serum albumin, 2.5 nM cobalt chloride) for 5--10 minutes, incubated with 50 μL rTdT buffer solution supplemented with 90 μL balanced buffer solution, 10 μL nucleotide-nucleoside mixture and 2 μL rTdT enzyme on ice, and then at 37°C for 60 minutes after slide placement. In the dark, sections were treated with 20 × standard saline citrate (1:10 dilution with deionized water) for 15 minutes at room temperature, washed with phosphate buffered saline, counterstained with VECTASHIELD^®^ Mounting Medium with DAPI (Aendi Biological Science and Technology Co., Ltd., Nanjing, China), and finally observed under a fluorescence microscope. ### Statistical analysis {#sec3-5} Statistical analysis was performed using SPSS 17.0 software, and the data were expressed as mean ± SD. One-way analysis of variance was performed for comparison between groups. A value of *P* \< 0.05 was considered statistically significant. We would like to thank all teachers from Department of Neurosurgery & Laboratory Animal Center, Second Affiliated Hospital of Xinjiang Medical University, China and Department of Pathology, Xinjiang Medical University, Urumqi, Xinjiang Autonomous Region, China for their technical support and generous help. **Funding:** The Foundation of Xinjiang Uygur Autonomous Region in China, No. 200821137; the National Natural Science Foundation of China, No. 81160153. **Conflicts of interest:** None declared. **Ethical approval:** The experimental procedure received approval by the Animal Ethics Committee, First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang Autonomous Region, China. (Edited by Xu H, Guo YQ/Song LP) [^1]: Ruijia Wang, Studying for master\'s degree, Department of Neurosurgery, Second Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region, China [^2]: **Author contributions:** Ruijia Wang was responsible for the study concept and design, analysis of experimental data, manuscript writing, and statistical analysis. All authors participated in the experiments. Xinping Luan was in charge of manuscript authorization and foundation.

Dysprosium orthoferrite DyFeO~3~ (DFO) has been investigated since the 1960s for its particular magnetic structure and properties[@b1]. Recently, DFO has drawn renewed attention because of, e.g., its laser or terahertz wave induced spin dynamics[@b2][@b3][@b4][@b5][@b6][@b7][@b8], magnetic-field- or uniaxial-stress-induced multiferroic state[@b9][@b10][@b11], and Mossbauer spectrum[@b12]. DFO, like the other rare-earth orthoferrites, is a distorted perovskite crystallizing in an orthorhombic lattice with the Pbnm space group. There are two magnetic sublattices in DFO, namely those associated with Fe- and Dy-ions, which provide three different types of magnetic interactions, namely, Fe^3+^- Fe^3+^, Fe^3+^- Dy^3+^, and Dy^3+^- Dy^3+^ couplings. The Fe^3+^- Fe^3+^ exchange energy determines the predominant magnetic ordering of the iron sublattice below the first Néel temperature (*T*~*N1*~) of about 650 K, that is, a G-type antiferromagnetic structure in which nearest-neighboring irons have anti-parallel spins. In addition, because of a specific Dzyaloshinskii-Moriya (DM) interaction mediated by the tilting of the oxygen octaheda[@b13], the Fe spins are not perfectly collinear but, instead, are slightly canted with respect to one another. Indeed, in the so-called Γ~4~ configuration of DFO, which is the most stable magnetic state for the temperature region ranging from *T*~*N1*~ down to 50 K, the G-type antiferromagnetic vector lies along the *a*-axis, and the aforementioned canting provides a weak ferromagnetic component (overt canting) along the *c*-axis, as well as a weak C-type antiferromagnetic component (hidden canting) along the *b*-axis[@b1][@b14]. At 50 K, a spin reorientation (SR) transition to a so-called Γ~1~ state occurs[@b15], in which the G-type antiferromagnetic vector now points along *b*, the overt canting vanishes, and two new hidden cantings of A and C types appear along the *a* and *c*-axes, respectively[@b1][@b14][@b16]. The antiferromagnetic transition temperature (*T*~*N2*~) of the Dy^3+^ sublattice is two order of magnitudes lower than *T*~*N1*~, namely about 4.5 K. The resulting Dy^3+^ spin configuration was reported to be G~x~A~y~ in a previous study on DyAlO~3~[@b17]. Moreover, when a magnetic field is applied along the *c*-axis in this particular state, the system exhibits a magnetic-field-induced phase transition and an electric polarization appears[@b18]. Furthermore, both weak ferromagnetism and ferroelectricity are also obtained when applying stress along the \[110\] direction to the low-temperature state with both Dy and Fe spins ordered. A specific interaction between Fe and Dy neighboring ions can also exist in the Γ~4~(G~x~, A~y~, F~z~) state, and is of the utmost importance since it can result in a net magnetization of Dy^3+^ ions being either parallel or antiparallel to the net Fe^3+^ sublattice moment aligned along the *c*-axis[@b19][@b20]. Previous magnetizing studies of the Γ~4~ phase of DFO single crystals reported that the magnetizations of Dy and Fe sublattices are parallel to each other[@b1][@b21]. However, these studies were carried out with a relative large applied field (about 500 Oe)[@b21], which may force both the overt canting component of Fe^3+^ sublattice, F~Fe~, and net Dy^3+^ moment component, F~Dy~, to arrange along the field's direction. Therefore, it is not clear whether such experiments shed light on the intrinsic, zero-field spin configurations and magnetizing behavior of the material. In order to uncover the basic spin configuration, in general, and reveal the relative arrangement of F~Fe~ and F~Dy~ in the Γ~4~ phase for different fields and temperatures, in particular, we employ here small as well as large applied fields at various temperatures. The present work revisits the pioneering studies of refs [@b1] and [@b21], as our results suggest that that F~Fe~ and F~Dy~ tend to align antiparallel to each other, and not parallel as previously proposed. It is also discovered that the application of magnetic fields can result in a magnetic transition, driving F~Dy~ to align parallel to F~Fe~. Importantly, an original temperature-versus-magnetic field phase diagram within the Γ~4~ phase is further provided here, with this diagram exhibiting three different regions, each possessing its own distinct behavior for the dependence of the weak magnetization as a function of the magnetic field (i.e., linear *versus* constant or, alternatively, presenting either behavior depending on the history of the sample). Our results also reveal the precise conditions required to induce the reversal of the magnetization of the Dy sublattice and the flip of the Fe spins. All these (previously overlooked) possibilities for controlling the magnetic state and response of DyFeO~3~ render a highly tunable material, with potential important applications in the context of spintronic devices. Results ======= Crystal Growth, Sample Preparation and Basic Characterization ------------------------------------------------------------- [Figure 1(a)](#f1){ref-type="fig"} shows the DFO single crystal grown in floating air, with a 6 mm diameter and a 85 mm length. The as-grown crystal rod presents a pair of parallel cleavage planes along the growth direction, whose normal vector is confirmed to be the *b*-axis by Laue camera. Further orientation results are displayed in [Fig. 1(b)](#f1){ref-type="fig"} by means of a sketch, indicating that the *c*-axis is slightly canted (\~13°) from the growth direction. We then cut this crystal rod into a cuboid with a length of 2.4 mm, 2.6 mm and 1.4 mm along the *a*, *b*, and *c* axes, respectively. [Figure 1(c--e)](#f1){ref-type="fig"} shows the X-ray Diffraction (XRD) patterns, which allow to identify characteristic peaks for each axis. Note that the insets of [Fig. 1(c--e)](#f1){ref-type="fig"} are Laue photographs for each crystallography axis corresponding to the XRD peaks. Moreover, [Fig. 1(f--h)](#f1){ref-type="fig"} show the XRD rocking curves for the (200), (020), and (004) peaks, indicating that the full-width at half-maximum (FWHM) is 0.11, 0.05 and 0.08 degree, respectively. Both the XRD and Laue data demonstrate that a high quality DFO single crystal sample was successfully grown under our conditions. Moreover, magnetic properties are displayed in [Fig. 1(i--k)](#f1){ref-type="fig"}, and measured via three different procedures -- i.e., ZFCH, FCC and FCH--all using an applied field of 100 Oe (see Methods to describe these procedures). As shown in [Fig. 1(i,j)](#f1){ref-type="fig"}, the *a*- and *b*-axis magnetizations, whose ZFCH, FCC and FCH curves all coincide, show a rapid upturn below 50 K, which corresponds to the SR transition from Γ~4~ to Γ~1~. All these magnetizations also exhibit a sharp decrease at *T*~*N2*~ \~ 4.5 K, due to the formation of the antiferromagnetic (G~x~A~y~) state of the Dy spins. The magnetization along the *c*-axis is different from the *a*- and *b*-axis magnetizations. For instance, a large *c*-axis magnetization is present at the highest studied temperatures, due to the weak ferromagnetism (WFM) of the Fe^3+^ sublattice, and an abrupt decrease occurs at 50 K as a result of the SR transition. Further cooling within the Γ~1~ state results in a rather negligible *c*-axis magnetization value. It is important to realize that [Fig. 1k](#f1){ref-type="fig"} shows that our measured c-axis magnetization in the ZFCH procedure (which coincides with the FCH curve) is approximately constant between 50 K and 200 K, which contrasts with previous studies reporting a gradually decrease with temperature above the SR transition[@b11][@b21]. This difference is likely due to the application of relatively large magnetic fields (well above our used value of 100 Oe) in these previous works. Moreover, another new result is found here, namely, the FCC curve is separated from the two heating curves (ZFCH and FCH); such a behavior is similar to the one observed for the *a*-axis magnetization of dysprosium samarium orthoferrite single crystals[@b14]. The Relative Orientation between Fe and Dy Sublattice Moments ------------------------------------------------------------- According to previous studies in rare-earth orthoferrites[@b14], the nearly temperature-independent behavior of the ZFCH magnetization curve of [Fig. 1k](#f1){ref-type="fig"} between 50 K and 200 K would be representative of an *antiparallel* coupling between the Fe^3+^ sublattice overt canting (F~Fe~) and its induced rare-earth net moment. On the other hand, the sharp increase of the FCC magnetization of [Fig. 1k](#f1){ref-type="fig"} when cooling the system from 200 K to 65 K is characteristic of a net Dy^3+^ moment (F~Dy~) being *parallel*, rather than antiparallel, to F~Fe~, as also consistent with previous studies where relatively large fields were applied[@b1][@b11][@b21]. The fact that the FCC magnetization exhibits a sudden jump at 65 K, which results in the convergence with the two curves obtained upon heating (ZFCH and FCH), can therefore be thought of as signaling a parallel-to-antiparallel (PA) transition. In other words, the WFM of the Dy sublattice evolves from being parallel to becoming antiparallel to the WFM associated with the Fe ions. Furthermore, [Fig. 2(a--e)](#f2){ref-type="fig"} explore the effect of the magnitude of the applied magnetic field on the *c*-axis magnetization in the FCC and FCH processes. One can see that there is no PA transition for a field of 50 Oe, as evidenced by the fact that the FCC curve is very similar to the ZFCH one for any investigated temperature. F~Dy~ therefore appears to be antiparallel to F~Fe~ both upon cooling and heating for that small field, thus suggesting that the antiparallel magnetic configuration is the most stable one for the Γ~4~ phase of DFO at zero field. On the other hand, a sudden departure of the FCC curves with respect to ZFCH curve does occur for magnetic fields of 75 Oe and 100 Oe; further, the temperature of the magnetization jump that we interpret as a PA transition gets reduced for increasing the magnetic field (e.g., we get 65 K for 75 Oe, and 57 K for 100 Oe). Another interesting feature is that, for an applied field of 200 Oe, the FCH curve does not display any plateau within the stability range of the Γ~4~ phase, but rather follows closely the FCC curve for any temperature. Such a result indicates that, for that larger field, F~Dy~ is always parallel to F~Fe~ both in the cooling and heating processes above the SR critical temperature. To ratify the naturally antiparallel alignment of F~Fe~ and F~Dy~, we conducted another experiment whose results are reported in [Fig. 3(a)](#f3){ref-type="fig"}. The demagnetized (please see the details in Methods) DFO was then placed into the chamber, cooling it down to 4 K and then heating it up. The magnetization measurements were performed both during the cooling (Zero-Field Cooling Measurement) and heating (Zero-Field Heating Measurement) processes. As shown in [Fig. 3(a)](#f3){ref-type="fig"}, upon cooling we measure a *negative* c-axis magnetization within the stability range of the Γ~4~ phase; for example, a value of 2 × 10^−3^μ~B~/f.u. is found at the highest investigated temperature of 300 K. This magnetization is about thirty times smaller than the one reported in [Fig. 1k](#f1){ref-type="fig"} (which is close to 6 × 10^−2^μ~B~/f.u.). Such small negative value arises from the fact that (i) our thermal demagnetization process favors a vanishing F~Fe~ (as a result of magnetic moments of iron ions being randomly oriented up and down, as schematized in [Fig. 3(c)](#f3){ref-type="fig"}, which is a direct consequence of the existence of different antiferromagnetic domains with differently oriented F~Fe~ moments, but (ii) the downward geomagnetic field slightly magnetizes the Fe sublattice, as modeled in [Fig. 3(b)](#f3){ref-type="fig"}). When cooling the system from 300 K to 210 K, the total magnetization remains small and negative but slightly increases in magnitude to reach 2.5 × 10^−3^ μ~B~/f.u., which we attribute to the increasing ordering of the Fe spins. Further cooling below 210 K and down to 50 K results in a total magnetization decreasing in strength until becoming rather small (about 1 × 10^−4^ μ~B~/f.u.). This stationary point at about 210 K likely indicates the appearance of F~Dy~, which is induced by, and antiparallel to, F~Fe~. This net moment of Dy ions, F~Dy~, then grows as the temperature is reduced down to 50 K, which explains the concomitant decrease of the magnitude of the *total* magnetization. The spin reorientation then happens, which yields an almost complete annihilation of the total magnetization below 50 K. Interestingly, [Fig. 3(a)](#f3){ref-type="fig"} further shows that the heating process significantly differs from the cooling one, as evidenced by the fact that the total magnetization upon heating is now *positive* after the Γ~1~ to Γ~4~ SR transition has occurred. Let us now discuss in more detail the magnetic response of DFO in the Γ~4~ phase, to see whether it offers additional hints on the spin structure and interplay between the Fe and Dy sublattices. [Figure 4(a--f)](#f4){ref-type="fig"} display the magnetization hysteresis loops (MH curves) measured at several temperatures in the relevant range. DFO is demagnetized before conducting each MH measurement, in order to get a nearly vanishing initial magnetization. We find that the magnetization increases when applying a magnetic field (starting from the M \~ H \~ 0 point) until reaching a plateau for the largest (positive) applied fields during the MH process. The existence of such a plateau shows that the WFM component of the total magnetization is saturated for these relatively large fields. Note that these saturated values are also consistent with the MT curves reported in [Fig. 2(e)](#f2){ref-type="fig"} for the FCC and FCH measurements corresponding to the application of fields up to 200 Oe. In [Fig. 4(g)](#f4){ref-type="fig"} we show the *initial part* of the MH curves (i.e., starting from a demagnetized sample and progressively increasing the field's magnitude) for all studied temperatures. These results indicate that the total magnetization first increases linearly with the field, and then departs from linearity at a certain field magnitude. We can conjecture that the response in the first segment is dominated by the relatively large F~Fe~ canting, while the relatively small F~Dy~ is probably still pointing along the opposite direction. Note that, in this regime, F~Dy~ will be subject to two competing forces that both scale linearly with the applied magnetic field: on one hand we have the direct action of the applied field, which drives both F~Fe~ and F~Dy~ pointing along the field's direction; on the other hand we have the anti-parallel coupling with F~Fe~, which itself grows linearly with the field. As the applied field grows, F~Fe~ eventually saturates, and F~Dy~ progressively aligns parallel to the applied field (and to F~Fe~) in the second segment of the response. Following this picture, we quantitatively analyzed the results of [Fig. 4(g)](#f4){ref-type="fig"}, in the following way: we first draw a (purple) line fitting the data at smaller fields and then another line fitting data at higher fields (see the inset of [Fig. 4(g)](#f4){ref-type="fig"} for the case of 52 K). The magnetization value associated with the intersection of these two lines is interpreted as being the *difference* between the saturated values of F~Fe~ and F~Dy~, while the plateau of the MT curves in [Fig. 4(g)](#f4){ref-type="fig"} corresponds to the *sum* of the saturated values of F~Fe~ and F~Dy~. Thus, for instance, the inset of [Fig. 4(g)](#f4){ref-type="fig"} shows that the saturated values of F~Fe~ and F~Dy~ at 52 K are 0.133 μ~B~/f.u. and 0.015 μ~B~/f.u., respectively. Such a procedure therefore allows us to extract the saturated values of F~Fe~ and F~Dy~ at *all* investigated temperatures, and the corresponding results are shown in [Fig. 4(h)](#f4){ref-type="fig"}. It is interesting to realize that F~Dy~ vanishes above 200 K, as consistent with the results in [Figs 1k](#f1){ref-type="fig"}, [2c,d](#f2){ref-type="fig"} and [3a](#f3){ref-type="fig"}. Moreover, the values of F~Fe~ + F~Dy~ and F~Fe~ − F~Dy~ thus obtained are also reported in [Fig. 2(a)](#f2){ref-type="fig"} by means of symbols; we find that the total saturated WFM values are consistent with the FCC MT curves for fields above 75 Oe, while the obtained difference of saturated moments agree well with data of the FCH MT curves for fields close to 100 Oe field. Note that the value of 100 Oe roughly corresponds to the field at which the slope of the MH curve changes (see inset in [Fig. 4(g)](#f4){ref-type="fig"}), supporting the validity of our analysis. Mapping of the WFM States ------------------------- Let us now focus on the hysteresis characteristics of DFO in the Γ~4~ phase, trying to understand the origin of the various behaviors obtained. For the purpose of this discussion, it is convenient to introduce what we denote as the *standard* MH hysteresis loops in [Fig. 4(a--f)](#f4){ref-type="fig"}, that is, the loops one obtains by starting from the maximum *positive* field of 250 Oe, then continuously decreasing this field down to a *negative* field of −250 Oe, and finally continuously varying again this field from the aforementioned negative to positive maximum fields to complete the cycle. Interestingly, and as shown in [Fig. 4](#f4){ref-type="fig"}, these standard loops adopt a square-like form for temperatures above 150 K. In particular, once the plateau is reached from the initial state, the MH curves never display a linear behavior again. In contrast, at temperatures below 150 K, we find that it is impossible to obtain the square-loop behavior: by application of a magnetic field we reach a plateau as in the cases above 150 K; yet, when the field decreases and eventually changes sign, at some point the material falls back (abruptly in some cases, like at 100 K in [Fig. 4(b)](#f4){ref-type="fig"}) into a state with a linear MH characteristic, and the transition to the state with large but opposite magnetization is ultimately gradual. To rationalize these behaviors, it is useful to distinguish three regions in the field-temperature phase diagram of the material, as shown in [Fig. 5](#f5){ref-type="fig"}(a): We have regions in which the material always presents a flat MH characteristic, a situation that we denote "hard WFM" state. The boundary of this region is given by the temperature-dependent magnetic field values at which the MH plateau is reached (see [Fig. 4](#f4){ref-type="fig"}); this boundary is marked by red lines and squares in [Fig. 5(a)](#f5){ref-type="fig"}. Then, we have a region of the phase diagram in which the material can present either a square-like MH curve or a linear one, depending on the history of the sample. This is what we call the "history-dependent WFM" region in [Fig. 5(a)](#f5){ref-type="fig"}. Finally, there is a region of the phase diagram within which the measured MH curves are always linear, independently of the history of the sample; this is what we call "soft WFM" region in [Fig. 5(a)](#f5){ref-type="fig"}. Such "soft WFM" MH behavior can only be found in low temperature region. Indeed, as can be seen in [Fig. 4](#f4){ref-type="fig"} and is reflected in the phase diagram of [Fig. 5(a)](#f5){ref-type="fig"}, for high enough temperatures (roughly above 90 K) we always obtain square MH characteristics even at zero field; in such conditions, the material can only be in the "history-dependent" or "hard" states, never in the "soft" one. Moreover, for temperatures above 150 K, [Fig. 4(c--f)](#f4){ref-type="fig"} further show that the magnetization within our standard hysteresis loops abruptly switches from positive to negative values, with the magnitude of this magnetization being conserved, when under a magnetic field. For instance, such a switching occurs for a magnetic field of −225 Oe at 300 K. These jumps are reported by means of blue squares in the magnetic field versus temperature phase diagram depicted in [Fig. 5(a)](#f5){ref-type="fig"}, and correspond to a transition between two "hard" WFM states having opposite long-range-ordered F~Fe~--since F~Dy~ is rather small above 150 K as shown in [Fig. 4](#f4){ref-type="fig"}(h). It is also interesting to realize that, for T = 100 K, the standard hysteresis loop switches from a positive value of the magnetization equal to 0.13 μ~B~/f.u (which corresponds to the F~Fe~ + F~Dy~ sum one can extract from [Fig. 4(h)](#f4){ref-type="fig"} at T = 100 K, and as characterizing a "hard" WFM state) to a negative one equal to −0.03 μ~B~/f.u (as representative of a "soft" WFM state, since the magnetization then linearly varies with H), for an applied magnetic field of about −10 Oe. Interestingly, this negative magnetization of −0.03 μ~B~/f.u is neither equal to the value of -(F~Fe~ + F~Dy~) = −0.128 μ~B~/f.u nor to the value of -(F~Fe~ − F~Dy~) = −0.113 μ~B~/f.u one can deduce from [Fig. 4(h)](#f4){ref-type="fig"}. Similarly, the jump between the "hard" and "soft" states for magnetic field close to 140 Oe at T = 52 K does not correspond to a change of magnetization from (F~Fe~ + F~Dy~) = 0.147 μ~B~/f.u to (F~Fe~ − F~Dy~) = 0.117 μ~B~/f.u one can obtain from [Fig. 4(h)](#f4){ref-type="fig"}. To explain such results, it is reasonable to assume that "soft" and even "hard" WFM states, *at and below 100* *K*, are formed by domains having opposite directions for the antiferromagnetic (AFM) vector of the Fe sublattice. When no field is applied and the total magnetization is vanishing (e.g., at the beginning of the initial hysteresis loop for T = 100 K and 52 K, but also during the standard hysteresis loop at T = 52 K, see [Fig. 4(a,b)](#f4){ref-type="fig"}), these AFM domains should also possess opposite directions for the magnetization of the Fe ions (positive or negative along the c-axis), since, within the Fe sublattice, the direction of the G-type antiferromagnetic vector dictates the direction of the WFM via a Dzyaloshinskii-Moriya (DM) interaction[@b11]. Moreover, these antiferromagnetic domains should also naturally favor opposite directions for the magnetization of the Dy ions (negative or negative along the c-axis), since F~Dy~ and F~Fe~ likely energetically prefer to be antiparallel to each other via another DM interaction[@b19]. However, at T = 100 K and H = −10 Oe during the standard hysteresis loop, the resulting "history-dependent" WFM state has a negative, small, but non-zero total magnetization, which can be explained by the facts that (i) there is a competition between the magnetic field desiring to make *all* the weak magnetizations of the Fe and Dy ions (i.e., belonging to the different AFM domains) aligned along its direction, and the aforementioned DM interactions[@b11][@b19] that prefer to induce the WFM of the Fe ions and/or the WFM of the Dy ions to be aligned opposite to the applied magnetic field in some AFM domains; and (ii) the magnetic field wins more and more this competition as its strength grows. Note that items (i) and (ii) can also imply that the "hard" WFM state is formed by AFM domains below 100 K too, but with the WFMs of the Fe and Dy ions now completely following the magnetic field (i.e., not anymore reacting to the DM interactions[@b11][@b19]) and therefore saturating in value. The analysis of the results of [Fig. 4](#f4){ref-type="fig"} therefore supports the idea that "soft" and even "hard" WFM states existing below 100 K can possess AFM domains, for which the ratio between domains with up magnetization and domains with down magnetization can be controlled by the magnitude and sign of the applied magnetic field. Note that, on the other hand, the "hard" WFM states above 150 K may be single monodomain states, which would explain the fact that the standard hysteresis loop has a perfect square shape and, therefore, that "soft" WFM states do not appear anymore in this standard loop. Note that the facts that it is possible to access the antiferromagnetic-monodomain at relatively high temperatures, while it becomes essentially impossible (for the applied magnetic fields) at temperatures of 100 K and below, are actually the behaviors to expect. As a matter of fact, achieving the transformation to a monodomain state should be easier (i.e., it should require smaller applied fields) when the antiferromagnetic order is not strongly developed and/or the material has enough thermal energy, i.e., at higher temperatures. Moreover and in order to complete the phase diagram of [Fig. 5a](#f5){ref-type="fig"}, we decided to perform additional experiments, that consist in measuring the *c*-axis magnetization-versus-temperature curves of a DFO sample that is first magnetized at 300 K by a positive field along the c-axis of 200 Oe (respectively, negative field of −200 Oe) and then cooled down to 4 K and then heated up to 300 K under negative fields of −50, −75 or −100 Oe (respectively, positive fields of +50, +75 and + 100 Oe). The resulting FCC and FCH MT functions are displayed in [Fig. 5](#f5){ref-type="fig"}(b, c, e--j). For the sake of completeness and for comparison, we also conducted similar measurements but for which no magnetic field is applied during cooling and heating after the DFO sample has been magnetized under a field of 200 Oe (see results in [Fig. 5(d)](#f5){ref-type="fig"}). Note that this initial application of a field of 200 Oe generates a total magnetization of 0.07~B~/f.u. at 300 K (that is similar to the saturated value shown in [Fig. 4f](#f4){ref-type="fig"}), and therefore results in the formation of a "hard" WFM state. One can see that for fields equal to or above 75 Oe in magnitude, the total FCC magnetization can suddenly switch and nearly completely reverts its value, via a first jump, for a specific field-dependent temperature that is always larger than 130 K. Such data allow us to extend the blue lines of [Fig. 5(a)](#f5){ref-type="fig"}, and correspond to the spin flip (SF) transition between two "hard" WFMs states having opposite and long-range-ordered F~Fe~ magnetization. Moreover, for these fields equal to or above 75 Oe in magnitude, a second type of jump of the FCC occurs at smaller temperature (e.g., equal to 72 K for H = 75 Oe), below which the FCC and FCH curves become identical. Unlike the first type of jump and as evidenced in [Fig. 5(b,f)](#f5){ref-type="fig"}, this second type of jump is also happening for fields of 50 Oe and even 0 Oe magnitude and corresponds to the aforementioned PA transition below which F~Dy~ naturally prefers to be antiparallel (rather than parallel) to F~Fe~. This second type of jumps therefore allows us to add more point to the green curve of [Fig. 5(a)](#f5){ref-type="fig"}, which is associated with the transition from "hard" WFM to "soft"/"history-dependent" WFM. Interestingly, the continuity one can see in [Fig. 5a](#f5){ref-type="fig"} between the data extracted from [Fig. 4](#f4){ref-type="fig"} and those obtained from [Fig. 5(b--j)](#f5){ref-type="fig"} demonstrates that the phase diagram of [Fig. 5(a)](#f5){ref-type="fig"} is universal, in the sense that it applies to MH measurements done at fixed temperature (as in [Fig. 4](#f4){ref-type="fig"}) but also to MT experiments conducted at fixed magnetic field (as in [Fig. 5](#f5){ref-type="fig"}). Discussion ========== Weak ferromagnetism has been systematically investigated in a high-quality single crystal of DFO grown by an optical floating zone method. Magnetic measurements reveal that the Γ~4~ magnetic state naturally prefers to present F~Fe~ and F~Dy~ moments being antiparallel to each other. We further found that relatively small magnetic fields can transform the antiparallel coupling into a parallel one, with the resulting state being coined "hard" WFM and for which the magnetization is nearly independent from the applied field. A magnetic field-*versus*-temperature phase diagram is constructed, mapping the "hard" WFM and "soft" WFM states but also a third state denoted as "history-dependent" WFM which can either be "hard" or "soft" depending on the history of the procedure used. This phase diagram also precisely predicts the sudden jumps of different natures (e.g., between two "hard" WFM states of opposite magnetization or between "soft" and "hard" WFM states), which is of obvious importance for devices exploiting spin switching phenomena. Methods ======= A single crystal DFO was grown by an optical-floating-zone method (Crystal System Inc., type FZ-T-10000-H-VI-P-SH). The compounds of feed and seed rods were prepared by the solid state reaction of the raw materials Dy~2~O~3~(99.9%), and Fe~2~O~3~(99.99%) with the proper cation stoichiometry, which was calculated by the target compound. During the growth process, the molten zone moved upwards at a rate of 3 mm/h, with the seed rod (lower shaft) and the feed rod (upper shaft) counter rotating at 30 rpm in flowing air. Employing an X-ray Laue photograph (Try-SE. Co, Ltd.), we determined the crystallographic orientations, and cut the crystal into a 2.4 × 2.6 × 1.4 mm^3^ sample. The orientations were further verified by means of θ-2θ linear scans, using a high-resolutionX-ray diffraction (Bruker D8 Discover with 4-bounce Ge(220) monochromator and Cu K~α1~ radiation (λ = 1.5406 Å)). The X-ray diffraction rocking curve on the (200), (020) and (004) diffraction peaks were also taken using this equipment. Measurements of the magnetization as a function of temperature and magnetic field were carried out using a Physical Property Measurement System (PPMS-9, Quantum Design) and a superconducting quantum interference device (SQUID) magnetometer (MPMS XL-5, Quantum Design), respectively, with the direction of the applied magnetic field being parallel to the corresponding crystallography axis. Different procedures were employed to acquire the temperature dependence of the magnetization (MT). For instance, we cooled the sample under no field down to 4 K, and then applied a magnetic field while heating the system; MT measurements were obtained during this latter heating process, which we denote as zero-field-cooling heating (ZFCH). We also cooled the system down to 4 K but now under an applied magnetic field, and measured the resulting MT curve during that process, which is coined field-cooling cooling (FCC) measurements. Finally, at the end of the FCC process, we also heated the system under the same magnetic field and measured the magnetization as a function of temperature. These measurements are denoted here as field-cooling heating (FCH). Furthermore, the magnetization *vs*. magnetic field isotherm curves were measured at several representative temperatures by changing the field at these temperatures. Note that before all of those measurements, both the sample and the MPMS chamber are pretreated: (1) the sample was demagnetized at 500 °C, which is higher than the *T*~*N*1~ to disorder the orientation of WFM components; (2) a Pd standard sample, which is commonly used in MPMS to perform a precise small field correction before the measurements, is employed to obtain a quasi-zero-field measurements chamber. Additional Information ====================== **How to cite this article**: Cao, S. *et al*. Tuning the Weak Ferromagnetic States in Dysprosium Orthoferrite. *Sci. Rep*. **6**, 37529; doi: 10.1038/srep37529 (2016). **Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work is supported by the National Key Basic Research Program of China (Grant No. 2015CB921600), the National Natural Science Foundation of China (NSFC, Nos 51372149, 11274221, 11274222, 11574194, 51572278), Eastern Scholar Program and Shuguang Program (Grant No. 12SG34) from Shanghai Municipal Education Commission, QiMingXing Project (14QA1402000). L.B. thanks the support of ARO Grant o.W911NF-16-1-0227. We also thank support from FNR Luxembourg through Grants FNR/P12/4853155/Kreisel (J.I. and H.J.Z.), and INTER/MOBILITY/15/9890527 GREENOX (L.B. and J.I.), and from the China Scholarship Council (P.C.). **Author Contributions** S.X.C., L.C. and W.Z. contributed equally to this work. S.X.C., L.C. and W.Z. designed and performed the experiments. K.X. and G.H.W. participated the crystal growth process. B.J.K. participated the magnetic measurements. Y.L.Y., H.J.Z. P.C. participated the data analysis. S.X.C., L.C., W.Z., A.S., R.-K.Z., J.C.Z., W.R., J.I. and L.B. discussed the theoretical model. L.C. and W.Z. co-wrote the paper. S.X.C., W.R., L.B. and J.I. further modified the manuscript. {#f1} {ref-type="fig"}. Zoom-in magnetization curves under the magnetic fields of 50, 75, 100 and 200 Oe are shown in (**b--e**), respectively, with the arrows indicating the spin reorientation (SR) and the parallel-antiparallel (PA) transition.](srep37529-f2){#f2} {#f3} {#f4} {ref-type="fig"}; (**b--j**) FCC c-axis magnetization-versus-temperature under various applied fields for magnetized DFO. FCH measurements are also reported in panels (**d--j**). The results of panels (**b--j**) allow to complete and confirm the proposed, original phase diagram of panel a.](srep37529-f5){#f5} [^1]: These authors contributed equally to this work.

1. Introduction {#sec1-jof-03-00020} =============== Onychomycosis, a fungal nail infection affecting either the toenails (more common) or fingernails, involving any or all components of the nail structure, is typically chronic and tough to treat. When the causative agent is a dermatophyte, the infection is termed tinea unguium. Common symptoms are thickening of the nails with discoloration, brittleness or deformity and prolonged infection may be accompanied by pain under the nail and while wearing shoes, due to nail thickening or hyperkeratosis. People with these infections have been found to suffer psychosocial stress due to the cosmetic repercussions of an unhealing infection. Tinea unguium due to dermatophytes (*Trichophyton*, *Epidermophyton* species) occurs more often in temperate regions, whereas onychomycosis due to non-dermatophyte moulds and yeasts like *Candida* and *Trichosporon* species are seen more commonly in the tropical regions of the world. Among the non-dermatophyte moulds, the common onychomycosis-causing fungi are *Scytalidium* (or *Neoscytalidium*), *Aspergillus*, *Fusarium*, *Scopulariopsis*, *Paecilomyces*, *Pseudoallescheria*, *Penicillium* and *Alternaria* species. Rarer species implicated are *Onychocola* species, *Pyrenochaeta* species, *Chaetomium globosum* and *Lasiodiplodia theobromae*. All fungi involved exhibit keratinolytic activity \[[@B1-jof-03-00020],[@B2-jof-03-00020],[@B3-jof-03-00020]\]. Some studies have stressed the significant prevalence of non-dermatophyte fungi (NDF) in onychomycosis. A 2015 study showed non-dermatophyte moulds emerging as a leading cause of onychomycosis in south-east Rajasthan in India \[[@B4-jof-03-00020]\]. In 2016, a growing incidence of non-dermatophyte onychomycosis was reported from Tehran, Iran with *Aspergillus* as the most common cause, followed by *Fusarium* \[[@B5-jof-03-00020]\]. NDF have also been seen to colonize damaged nail plates \[[@B6-jof-03-00020]\]. Predisposing risk factors to onychomycosis include trauma, frequent contact with water, lack of foot hygiene, tinea pedis, immunodeficiency, genetic predisposition, psoriasis, diabetes mellitus and old age (nail ischaemia). Traditionally, onychomycosis has been classified as distal subungual onychomycosis, proximal subungual onychomycosis, white superficial onychomycosis and *Candida* infections of the nail, while end-stage nail disease is termed total dystrophic onychomycosis \[[@B1-jof-03-00020],[@B2-jof-03-00020],[@B3-jof-03-00020]\]. In 2011, the Onychomycosis Severity Index (OSI) was created, a simple grading and prognostic tool showing high consistency and reliability, in which non-dermatophyte moulds and yeasts as causative agents were included as one of the factors indicating poor prognosis \[[@B7-jof-03-00020]\]. Currently, the preferred drugs for treating onychomycosis are oral itraconazole and terbinafine, in combination with topical nail lacquers such as ciclopirox and amorolfine \[[@B8-jof-03-00020]\]. In spite of the combination therapy, cure rates are considerably low. Factors contributing to ineffective treatment may include the existing hyperkeratosis in the infected nail that may limit drug penetration. In addition, prolonged treatment regimens of six to twelve months may deter the patient from being compliant, especially when the recurrence rates after apparent cure are quite high. Furthermore, the development of antifungal resistance is an emerging concern. Terbinafine resistance mechanisms have now been identified in *Trichophyton rubrum*, *Aspergillus nidulans* and *Aspergillus fumigatus* \[[@B9-jof-03-00020],[@B10-jof-03-00020],[@B11-jof-03-00020]\]. Most of the anti-onychomycosis drugs target the pathway of fungal ergosterol biosynthesis, an essential structural component of the fungal cell membrane. Itraconazole, like all azole antifungals, inhibits lanosterol 14α-demethylase, an enzyme of the cytochrome P450-type. Caution is warranted due to possible drug--drug interactions, especially in the elderly who may be on many medications simultaneously. Possible hepatotoxicity requires monitoring of liver enzymes during the treatment period. Itraconazole differs from the other azole drugs, because in addition to the above, its mechanism of action includes inhibition of the hedgehog signaling pathway \[[@B12-jof-03-00020]\] and angiogenesis, giving it anticancer properties as well as antifungal. Terbinafine exhibits fungicidal activity by potent non-competitive inhibition of squalene epoxidase enzyme, leading to the accumulation of intracellular squalene and decreased ergosterol. As squalene epoxidase is not an enzyme of the cytochrome P450-type, this class of enzymes is not inhibited and cholesterol biosynthesis in vivo is also unaffected, leading to lesser toxicity associated with prolonged treatment. Notably however, liver toxicity has been occasionally linked to Terbinafine \[[@B13-jof-03-00020]\]. The mechanism of action of topical ciclopirox is believed to involve its high affinity to trivalent metal cations, leading to the inhibition of essential co-factors to catalase and peroxidase enzymes and resulting in the loss of enzyme function. Amorolfine is a morpholine topical antifungal drug that interferes with ergosterol biosynthesis at two steps, leading to the accumulation of an intermediate compound in the cell membrane while ergosterol is depleted \[[@B1-jof-03-00020],[@B2-jof-03-00020],[@B3-jof-03-00020],[@B8-jof-03-00020]\]. To be effective against onychomycosis, adequate amounts of all the aforementioned drugs must penetrate the nail bed and remain active within the keratin matrix of the nail and nail bed. Keratin-bound drugs have been found to have decreased penetration, resulting in drug accumulation in surface nail layers with little or no amounts in the deeper layers and nail bed. A low affinity to keratin is desirable when choosing the ideal drug to treat onychomycosis. Efinaconazole is a relatively new antifungal synthesized as an azole amine derivative by Kaken Pharmaceutical Co. The drug was approved and launched in Canada (2013), the US and Japan (2014) for the topical treatment of onychomycosis. Formulated as a 10% solution containing 100 mg of efinaconazole per gram in a clear, colorless to pale yellow solution \[[@B14-jof-03-00020]\], it acts by inhibiting lanosterol 14α demethylase and its in vitro activity was found to be minimally affected by keratin. The methylene-piperidine group at the C-4 position of its molecular structure may be responsible for its low keratin binding. High nail penetration and potent fungicidal activity in the presence of keratin were observed both in vitro and in vivo. This property makes it almost ideal for the treatment of onychomycosis. It has low surface tension, which aids in penetration and spreading, and is sparingly soluble in water. The drug accesses the site of infection by both transungual delivery and spreading through the subungual space \[[@B15-jof-03-00020],[@B16-jof-03-00020],[@B17-jof-03-00020],[@B18-jof-03-00020],[@B19-jof-03-00020]\]. In a guinea pig onychomycosis model, topically applied efinaconazole was found to be more effective in reducing toenail fungal burden than were amorolfine and terbinafine \[[@B20-jof-03-00020]\]. Efinaconazole has also been shown to possess a broader spectrum of activity than existing antifungals against dermatophyte and non-dermatophyte moulds and yeasts, including *Fusarium* species, which cause nail infections that respond poorly to oral drugs \[[@B21-jof-03-00020],[@B22-jof-03-00020],[@B23-jof-03-00020]\]. In the present study, we tested the in vitro activities of two drugs, itraconazole and efinaconazole, against 44 isolates of the most common non-dermatophyte fungi causing onychomycosis. 2. Materials and Methods {#sec2-jof-03-00020} ======================== 2.1. Study Isolates {#sec2dot1-jof-03-00020} ------------------- Forty-four clinical isolates of non-dermatophyte fungi commonly causing onychomycosis were collected from Sri Ramachandra Medical College and Research Institute (Chennai, India), LV Prasad Eye Institute (Hyderabad, India) and Kempegowda Institute of Medical Sciences (Bengaluru, India) and used for in vitro susceptibility testing against itraconazole and efinaconazole. In total, 12 *F. falciforme*, 4 *F. keratoplasticum*, 2 unnamed members of the *Fusarium solani* species complex, 2 *F. delphinoides*, 1 *F. incarnatum*, 10 *A. flavus*, 2 *A. terreus*, 5 *A. niger*, 3 *A. fumigatus*, 1 *Alternaria* species, 1 *Penicillium* species, and 1 *Candida albicans* were tested. The isolates were cultured on Sabouraud Dextrose Agar (SDA), Potato Dextrose Agar (PDA) and Oatmeal Agar (OA) (HiMedia Laboratories Pvt. Ltd., Mumbai, India). Macroscopic observations including growth rate, and features of colony morphology such as texture, colour and production of diffusible pigment were noted. The microscopic features like conidia formation, presence of macroconidia (with number/nature of septa), presence of microconidia and chlamydospores were observed in lactophenol cotton blue wet mounts. Speciation was done accordingly. For *Fusarium* isolates, speciation was done by DNA sequencing of TEF-1α (Translation Elongation Factor 1-alpha) using primers EF1 (forward primer; 5′-ATGGGTAAGGA(A/G)GACAAGAC-3′) and EF2 (reverse primer; 5′-GGA(G/A)GTACCAGT(G/C)ATCATGTT-3′) \[[@B24-jof-03-00020]\] and BLAST (Basic Local Alignment and Search Tool) matching at NCBI GenBank (<http://www.ncbi.nlm.nih.gov/BLAST/>) and Fusarium-ID (<http://isolate.fusariumdb.org/index.php>) databases. 2.2. Antifungal Agents {#sec2dot2-jof-03-00020} ---------------------- Antifungal drugs were obtained in pure powder form; itraconazole (Sigma-Aldrich, St. Louis, MO, USA) and efinaconazole (Hwasun Technologies, Shanghai, China) and were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to make stock solutions of concentrations 100X the highest concentration to be tested. Itraconazole was tested in the range of 0.06125--32 µg/mL for *Fusarium* species and in the range of 0.0078--4 µg/mL for *Aspergillus* and other species. Efinaconazole was tested in the range of 0.0078--4 µg/mL for all isolates. 2.3. In Vitro Antifungal Susceptibility Testing {#sec2dot3-jof-03-00020} ----------------------------------------------- Antifungal susceptibility testing was done according to the CLSI-M38-A2 guidelines following the broth microdilution method described by the Clinical and Laboratory Standards Institute (CLSI) \[[@B25-jof-03-00020]\]. The fungal growth medium used was the synthetic broth medium RPMI-1640 with L-glutamine, without sodium bicarbonate and with phenol red as a pH indicator (HiMedia Laboratories, Mumbai, India) buffered to a pH of 7.0 at 25 °C using MOPS (3-\[*N*-morpholino\] propanesulfonic acid) buffer (Sigma-Aldrich, St. Louis, MO, USA). A saline spore suspension of a ten-day-old well-sporulated culture on Potato Dextrose Agar (PDA) slant (HiMedia Laboratories Pvt. Ltd., Mumbai, India) was used for inoculum preparation. With the turbidity of the saline spore suspension adjusted to 0.5 McFarland Turbidity Standard (Appendix C section of CLSI M38-A2)., it was then further diluted 1:50 times (for moulds) and 1:500 (for yeasts) in RPMI-1640 with [l]{.smallcaps}-glutamine and without sodium bicarbonate This served as the final inoculum and 0.1 mL i.e., 100 µL of this inoculum suspension and 100 µL of the antifungal drug dilutions were added to the wells of the microdilution plates. Each drug was designated one row in the plate with each well in the row for a particular drug concentration. The growth control well contained 100 µL of the inoculum and 100 µL of the drug diluent without antifungal agent. Sterility control was also included with plain RPMI-1640 medium without inoculum. *Paecilomyces variotii* CBS 132734 was included in the tests as a quality control strain. The plates were incubated at 35 °C and the readings were taken after 48 h of incubation. The first well showing 100% inhibition of growth in the case of moulds and 50% inhibition of growth in the case of yeasts were taken as the Minimum Inhibitory Concentration (MIC). Susceptibility testing was performed in triplicate and the modal MIC for each drug was selected as the MIC value of the drug for the particular isolate. 2.4. Statistical Analysis {#sec2dot4-jof-03-00020} ------------------------- All MIC data for efinaconazole and itraconazole were recorded and analyzed using Microsoft Excel 2010. Geometric mean MIC, MIC50, MIC90 and cumulative MIC frequency of both drugs were calculated. 3. Results {#sec3-jof-03-00020} ========== 3.1. Susceptibility Pattern of Test Isolates against Efinaconazole and Itraconazole {#sec3dot1-jof-03-00020} ----------------------------------------------------------------------------------- [Table 1](#jof-03-00020-t001){ref-type="table"} concisely represents the MIC range and geometric mean MIC of all 44 test isolates of the present study. 3.2. Fusarium Species {#sec3dot2-jof-03-00020} --------------------- Efinaconazole inhibited fungal growth at concentrations ranging from 0.03125--2 µg/mL ([Table 1](#jof-03-00020-t001){ref-type="table"}). However, based on the MIC90 for the genus (not shown in [Table 1](#jof-03-00020-t001){ref-type="table"}), most of the *Fusarium* isolates were inhibited between 0.25--0.5 µg/mL. *Fusarium dimerum* species complex showed the least MIC for efinaconazole (geometric mean MIC 0.088 µg/mL). The cumulative MIC frequency of efinaconazole against 21 *Fusarium* species tested is shown in [Figure 1](#jof-03-00020-f001){ref-type="fig"}. In contrast, itraconazole failed to exhibit inhibitory action against *Fusarium* with high MICs 16--\>32 µg/mL. There were no differences in susceptibility patterns by geographical location. 3.3. Aspergillus Species {#sec3dot3-jof-03-00020} ------------------------ Efinaconazole showed uniformly excellent activity against all *Aspergillus* species tested, with an MIC equal to 0.0078 µg/mL, the lowest concentration of the drug tested. Itraconazole also showed good activity against *Aspergillus* with an MIC range of 0.0078--1 µg/mL, with most isolates being inhibited between 0.25--1 µg/mL, based on the MIC90 data for the genus (not shown in [Table 1](#jof-03-00020-t001){ref-type="table"}). *Aspergillus flavus* showed the maximum susceptibility to itraconazole among the *Aspergillus* species, with a geometric mean MIC of 0.22 µg/mL. The cumulative MIC frequency of itraconazole against 20 *Aspergillus* species tested is shown ([Figure 2](#jof-03-00020-f002){ref-type="fig"}). There were no differences in susceptibility patterns by geographical location. 3.4. Other Species {#sec3dot4-jof-03-00020} ------------------ Three isolates, one each of *Penicillium* sp., *Alternaria* sp. and *Candida albicans* that were tested showed an MIC of 0.016 µg/mL for efinaconazole and and MIC of 0.25 µg/mL (*Alternaria* sp.) and 0.125 µg/mL (*Candida albicans* and *Penicillium* sp.) for itraconazole. 4. Discussion {#sec4-jof-03-00020} ============= In the present study, itraconazole MICs of test isolates were in the range as established by previous studies \[[@B21-jof-03-00020],[@B22-jof-03-00020],[@B26-jof-03-00020],[@B27-jof-03-00020],[@B28-jof-03-00020]\]. MICs of efinaconazole were significantly lower than that of itraconazole in all 44 isolates tested (*p* \< 0.001, unpaired *t*-test). Especially for *Fusarium* species, which are known to cause recalcitrant infections \[[@B29-jof-03-00020]\], efinaconazole showed roughly eight-fold more activity than itraconazole based on the geometric mean MICs of both the drugs. Itraconazole MICs were above the epidemiological cut-off values (ECVs) recently established for *Fusarium* \[[@B30-jof-03-00020]\]. A few researchers have also reported similar findings regarding *Fusarium* MICs to efinaconazole \[[@B23-jof-03-00020]\]. In other studies \[[@B16-jof-03-00020]\], two- to three-fold greater mycological and complete cure rates were observed clinically with efinaconazole 10% topical solution than those with other topical antifungal drugs. Based on geometric mean MICs, efinaconazole showed 74-fold more activity than itraconazole for *Aspergillus* species in the present study, comparable to other studies \[[@B23-jof-03-00020]\]. From the MIC data, efinaconazole is seen to be more potent than itraconazole against different non-dermatophyte fungi causing onychomycosis and can be recommended as an effective drug for use in treatment. Early treatment of onychomycosis to avoid disease progression to other toenails is important. An infected nail also serves as a mycotic reservoir and a potential portal of entry for systemic dissemination \[[@B1-jof-03-00020],[@B2-jof-03-00020],[@B3-jof-03-00020]\]. The low keratin affinity of efinaconazole contributes to the drug's nail penetration and fungicidal activity in onychomycosis treatment \[[@B16-jof-03-00020]\]. The morphological changes seen in fungal structure due to exposure to efinaconazole include shortening of interseptal distance, globular swellings, non-uniform widths and flattening. The ultrastructural changes consist of thickening of the cell wall, separation of plasma membrane from the cell wall, accumulation of electron-dense granules in the space between the cell wall and plasma membrane, and discontinuity of the plasma membrane along with degeneration of intracytoplasmic organelles. All these changes are likely to be responsible for cell death \[[@B15-jof-03-00020]\]. Prolonged antifungal drug exposure can induce a stress response in fungal cells, leading to a decrease in drug susceptibility via mechanisms such as the induction of drug efflux pumps. However, a study by Iwata et al. \[[@B31-jof-03-00020]\] observed no evidence of efinaconazole resistance under experimental conditions among dermatophytes, suggesting that efinaconazole has low potential to induce drug resistance, at least among dermatophyte species. Efinaconazole 10% topical solution marketed as Jublia (the US and Canada) and Clenafin (Japan) has a better safety profile than oral itraconazole therapy which requires blood monitoring \[[@B32-jof-03-00020]\]. It is labeled pregnancy class C with a caution from the FDA (Food and Drug Administration) regarding use in breastfeeding women, although it is unknown if it is excreted into human milk. The typical duration of treatment is an application once daily with a flow-through brush applicator for 48 weeks. As the drug is topical, patient compliance is better and associated toxicities are lesser than those with oral itraconazole. Many cases of onychomycosis are of mixed-pathogen nature, and the broad spectrum of activity of efinaconazole is useful in treating such cases. Unfortunately, efinaconazole is still not available in the Indian market \[[@B33-jof-03-00020]\]. Thousands of patients could benefit from this drug if it is made available in India at the earliest. 5. Conclusions {#sec5-jof-03-00020} ============== Efinaconazole proves to be a more potent drug than itraconazole for the treatment of onychomycosis due to non-dermatophyte fungi including *Fusarium* species. Its topical route of administration and low keratin binding make it more suitable for prolonged treatment than the oral drugs currently in use. Efinaconazole needs to be made available for patients in India. This work was supported by the Indian Council of Medical Research. Ananya Tupaki-Sreepurna, Anupma Jyoti Kindo and Murugan Sundaram conceived experiments; Anupma Jyoti Kindo, Murugan Sundaram, Savitri Sharma and Anjana Gopi designed experiments; Ananya Tupaki-Sreepurna, Bhavna T. Jishnu and Vijayakishore Thanneru performed experiments; Ananya Tupaki-Sreepurna, Bhavna T. Jishnu, Vijayakishore Thanneru, Anupma Jyoti Kindo, Savitri Sharma and Anjana Gopi analyzed data; Ananya Tupaki-Sreepurna, Bhavna T. Jishnu and Vijayakishore Thanneru wrote parts of the manuscript; Anupma Jyoti Kindo and Murugan Sundaram edited the manuscript. The authors declare no conflict of interest. {#jof-03-00020-f001} {#jof-03-00020-f002} jof-03-00020-t001_Table 1 ###### In vitro antifungal activities of efinaconazole and itraconazole against common non-dermatophyte fungal agents of onychomycosis. Organism (Number of Isolates) Drug MIC (µg/mL) ----------------------------------------------------------- --------------- ------------- -------- *Fusarium falciforme* (12) Efinaconazole 0.063--1 0.19 Itraconazole \>32 \>32 *Fusarium keratoplasticum* (4) Efinaconazole 0.032--2 0.18 Itraconazole \>32 \>32 *Fusarium solani* species complex --- unnamed members (2) Efinaconazole 0.50--1 0.70 Itraconazole \>32 \>32 *Fusarium dimerum* species complex (2) Efinaconazole 0.032--0.25 0.088 Itraconazole 16--32 22.63 *Fusarium incarnatum equiseti* species complex (1) Efinaconazole 0.50 0.50 Itraconazole \>32 \>32 *Aspergillus flavus* (10) Efinaconazole 0.0078 0.0078 Itraconazole 0.0078--1 0.22 *Aspergillus niger* (5) Efinaconazole 0.0078 0.0078 Itraconazole 0.25--1 0.57 *Aspergillus fumigatus* (3) Efinaconazole 0.0078 0.0078 Itraconazole 1 1 *Aspergillus terreus* (2) Efinaconazole 0.0078 0.0078 Itraconazole 0.25 0.25 *Penicillium* species (1) Efinaconazole 0.016 0.016 Itraconazole 0.13 0.13 *Alternaria* species (1) Efinaconazole 0.016 0.016 Itraconazole 0.25 0.25 *Candida albicans* (1) Efinaconazole 0.016 0.016 Itraconazole 0.13 0.13

Introduction {#sec1} ============ COVID-19 caused by SARS-CoV-2 produced an international outbreak at the end of 2019, and on 11 March 2020 the World Health Organization declared it a global pandemic. The pandemic spread to the UK by late January 2020, and on 23 March, the UK government instituted a lockdown on the whole population. In other respiratory tract infections, it is well documented that the risk of stroke is increased by a factor of 2.3--7.82 within the first 3 days of infection.[@bib1] Although early evidence suggests COVID-19 also confers an increased risk of acute ischaemic stroke (AIS), the underlying pathological mechanism remains uncertain, although multiple reports suggest infected patients can develop a hypercoagulable condition[@bib2], [@bib3], [@bib4]; D-dimer levels are reported to be up to 12-fold higher than normal.[@bib2] In a study of 221 consecutive patients admitted to one hospital in Wuhan, China, with confirmed COVID-19, AIS occurred in 11 (5%) of patients with a range of stroke subtypes.[@bib2] COVID-19 causes the most severe illness in the elderly, the immunocompromised, and those with other significant comorbidities[@bib5] ^,^ [@bib6]; most patients with COVID-19-related AIS fall into one or more of these categories. Mechanical thrombectomy (MT) alongside intravenous thrombolysis (if not contraindicated) is the first-line treatment for patients with AIS and occlusion of a large cerebral artery demonstrated by computed tomography (CT) angiography (CTA) or magnetic resonance angiography (MRA).[@bib7] The COVID-19 pandemic has presented new and varied challenges to the still-evolving UK MT services. Practices within interventional neuroradiology (INR) theatres have had to be significantly modified to protect both staff and patients. National and international interventional and neuro-interventional societies have issued guidelines regarding recommended changes in practice, some of which have contributed to forming a framework for current clinical practice.[@bib8] ^,^ [@bib9] As the UK emerges from the worst of the initial peak of the pandemic, the authors, on behalf of the British Society of Neuroradiologists (BSNR) and the UK Neurointerventional Group (UKNG), sought to review the initial challenges to the UK\'s MT service and its response in order to evaluate and disseminate the lessons learned. Materials and methods {#sec2} ===================== An online survey (Google Forms) was sent out on 1 May 2020 to all 28 UK neuroscience centres that have the potential capability to perform MT (Electronic Supplementary Material S1: Survey). Standard data and statistical analysis (*t*-test and Fisher\'s exact test) was carried out using GraphPad Prism (GraphPad Software). Statistical significance was set at p\<0.05. Number of MTs performed {#sec2.1} ----------------------- To gain insight into MT service provision across the UK, the survey asked for the number of patients undergoing MT during each month from January to April 2020, in order to document the rate of MT procedures carried out immediately before and during the first 3 months of the COVID-19 pandemic in the UK. MT patient pathway {#sec2.2} ------------------ To establish changes made to the MT triage protocols for patients with AIS during the COVID-19 pandemic, in the absence of a positive COVID-19 test result, the survey requested information regarding what screening measures for COVID-19 had been introduced for MT patient referrals and whether all MT-eligible patients were managed as if they had suspected COVID-19. Data were requested regarding whether there had been any tendency in the UK to narrow the acceptance criteria for AIS patients referred for MT in an effort to (a) mitigate the spread of COVID-19 to neuroscience centre patients, healthcare workers, and their families, (b) to target therapies to those patients in most need, and (c) to optimise allocation of healthcare resources including personal protection equipment (PPE). Avoiding delays between stroke onset and recanalisation of large vessel occlusion (LVO) is of paramount importance in achieving optimal outcomes for AIS patients; however, additional safety measures as a result of the COVID-19 pandemic inevitably hindered normal workflow patterns before, during, and after MT. The survey also assessed whether additional precautions at several stages of MT patient flows caused delays in treatment, and what were considered the primary contributing factors. Furthermore, hospital visitors are largely prohibited during the pandemic, which presents challenges to obtaining an accurate history and conducting informed consent. The survey sought responses to ascertain whether the pandemic had affected the patient consent process prior to MT and how the absence of relatives accompanying the patient had changed established working practices. Intra-procedural additional safety precautions are essential to minimise exposure of healthcare staff to respiratory secretions from patients with confirmed or suspected COVID-19 and those patients in whom the COVID-19 status is uncertain. The survey was used to establish the rates of local (LA) and general anaesthetic (GA) used for patients undergoing MT anaesthetic prior to the COVID-19 outbreak, whether or not it was routine practice to intubate patients in theatre prior to bringing the patient into the angiography room for MT, and determine if practice had changed as a result of the pandemic. It is important to ensure safe levels of PPE are used by all members of staff within the angiography suite. It is also essential to minimise the number of personnel in the room who are potentially exposed to patient respiratory aerosol. The survey also assessed the level of PPE used during MT procedures in the UK under the conditions of either local or general anaesthesia. There have been anecdotal reports of procedural differences experienced whilst treating COVID-19 patients with AIS including rate of successful recanalisations, clot location, consistency, and burden of thrombus. Centres were asked to provide the number of successful recanalisations, defined as thrombolysis in cerebral infarction (TICI) scores of 2B or above, during the months from January to April 2020. Additionally, the survey sought to evaluate whether UK INRs had noticed procedural differences in treating COVID-19 patients in comparison with non-COVID-19 patients and whether clinical outcomes and mortality rates had changed. Post-procedurally, for suspected or confirmed COVID-19 patients, data were required regarding whether MT centres had changed their practice around recovery or routine post-MT brain imaging, and if there had been any issues with regards to repatriation of MT patients to the referring primary stroke centre. Effects on training and working {#sec2.3} ------------------------------- UK MT centres were asked whether or not the COVID-19 pandemic had affected training opportunities for INR specialist registrars (SpR), and if yes, what the reasons were. To determine the impact of the pandemic, the survey questioned whether UK MT centres had split INRs into separate teams and to provide details on how they had arranged their rota accordingly. Additional responses were gathered in relation to any other changes to INR working patterns and if any centres had noticed an adverse impact on the mental health of INRs and the wider MT team. Impact on MT service development {#sec2.4} -------------------------------- Respondents were asked to indicate whether the COVID-19 pandemic had hampered their centre\'s MT service development. Results {#sec3} ======= Responses were received from 27/28 MT-capable centres (96%) via an online web link (see Acknowledgements). Of the 27 responders to the UKNG/BSNR survey, three UK neuroscience centres do not currently provide MT services. Following lockdown on 23 March 2020, there was a 27.7% reduction in MTs performed during April 2020 compared with the first 3 months of the year ([Fig 1](#fig1){ref-type="fig"} ).Figure 1Number of MTs performed across the UK in 2020 (*t*-test).Figure 1 Twenty of the 24 MT-active centres that responded considered all MT patients as COVID-19 suspicious/positive unless or until proven otherwise. Sixteen of the 24 centres used symptomatic screening questionnaires at the point of referral. Four of the 24 centres introduced a CT chest as an additional screening test to the patient referral pathway. Seven of the 24 centres used no additional COVID-19 pre-MT screening procedures ([Fig 2](#fig2){ref-type="fig"} ).Figure 2COVID-19 pre-MT acceptance screening procedures.Figure 2 Eleven of 24 MT-active UK centres had changed their case selection towards stricter adherence to national MT patient acceptance criteria and some had introduced an age threshold. Reasons given included additional delays in patient transfer potentially resulting in futile procedures. Twenty-two out of 24 centres reported delays to the patient pathway. More than one response per centre was permitted to facilitate documentation of multiple possible contributory reasons. The most common reasons for delay were delayed presentation from stroke onset (16/24), delayed investigation of MT patients (11/24), delayed referral to MT centre (9/24), and delayed inter-facility transfer (9/24). Limited resources were reported as a contributing factor in 16/24 centres including availability of an appropriate bed, lack of anaesthetic availability, angiography room occupancy for patient recovery, and/or delays from additional COVID-19-related angiography room cleaning. Twelve of 24 centres reported having to change their consent process and or their practices for communicating with relatives. There had been no significant change to the proportion of MTs performed under GA during the COVID-19 pandemic (range from 72.7% to 64.3%; [Fig 3](#fig3){ref-type="fig"} ). Prior to the pandemic 2/24 centres routinely performed endotracheal intubation in general theatres prior to transferring patients to the angiography suite; during the pandemic 5/24 centres did.Figure 3Number of MTs performed under GA (Fisher\'s exact test).Figure 3 For MT performed under LA, 16/24 centres used enhanced PPE, 7/24 used safe PPE (fluid-resistant surgical mask, face shield/goggles, and gloves) and 1/24 did not employ additional measures to standard theatre operating kit. For MT performed under general anaesthesia, 16/24 centres used enhanced PPE and 8/24 used safe PPE. There was no significant change to the proportion of MT procedures that resulted in successful recanalisation (defined as TICI2B--3 inclusive; range from 84.2% to 79.1%; [Fig 4](#fig4){ref-type="fig"} ); however, six of 24 centres reported (subjectively) more friable and or adverse clot consistency in COVID-19 patients. Three centres reported a decrease in National Institutes of Health Stroke Scale (NIHSS) at 24 h post-MT in COVID-19 patients and one centre reported an increase in mortality post-MT in COVID-19 patients.Figure 4Successful recanalisation defined as TICI 2B/2C/3 (Fisher\'s exact test).Figure 4 Ten of the 24 MT-active centres changed to recovering suspected or confirmed COVID-19 patients within the angiography suite following their MT procedure. No centre reported a change to their standard post-MT imaging protocol. Six of the MT-active 24 centres reported additional delays to patient repatriation to the primary stroke centre post-MT, the most commonly cited reason being a lack of availability of appropriate isolation beds (n=4). Seventeen of the 24 centres reported that the COVID-19 pandemic had reduced training opportunities for INR SpRs. Reduced caseload (15/24), infection-control measures mandating minimum staff being present for procedure (8/24), and trainee service re-deployment (6/24) were the main reasons given. As a result, 3/24 centres said they planned to extend training programmes for affected SpRs. Nine of the 24 centres had split their INR consultant staff into teams during the pandemic, mostly into 1-in-2 or 1-in-3 rotas. Other changes to the normal working patterns included virtual multidisciplinary team meetings (MDT), (partial) re-deployment to cover diagnostic radiology rotas, reduced out-of-hours services due to reduced nursing support, and working from home (remote reporting of neurological imaging). In the present survey, 10/24 centres reported an adverse effect on the mental health of the wider angiography team, and 8/24 centres reported an adverse effect on the mental health of INRs. Fourteen of the 24 centres reported that the pandemic had hampered their development plans for their local or regional MT service. Discussion {#sec4} ========== Early evidence from Chinese studies demonstrated that COVID-19 can cause neurological deficits in infected patients and AIS in approximately 5%.[@bib2] ^,^ [@bib4] Despite the Chinese data indicating high stroke incidence in COVID-19 patients, reports from most countries affected by the pandemic suggest that referrals to hospital for stroke treatment including MT actually fell, at least initially, following government-imposed lockdown measures. In a prospective study of 1,513 patients at 32 centres in all the administrative regions of France, there was a 21% decrease in MT case volumes during the initial stages of the COVID-19 epidemic.[@bib10] Although some cities, notably New York, noticed a surge in MT numbers, including for AIS in younger patients,[@bib11] a national survey of 856 hospitals across the USA found a 39% decline in the number of patients receiving stroke imaging.[@bib12] The present survey indicates a similar trend with a 27.7% reduction in MTs performed in April 2020 across the UK. The efficacy of MT is inversely proportional to the delay between stroke onset and LVO recanalisation. There are anecdotal accounts of patients being reluctant to present to hospital during the pandemic. Once admitted, additional safety measures were, of necessity, imposed onto the patient journey including added precautions on the stroke ward, in the CT machines, during ambulance transfer, during airway preparation, within the angiography suite, and during warding and repatriation. In France, significantly increased delays were found between imaging and groin puncture, overall and in transferred patients in particular.[@bib10] The majority of the UK centres encountered delays to multiple steps in the patient pathway. Given the uncertainty over the duration of the pandemic, these issues need to be urgently addressed to ensure the best possible functional outcomes for patients.[@bib13] One of the challenges in identifying COVID-19 in AIS is an inability to obtain an accurate history of clinical symptoms due to underlying speech problems and/or confusion. Furthermore, at present COVID-19 status cannot be confirmed or excluded using antigen or antibody testing within the timeframe required for initial evaluation and decision-making regarding time-critical therapies for patients with AIS. A significant rate of false-negative antigen tests is known to occur. The Society of NeuroInterventional Surgery (SNIS) recommendation was that, for MT patients without a documented "negative" COVID-19 status, healthcare staff in the angiography room should wear enhanced PPE at all times, including N-95 or FFP2/FFP3 air-filtration mask.[@bib9] Discordant pre-MT screening practices and variable use of PPE across the country was likely a reflection of initially confusing and rapidly changing guidelines at local, regional, and national level. Given that the majority of patients presenting for MT will have unknown COVID-19 status, the Society for Neuroscience in Anesthesiology & Critical Care (SNACC), SNIS and European Society of Minimally Invasive Neurological Therapy (ESMINT) strongly recommended a lower threshold for intubation, especially if the anaesthetist or INR have any concerns for possible conversion from LA to GA.[@bib8] ^,^ [@bib9] ^,^ [@bib14] It is further recommended that intubation should not take place within the angiography suite, but rather in a negative-pressure environment, regardless of its location, prior to transfer to the angiography suite. Fiorella *et al.* cited The Anaesthesia Patient Safety Foundation recommendation that suspected or confirmed COVID-19 patients should not be brought back to post-acute care units, and those requiring extubation should not have this performed in the angiography suite.[@bib15] In preparation for potential future pandemics, and in the interest of infection control in general, it is preferable to have negative-pressure angiography rooms and/or a separate area for anaesthetic induction and post-MT recovery within the interventional radiology theatres. Working during the pandemic has brought many challenges; however, UK centres have adapted local processes at pace to ensure ongoing provision of this vital health service with no significant changes to the reported rate of successful recanalisation. Going forward, the adverse impact on service development, training for SpRs, and the effect on the mental health of INR and wider teams should be acknowledged. Some limitations of this survey need to be acknowledged. The qualitative assessment of patient delays provides an overall insight to the issues faced at UK MT centres; however, further analysis on patient outcome could not be ascertained. The subjective binary self-reporting of mental health of UK MT teams during the pandemic limits deeper understanding of its impact. The changes to training were assessed by clinical INR leads at each centre rather than relying on responses from SpRs themselves. In conclusion, the present survey has highlighted a trend of decreasing cases and delays in the patient pathway during the early stages of the COVID-19 pandemic across UK centres. Patient anxiety and initial confusion amongst stroke/INR teams may have initially contributed to reduced numbers of MT procedures. Further and larger studies would help advance knowledge of how changes in practice for this essential time-critical service can improve the management of MT-eligible patients during this or any future pandemic. Conflict of interest {#sec5} ==================== The authors declare no conflict of interest. Appendix A. Supplementary data {#appsec1} ============================== The following is the Supplementary data to this article:Multimedia component 1Multimedia component 1 The authors thank the INR teams at Barts Health NHS Trust, Belfast Trust Hospitals, Brighton and Sussex NHS Trust, Newcastle Upon Tyne Hospitals NHS Foundation Trust, Leeds Teaching Hospitals Trust, Cardiff and Vale University Health Board, Liverpool University Hospitals NHS Foundation Trust, North Bristol NHS Trust, Lancashire Teaching Hospitals NHS Foundation Trust, Salford Royal NHS Foundation Trust, University Hospital of North Midlands NHS Trust, Hull University Teaching Hospitals NHS Trust, University Hospitals Plymouth NHS Trust, Imperial College Healthcare NHS Trust, St George\'s University Hospitals NHS Foundation Trust, King\'s College Hospital NHS Foundation Trust, University College London Hospitals NHS Foundation Trust, University Hospital Southampton NHS Foundation Trust, Oxford Health NHS Foundation Trust, University Hospitals Birmingham NHS Foundation Trust, Sheffield Teaching Hospitals NHS Foundation Trust, Coventry and Warwickshire Partnership NHS Trust, Cambridge University Hospitals NHS Foundation Trust, NHS Lothian (and on behalf of NHS Greater Glasgow and Clyde) and South Tees Hospitals NHS Foundation Trust for their cooperation with this UK Neurointerventional Group (UKNG) and the British Society of Neuroradiologists (BSNR) national survey. N.M. has undertaken educational consultancy work for Microvention-Terumo. P.W. has received institutional research grants form Microvention and Medtronic and unrestricted institutional educational grants form Stryker, Penumbra and Medtronic in addition to having undertaken educational consultancy work for Microvention-Terumo. R.C. has undertaken educational consultancy work for Microvention-Terumo and Stryker. W.I. has undertaken educational consultancy work for Stryker. Supplementary data to this article can be found online at <https://doi.org/10.1016/j.crad.2020.07.001>.

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Introduction {#S1} ============ Executive Functions (EF) can be understood as a variety of interrelated processes that help to direct and control mental abilities to accomplish a task or goal ([@B64]). [@B53] propose a hierarchical model in which EF is considered as a unitary construct with three main components: (1) inhibition, (2) updating, and (3) shifting. Inhibition is the ability to suppress one automatic or prepotent response in favor of another, or to suppress the response altogether, known as response inhibition. Another aspect of inhibition is interference control, which is required to select relevant stimuli when a distractor appears ([@B53]; [@B21]; [@B85]; [@B78]). This process is one of the first stages to develop and is thought to be responsible for changes in other EF components ([@B20]; [@B31]). Updating is the ability to retain and manipulate information during a short period of time ([@B53]; [@B44]). This ability is essential for learning ([@B14]). Finally, shifting is the ability to change from "one mental set" to another ([@B53]). These components are involved in several everyday activities ([@B21]). Previous studies have found a relation between EF and intelligence ([@B1]; [@B54]; [@B42]); however, EF is even more predictive of academic success than IQ ([@B33]; [@B7]). Apart from academic success, EF also seems to have an impact on social adjustment ([@B9]). "Social adjustment is defined as the degree to which children get along with their peers; the degree to which they engage in adaptive, competent social behavior; and the extent to which they inhibit aversive, incompetent behavior" ([@B16], p.82). Difficulties in EF are present in social maladjustment ([@B57]; [@B7]). EF components are impaired in various childhood disorders ([@B3]), such as ADHD ([@B63]; [@B34]), autism ([@B12]), obsessive-compulsive disorder ([@B25]), and behavioral disorders ([@B63]). For these reasons, studies on EF interventions in children and the mechanisms involved in their development are relevant. This knowledge can be applied to EF programs aimed at school settings for typically developing children as a protective factor or in clinical contexts for those with EF difficulties as part of the intervention. If inhibition is one the core components of EF, the intensity domain of attention is the core component of attention ([@B75]). The intensity domain involves alertness, sustained attention and vigilance as the basis of attention ([@B37]). Tonic alertness is thought of as a top-down control function of the arousal system without the influence of external stimuli, whereas phasic alertness is the capability to respond following a warning stimulus ([@B76]). Sustained attention involves the detection of changes over a long period with a high rate of relevant stimuli. In contrast, vigilance, a state of sustained alertness, involves the detection of changes when only a low rate of relevant stimuli exists ([@B37]). Some aspects of attention overlap with certain components of EF ([@B69]), which explains the high degree of interaction between attention and EF. The core processes of attention and EF are related; for instance, inhibition is fundamental for attentional maintenance ([@B60]). Furthermore, previous research has found that children with higher levels of sustained attention present high levels of inhibitory control ([@B64]). Sustained attention and behavioral inhibition interact throughout child development. A longitudinal study (testing attention at 9 months and studying behavioral inhibition until adolescence) demonstrated that sustained attention is related to inhibitory control. Individuals with lower levels of sustained attention presented increased levels of behavioral inhibition during childhood and social discomfort during adolescence ([@B58]). Apart from sustained attention, vigilance and inhibitory control are closely related ([@B51]). Studying the attentional element involved in EF tasks, procedural metacognitive strategies (including self-regulatory strategies) and related skills may help us to design EF training strategies and interventions based on scientific data. Attention is strongly needed in EF tasks, and EF and self-regulation share resources ([@B40]). Some attention training has shown benefits in EF tasks. One study demonstrated how attention training in children with ADHD not only reduced symptoms of inattentiveness, but also enhanced EF, specifically, by shifting attention ([@B45]). Studies on attention span and working memory have shown how training benefits participants with ADHD with regard to EF ([@B44], [@B43]; [@B4]). In our view, due to the interaction between attention, EF and self-regulation, training that combines these processes may produce more transfer effects than just training EF alone. Following this hypothesis, our team developed Nexxo-training, which aims to improve vigilance, inhibition and procedural metacognitive strategies in typically developing children. Most cognitive training can be classified into two categories: process-based training and strategy-based training ([@B55]; [@B39]). Both approaches involve practice or intentional instruction to improve cognitive skills. The main difference is that strategy-based training uses more explicit task instructions than process-based training ([@B39]). Regarding attention and EF training, a few process-based training methods have shown positive effects in typically developing children, either in terms of attention ([@B80]) executive attention ([@B70]), fluid intelligence ([@B43]; [@B49]), or academic performance ([@B17], [@B18]; [@B38]). Nevertheless, the limitations of process-based training have been found in the far transfer or generalization of the training in the user's everyday life. Similarly, limitations have been found in long-term effects ([@B67]). The aim of EF training should be the generalization of the training in children's daily life, in cognitive skills, academic performance, and social adjustment, which are considered "far transfer." A significant number of previous studies on EF training efficacy fail to find or examine these types of transfer results ([@B67]). To overcome this limitation of traditional process-based training, strategy-based training provides guidance with the tasks which help users to identify the strategies needed to perform those tasks. An example of this kind of guidance is scaffolding, or metacognitive strategies, designed in combination with the training ([@B61]). Indeed, strategy-based training has yielded positive results. [@B61] compared two groups with executive attention training in typically developing children with an active control group. One of the training groups followed traditional attention and EF protocol, whereas the other underwent metacognitive strategies. The children in the metacognitive group showed not only greater gains in intelligence, but also significant increases in conflict processing, measured through electrophysiological techniques. In addition, changes in brain activity regarding conflict processing predicted gains in intelligence in this group. The EF and attention intervention program that we analyze, called Nexxo-training, combines inhibition and vigilance training through a touchscreen application with strategies of "procedural metacognition" directed by a single instructor. This strategy-based training consists of repeating a task in combination with strategies to improve performance tasks. The unique feature of this specific strategy-based training is that the training provides not only procedural metacognitive strategies (i.e., general strategies for the whole group), but also compensatory strategies for participants who experience greater difficulty during the training. In this way, the developmental processes involved in the attention and EF training task can be easily improved and generalized. A previous study of Nexxo-training, a randomized-controlled study, showed far transfer after training in supervision, attention and EF as reported by parents (Rossignoli-Palomeque et al., submitted). Far transfer occurs when training effects are produced in tasks or constructs that have not been directly trained. By contrast, near transfer occurs when the effects are reflected in similar tasks to those that have been directly trained ([@B42]). Further research on this type of training is crucial as it offers a new direction for cognitive training interventions. In addition, to plan any form of attention and EF intervention, developmental factors must also be considered. In general, the initial manifestations of EF occur during the 1st year of life, with accelerated development in childhood ([@B10]). EF development may be a pyramidal process. Certain basic components, such as inhibition, will later support the development of other more complex processes, such as flexibility ([@B29]). Nevertheless, other components, such as planning, do not reach adult levels until approximately the age of 12 years old while others, such as abstraction, will continue to develop into adulthood ([@B90]) reaching peak performance at around 20--30 years of age ([@B8]). Regarding attention, conscious control of attention increases between 2 and 6 years of age ([@B68]; [@B22]). There is a second significant improvement in cognitive control of attention at around 9--12 years of age ([@B62]). Meanwhile, sustained attention improves significantly between the ages of 3 and 5 years old ([@B32]) and continues to develop progressively throughout a child's school years. There are significant changes in sustained attention from 6 to 7 years of age in comparison with 10- to 11-year-olds ([@B48]). Inhibition and attention are relevant cognitive abilities. In terms of development, go/no-go tasks have demonstrated a significant improvement in response inhibition and sustained attention between the ages of 6 and 8 years old, while these changes are more subtle from 8 to 11 years of age ([@B47]). Previous studies, using go/no-go tasks for assessment, support the same idea that there is an improvement in response inhibition abilities between the ages of 6 and 8 years ([@B5]). Inhibition is a process that develops particularly between the ages of 5 and 10 years ([@B84]). Apart from the relation between attention, EF and developmental factors, it is also worth considering what other skills and strategies may be involved in performing attention and EF tasks successfully. Previous studies have shown that inhibition training in preschoolers produced a trend-level improvement in reasoning and neural changes in the experimental group ([@B49]). Other authors suggest that students with a high IQ also perform well in EF tasks, specifically in inhibition and flexibility ([@B72]). On the other hand, lower vigilance performance has been linked to a lower IQ in children who are at risk of learning disabilities ([@B77]). Therefore, if attention, EF and intelligence are related, which specific cognitive abilities are involved, and which are better at predicting attention and EF performance? These crucial questions must be addressed by attention and EF training developers. Regarding schoolchildren's use of procedural metacognitive strategies in inhibitory tasks, it seems that verbal strategies (e.g., verbalizations of what to do/not do) and motor strategies (e.g., moving away, shaking their heads, covering their mouths, etc.) are used by preschoolers to inhibit themselves ([@B26]). The combination of both types of strategies seems to produce better inhibitory results ([@B52]). The development of these strategies depends on the child's age. For instance, verbalizations and inner speech evolve between 2 and 8 years of age, from irrelevant speech to self-directed verbalizations, both of which are relevant to the task ([@B88]). Another type of strategy, which seems to promote better results in EF tasks in older students and adults, are self-instructions (e.g., saying out loud what to do, how to do it, etc.) ([@B41]). The development of these strategies varies throughout child development ([@B86]; [@B6]) and is also based on the level of task difficulty ([@B27]). Nexxo-training strategies consist of procedural metacognitive strategies. These strategies involve self-regulation (motor and verbal strategies), instructional comprehension, and self-instruction strategies, according to the participant's development. Self-instruction and instructional comprehension involve three phases: (1) forethought (establish goals, "what do I have to do?"), (2) performance/volitional control (planning, monitoring and controlling cognition, "how am I going to do it?") and, (3) self-reflection (self-evaluation and cognitive flexibility to make adjustments if required). These three phases are metacognitive strategies that can be applied in self-regulated learning ([@B23]). EF and procedural metacognition (such as the strategies mentioned above) share common theoretical characteristics, developmental paths, and even brain regions. Therefore, the student's control of their own learning is crucial ([@B66]). To our knowledge, this is the first EF training that offers these strategies for school-aged students. The primary focus of this study was to analyze the strategies that students (aged 6--8 years old) use when confronted with challenging strategy-based EF and attention training ("Nexxo-training"). This training, delivered through an online application, combines inhibition and vigilance training with procedural metacognitive strategies. The study also analyzes the cognitive skills and developmental factors that may modulate task performance. The study objectives are as follows: (1) to determine whether procedural metacognitive strategies have an impact on task performance and which ones are relevant; (2) to ascertain whether age moderates the use of strategies and task performance; (3) to identify which cognitive skills are related to task performance as possible predictors; and (4) if cognitive skills are predictive of task performance, the final objective is to test whether this relation is sustainable when the lowest and highest levels of performance are compared. This information is crucial to the scientific development of new training technologies for EF and attention interventions. Materials and Methods {#S2} ===================== Ethics Statement {#S2.SS1} ---------------- In accordance with the Declaration of Helsinki, written informed consent was obtained from each parent's participant. This study was approved by the ethics committee of the San Carlos Hospital (n° 15/315-E) in June 2015. Participants {#S2.SS2} ------------ The study participants were recruited from two schools after receiving their parents' consent. Forty-six typically developing children aged between 6 and 8 years old (24 girls and 22 boys) participated in the study. The selected children were in the 1st grade (*n* = 28, $\overline{x}$ = 78.32 ± 4.037 months) or 3rd grade of primary education (*n* = 18, $\overline{x}$ = 102.11 ± 3.445). The parents' average professional range was $\overline{x}$ = 2.59 ± 0.53 (0 = low level, 1 = medium-low, 2 = medium, 3 = medium-high, and 4 = high) according to the "National Institute of Professional Range" (Spain). The inclusion criteria were as follows: (1) between the ages of 5--7 and 8--9 years; (2) no previous diagnosis of diseases or disorders related to developmental delays; (3) no psychological or speech therapy treatment required at the time of the study or earlier; (4) Spanish-speaking (monolingual); and (5) no diagnosis of learning difficulties or repetition of school year. Criteria 1--5 were obtained through a parents' questionnaire. [Table 1](#T1){ref-type="table"} shows the sociodemographic description of the participants. ###### Sociodemographic description of participants. **Female (*n* = 24)** **Male (*n* = 22)** **Total (*n* = 46)** ----- ----------------------- --------------------- ---------------------- ----- ------ ------- ---- ----- ------ ------ ---- ----- Age 7.04 1.06 6 9 6.62 0.973 5 8 6.85 1.03 5 9 IQ 104 13.9 79 131 106 16.1 78 130 105 14.8 78 131 SD = standard deviation; IQ = intelligence quotient measured by Reynold Intellectual Screening Test (RIST); Min = minimum; Max = maximum. Assessments {#S2.SS3} ----------- ### Standardized Tests Were Used to Assess the Following Dimensions: {#S2.SS3.SSS1} Cognitive skills through individual cognitive assessments (40--45 min): attention using the DIVISA-R "Trees Simple Visual Discrimination Test -- Revised" ([@B71]), intelligence using the Reynolds Intellectual Screening Test (RIST) ([@B65]), the Five Digit Test (FDT) ([@B73]) to measure inhibition and cognitive flexibility, and, processing speed assessment through the Wechsler Intelligence Scale for Children-fourth edition (WISC-IV) ([@B87]). The DIVISA-R ([@B71]) is a computer-based test in which the participant is required to tap the same trees as the model as quickly as possible. It takes approximately 15 min and is suitable for children aged 6--12 years. It provides five main indexes: distraction-precipitation, commission errors, omission errors, processing speed, and a global attention score. The reliability is based on Cronbach's alpha ≥0.77 for all scales. The RIST ([@B65]) is a screening intelligence test. It contains two subscales: "guess what," to assess verbal intelligence, and "odd-item-out," to assess non-verbal intelligence. The sum of both subscales determines a general index of intelligence ($\overline{x}$ 100 ± 15). The reliability based on Cronbach's alpha is 0.91. The FDT ([@B73]) is a test to measure certain aspects of EF (inhibition and cognitive flexibility). It contains four subscales: decoding, counting, election and alternative. It provides measures of inhibition and flexibility. In the inhibition subscale, the participant is required to count the numbers in a box instead of reading the numbers (automatic response). In the flexibility subscale, the participant must change strategy (from counting the numbers in a box to reading the number seen in the box), indicated by boxes in a blue frame. The Spearman-Brown coefficient ranges between 0.92 and 0.95. The WISC-IV ([@B87]) implemented in this study included the Index of processing speed PSI (Coding and symbols searching). In coding, the participant is required to transcribe a digit-symbol code as quickly as possible for 2 min. In symbol searching, the participant is asked to decide whether target symbols appear in a row of symbols or not. These subscales were used to assess processing speed. The average internal consistency coefficient for PSI is 0.88. Inhibition and vigilance through go/no-go and stop signal task performance: the Nexxo application provides a score of task performance for inhibition and vigilance for each session according to the number of errors (omissions and commissions) and successes. At the end of the training, the scores for each session in the different blocks are added up to obtain an overall score for the intervention, which is used to as a measure of task performance in inhibition and vigilance for each participant. Task {#S2.SS4} ---- ### Go/No-Go and Stop Signal Tasks {#S2.SS4.SSS1} The Nexxo application is based on neuropsychological models known as "go/no-go" and "stop signal" tasks ([@B74]; [@B50]), which involve a suppression of an ongoing response (inhibition), "n-back," a typical task involving the temporary storage, manipulation, and selection of information ([@B83]) by deciding whether to make a response or not depending on whether a sequence is fulfilled (working memory), and, vigilance, in which changes are to be detected when only a low rate of relevant stimuli are presented ([@B75]). As there is a low presence of these types of games (n-back) in level 1 of the Nexxo app (i.e., the one used in the study), we excluded them to focus on inhibition and vigilance processes. The game had two different blocks: vigilance vs. inhibition. In the vigilance block, the user had to tap the screen sporadically (differentiating between possible distractors and thus maintaining a state of alertness, also known as "vigilance"), whereas in the inhibition block, the user had to tap very frequently (holding back an automatic response, which is known as "inhibition or self-control"). The mechanics of the game included requirements to touch the screen when a specific stimulus was present, for example: "tap when you see that the figures on the screen are the same." The screen turned green when the user tapped correctly and red when the user tapped incorrectly. The instructor applied compensatory strategies if the user displayed difficulties in carrying out the task. [Figure 1](#F1){ref-type="fig"} shows an example of a Nexxo activity. {#F1} Each game has a different command and stimulus presentation. In the vigilance block, the rate of target presence was less than 30% (70% no-go probability), whereas in the inhibition block the rate of target presence was over 70% (30% no-go probability). After each game, the participants were shown on the screen how many stars they had received as a reinforcement (0--3 depending on the level of performance). The participants played 30 games divided into two different blocks (15 vigilance games and 15 inhibition games) in the first level. There were 15 session in total (three games per session/each game was done twice) with each session lasting approximately 15 min. Additionally, Nexxo was developed to train processing speed (as the screen transition was set at one second, stimulus processing and the decision to tap or not tap required perceptual-motor agility). The Nexxo application also requires visual and auditory discrimination skills due to the presence of both types of stimuli in the form of targets and distractors (e.g., game V7 level 1 instruction: "tap each time you see a yellow circle with this sound"). Finally, Nexxo records the types of errors committed by the user: commission errors (the user tapped the screen when a response should have been withheld) and omission errors (the user did not tap when a response was required). ### Procedural Metacognitive Strategies {#S2.SS4.SSS2} The training also involved self-regulatory and self-monitoring strategies inspired by [@B59], which were directed by the instructor and recorded for each participant in each session, as follows: (1) general instructions (for all participants): an instruction to get ready for the session (the participants had to put their hands over two fixed stickers when they heard "in position" and wait for the instructor to give further instructions), "visual self-instruction" (wait-see-tap), a visual reminder of how to perform the games in order to foster self-control, and verbal self-instructions: "I am a good observer, I do not fall into traps," instructional comprehension/self-instruction (goal setting and planning): the instructor reads the instructions of the game out loud and asks the participants to say when and how they have to tap in each game though fixed questions (e.g., "when do we have to tap?" (the instructor) "we have to tap when..." (the participants) "how are we going to do it?" (the instructor) "we have to wait, see and tap"), and, verbal reinforcement after the games (e.g., "very good"); and (2) compensatory strategies (for participants who presented difficulties while performing the task): individual reinforcement if required (repeating the instruction to get ready, repeating self-instruction, repeating instructions, child verbalizations during the game (saying out loud what appears on the screen), or, in the latter case, instructor verbalizations (saying out loud what appears on the screen), and positive reinforcement through gestures (saying "well done" out loud). More information about strategies applied can be seen in [Supplementary Material](#SM1){ref-type="supplementary-material"}. Procedures {#S2.SS5} ---------- The Nexxo-training intervention combines the repetition of EF and attentional tasks in addition to strategies to enhance the tasks. We refer to these strategies as "procedural metacognitive strategies." In addition to general strategies aimed at the whole group, Nexxo-training provides compensatory strategies to individual participants who experience greater difficulties during training. The Nexxo application (go/no-go and stop signal tasks) was designed between 2012 and 2014, and a pilot version was developed for the study in October 2015 ([@B79]). Written informed parental consent was obtained from each participant. The participants underwent a neuropsychological assessment conducted by an examiner, which included individual tests to measure intelligence, attention, inhibition and flexibility, working memory, and processing speed. The examiners were trained psychologist who participated in the data collection. The group received a 5-week intervention conducted by a psychologist (groups of eight participants) using a special training script provided by each instructor. The Nexxo intervention was carried out over a 5-week intervention period (two sessions per week/15 min each/three games repeated twice in each session). Regarding inhibition training, a previous study of a go/no go task using a touchscreen application with preschoolers showed a trend-level improvement in reasoning and neural changes in the experimental group after 3 h of training ([@B49]). This is the reason why we decided to set the Nexxo-training duration at 3 h. The complementary strategies aimed at procedural metacognitive strategies were inspired by [@B59]. The complementary strategies were implemented by an instructor and recorded for each participant. [Figure 2](#F2){ref-type="fig"} shows a description of the Nexxo-training. {#F2} Data Analysis {#S2.SS6} ------------- Statistical analyses were performed using IBM SPSS Statistics 23. [Table 2](#T2){ref-type="table"} shows the frequency of participants with whom compensatory strategies were used at some point during the training. The "positive reinforcement" strategy was excluded from the following analyses because only two used it once. ###### Frequency of participants with whom compensatory strategies were used at some point during the training. **Total *N*** **1st grade *n*** **3rd grade *n*** -------------------------------------- --------------- ------------------- ------------------- Repeat warning to get ready 21 (45.65) 19 (67.86) 2 (11.11) Repeat self-instructions 13 (28.26) 12 (42.86) 1 (5.56) Instructional comprehension 35 (76.09) 25 (89.29) 10 (55.56) Positive reinforcement 2 (4.35) 2 (7.14) 0 (0) Child verbalizations 26 (56.52) 19 (67.86) 7 (38.89) Instructor verbalizations 16 (34.78) 11 (39.29) 5 (27.78) Total set of compensatory strategies 37 (80.43) 26 (92.86) 11 (61.11) \% = percentage. [Table 3](#T3){ref-type="table"} shows the scores in inhibition and vigilance tasks recorded by the Nexxo App, the number of total compensatory strategies applied and recorded by the instructor for children who experienced difficulties during the tasks, and the number of strategies applied of each subtype. These scores were reported for the total sample and, also, separately for the 1st and 3rd grade groups. ###### Indicators of performance in inhibition and vigilance, and compensatory strategies. **Mean** ***SD*** **Minimum** **Maximum** ------------------------------------------ ---------- ---------- ------------- ------------- **Inhibition** Total 92.5 5.93 79 100 1st grade 89.82 5.88 79 100 3rd grade 96.78 2.67 91 100 **Vigilance** Total 69.7 14.3 38 97 1st grade 61.79 10.73 38 85 3rd grade 82.11 9.45 60 97 **Repeat warning to get ready** Total 0.674 0.871 0 3 1st grade 1 0.9 0 3 3rd grade 0.17 0.51 0 2 **Repeat self-instructions** Total 0.609 1.42 0 8 1st grade 0.96 1.73 0 7 3rd grade 0.06 0.24 0 1 **Instructional comprehension** Total 2.59 2.29 0 7 1st grade 3.43 2.33 0 8 3rd grade 1.28 1.49 0 4 **Child verbalizations** Total 0.891 1.1 0 5 1st grade 1.18 1.25 0 5 3rd grade 0.44 0.62 0 2 **Instructor verbalizations** Total 0.609 1.11 0 5 1st grade 0.79 1.32 0 5 3rd grade 0.33 0.59 0 2 **Total set of compensatory strategies** Total 5.43 5.39 0 26 1st grade 7.46 5.81 0 26 3rd grade 2.28 2.42 0 8 SD = standard deviation. For cognitive skills, we used T-scores provided by the instruments, with the exception of FDT since part of our sample was younger than the norm-based scores provided by the instrument. In this case, we calculated T-scores for our sample (1st graders and 3rd graders, separately); the higher the T-score, the lower the FDT performance. For all the statistical analyses, the significance threshold was set at 0.05. In linear regressions, standardized β and adjusted *R*^2^ are reported. Results {#S3} ======= Compensatory Strategies and Task Performance {#S3.SS1} -------------------------------------------- We used partial correlation analysis to detect the possible relation between performance and compensatory strategies, controlling for age (in months) to eliminate possible moderation due to development. After controlling for age, there was a significant correlation between inhibition and vigilance performance: the participants with a higher level of performance in inhibition games also demonstrated a higher level in vigilance games (*r* = 0.517, *p* \< 0.001). The correlations between performance in both types of tasks and compensatory strategies were significantly negative for "repeat self-instructions" and "instructional comprehension" (see [Table 4](#T4){ref-type="table"}), meanwhile they were marginally significant between performance in "vigilance" and "instructor verbalizations" (*r* = −0.29, *p* = 0.053). Those who obtained lower scores in the tasks (either inhibition or vigilance) required more compensatory strategies. [Table 4](#T4){ref-type="table"} shows the correlations between inhibition and vigilance performance and compensatory strategies. ###### Partial correlation, controlling for age in months, between performance in inhibition and vigilance, and compensatory strategies. **Repeat warning** **Repeat** **Instructional** **Child** **Instructor** **Total set of** ------------ ------------- -------------------- ------------ ------------------- ----------- ---------------- ------------------ Inhibition Pearson's r --0.229 −0.354^∗^ --0.561^∗∗∗^ --0.110 --0.256 --0.475^∗∗^ *p*-value 0.130 0.017 \<0.001 0.472 0.090 0.001 Vigilance Pearson's r --0.196 −0.362^∗^ −0.342^∗^ --0.073 --0.290 --0.387^∗∗^ *p*-value 0.197 0.014 0.022 0.635 0.053 0.009 ∗ p \< 0.05, ∗∗ p \< 0.01, and ∗∗∗ p \< 0.001. Compensatory Strategies and Task Performance in Relation to Age {#S3.SS2} --------------------------------------------------------------- Using the participants' age in months as an independent variable in a linear regression showed that age predicts better performance in both inhibition (β = 0.613, *p* \< 0.001, adjusted *R*^2^ = 0.361) and vigilance (β = 0.706, *p* \< 0.001, adjusted *R*^2^ = 0.487), with a steeper slope for vigilance: older participants have better results (see [Figure 3](#F3){ref-type="fig"}). {#F3} Regarding the relation between age (in months) and compensatory strategies, statistically negative correlations were found with the total set of compensatory strategies, and the subtypes "repeat the warning for starting," "instructional comprehension," and "child verbalization" (see [Table 5](#T5){ref-type="table"}). ###### Correlations between age in months and compensatory strategies. **Repeat warning** **Repeat** **Instructional** **Child** **Instructor** **Total set of** ----------------- ------------- -------------------- ------------ ------------------- ----------- ---------------- ------------------ Age (in months) Pearson's r --0.510^∗∗∗^ --0.276 --0.484^∗∗∗^ −0.329^∗^ --0.174 --0.473^∗∗∗^ *p*-value \<0.001 0.063 \<0.001 0.026 0.248 \<0.001 ∗ p \< 0.05, ∗∗ p \< 0.01, and ∗∗∗ p \< 0.001. Cognitive Skills and Task Performance {#S3.SS3} ------------------------------------- Stepwise multiple linear regression analysis was used to identify which cognitive skills scales (DIVISA, RIST, WISC and FDT indexes) (independent variables) better predict performance in inhibition and vigilance tasks (dependent variables). For inhibition tasks, all the independent variables were non-significant. For vigilance tasks, the results showed that higher scores in odd-item-out from RIST (β = 0.389, *p* = 0.002) and lower scores in omissions from DIVISA (β = −0.479, *p* \< 0.001) and flexibility from FDT (β = −0.279, *p* = 0.02) predicted better performance. [Table 6](#T6){ref-type="table"} shows the complete regression model. ###### Regression model predicting performance in vigilance. **Unstandardized** **Standardized** ------------------------------------------------ -------------------- ------------------ --------- -------------- Intercept 79.367 11.072 -- 7.168^∗∗∗^ DIVISA-R: omissions --0.199 0.048 --0.479 --4.417^∗∗∗^ RIST: odd-item-out 0.46 0.137 0.389 3.356^∗∗^ FDT flexibility --0.407 0.167 --0.279 −2.431^∗^ *F*(3,38) = 13.11^∗∗∗^, adjusted *R*^2^ = 0.47 ∗ p \< 0.05, ∗∗ p \< 0.01, and ∗∗∗ p \< 0.001. DIVISA-R = test of simple visual discrimination of trees -- revised; RIST = reynold intellectual screening test; FDT = five digit test. To ascertain if this relation is present when comparing children with low and high performance in inhibition and vigilance tasks, the sample was divided into four groups using quartiles. The groups with the best performance (Q1, superior quartile) and worst performance (Q4, inferior quartile) for each task were selected for the analysis (see data in [Table 7](#T7){ref-type="table"}). ###### Data from Q1 and Q4 groups for Inhibition and Vigilance performance. ***n*** **Mean age** **SD age** **Score** **Mean** **SD** ------------ ---- --------------- -------------- ------------ ----------- ---------- -------- Inhibition Q1 14 (7 M;7 F) 95.07 11.38 ≥97 98.6 1.28 Q4 11 (5 M; 6 F) 78.27 4.41 ≤87 84 3 Vigilance Q1 12 (6 M; 6 F) 101.33 7.34 ≥82 87.8 4.37 Q4 11 (4 M; 7 F) 80.82 9.15 ≤60 51.6 7.85 M = Males; F = Females; SD = standard deviation. Because the sample size of the groups was small, and the normality assumption was not met, a non-parametric Mann--Whitney U test was carried out to compare the differences between the Q1 and Q4 groups. [Tables 8](#T8){ref-type="table"}, [9](#T9){ref-type="table"} show the results for Inhibition and Vigilance, respectively. ###### Mann--Whitney U test in inhibition. **Q1 Mdn** **Q4 Mdn** **Mann--Whitney U** ***p*-Value** ---------------------------- ------------ ------------ --------------------- --------------- **DIVISA-R** General attention index 3 5 44.5 0.345 Commissions 85 75 55.5 0.841 Omissions 45 85 43 0.299 Organization 50 25 49 0.523 Distraction 15 10 36.5 0.131 **RIST** Guess what 55.5 53 51 0.153 Odd-item-out 54.5 51 70.5 0.721 General intelligence index 107.5 100 62.5 0.427 **WISC-IV** Symbol search 10.5 11 67 0.579 Coding 9.5 10 72.5 0.8 Digit span 12 10 54 0.201 Digit forward 11 11 56 0.229 Digit backward 12.5 12 68.5 0.639 Processing speed index 104.5 104 67.5 0.602 **FDT** Inhibition 45.17 53.30 52 0.171 Flexibility 45.61 50.96 49 0.134 ∗ p \< 0.05, ∗∗ p \< 0.01, and ∗∗∗ p \< 0.001. Mnd = median; DIVISA-R = test of simple visual discrimination of trees -- revised; RIST = reynold intellectual screening test; WISC-IV = wechsler intelligence scale IV; FDT = five digit test. ###### Mann--Whitney U test in vigilance. **Q1 Mdn** **Q4 Mdn** **Mann--Whitney U** ***p*-Value** ---------------------------- ------------ ------------ --------------------- --------------- **DIVISA-R** General attention index 10 2.5 28.5 0.058 Commissions 85 88 50 0.723 Omissions 20 89 22.5 0.021^∗^ Organization 35 35 46.5 0.547 Distraction 15 5 18 0.008^∗∗^ **RIST** Guess what 53 50 57 0.578 Odd-item-out 60 41 14.5 0.002^∗∗^ General intelligence index 113.5 91 29.5 0.024^∗^ **WISC-IV** Symbol search 10 8 45.5 0.201 Coding 9.5 9 49 0.283 Digit span 12 10 51 0.350 Digit forward 11 11 63 0.847 Digit backward 13 12 46.5 0.226 Processing speed index 106 96 38.5 0.09 **FDT** Inhibition 45.57 54.97 40 0.109 Flexibility 44.49 50.96 33 0.042^∗^ ∗ p \< 0.05, ∗∗ p \< 0.01, and ∗∗∗ p \< 0.001. Mnd = median; DIVISA-R = test of simple visual discrimination of trees -- revised; RIST = reynold intellectual screening test; WISC-IV = wechsler intelligence scale IV; FDT = five digit test. Concerning inhibition tasks, no differences were found between the Q1 and Q4 groups in any of the skills assessed. Nevertheless, for vigilance tasks, the scores were significantly higher for Q1 in distraction from DIVISA (*U* = 18, *p* = 0.008), odd-item-out subtest (*U* = 14.5, *p* = 0.002) and general index (*U* = 29.5, *p* = 0.024) from RIST, and lower in omissions from DIVISA (*U* = 22.5, *p* = 0.021) and flexibility from FDT (*U* = 33, *p* = 0.042). Discussion {#S4} ========== Nexxo-training is an innovative strategy-based training for attention and EF. Strategy-based training combines the repetition of a task with strategies (e.g., scaffolding or metacognitive strategies) to improve performance ([@B55]; [@B39]). In this study, the Nexxo-training involved computer-based training through "go/no-go" and "stop signal" tasks, in combination with procedural metacognitive strategies for the whole group, adapted to the participants' developmental stage, as well as compensatory strategies for those who presented greater difficulties during the training. The tasks were developed using an application ("Nexxo" iPad application). As touchscreens and applications are appealing to children ([@B46]), this approach can motivate them to participate in the training. This new training approach has demonstrated positive results in school-age students in terms of attention and EF (Rossignoli-Palomeque et al., unpublished). To our knowledge, this is the first (strategy-based) cognitive training that provides, compensatory strategies for participants who experience greater difficulties. Considering the proportion of participants who required compensatory strategies at some point in the training period (80.43%), it seems that compensatory strategies are relevant over the course of the training process. The most commonly used compensatory strategy was instructional comprehension (76.05%), followed by child verbalizations (56.52%), repeating warning for starting (45.65%), instructor verbalizations (34.78%), repeating of self-instructions (28.26%), and gestures reinforcement (4.35%). Instructional comprehension (i.e., verbalizations of what to do) was the strategy most commonly required by both 1st-grade and 3rd-grade participants. This strategy is fundamental in self-regulated learning ([@B23]). As shown in [Table 2](#T2){ref-type="table"}, the younger participants displayed a greater need for repeating instructions to get ready (67.86% in 1st grade vs. 11.11% in 3rd grade), child verbalizations (67.86% in 1st grade vs. 38.89% in 3rd grade), and self-instructions (42.86% in 1st grade vs. 5.56% in 3rd grade). These results may be due to a greater development of attentional control and inner speech around the 3rd grade. As suggested by [@B88], inner speech evolves from irrelevant speech to self-directed verbalizations that are relevant for the task. Strategy-based attention and EF training with compensatory strategies is a new direction, and further research on attention and EF training should focus on strategies that are more likely to improve task performance and far transfer. Indeed, it is crucial to conduct this type of training research on strategies used by students while performing attention and EF tasks. Cognitive training should be designed based on neuropsychological models. The Nexxo application is founded on well-known attention and EF paradigms ([@B74]; [@B50]). In addition, the strategies, self-regulation strategies (motor and verbal strategies), instructional comprehension, and self-instruction have been designed considering developmental factors ([@B86]; [@B6]). As reviewed in scientific literature, verbal and motor strategies are used by preschoolers to inhibit themselves ([@B26]; [@B52]), and internal verbalizations evolve from irrelevant speech (at 2 years of age) to self-directed instructions that are relevant to the tasks (at 8 years of age) ([@B88]). Thus, it seems reasonable to use self-directed instructions as a verbal strategy in school-age students in combination with motor strategies for self-control. Finally, Nexxo-training also involves procedural metacognitive strategies, such as self-instruction and instructional comprehension strategies, to promote self-control and attention. As cognition and self-regulation are viewed as an integral unit ([@B86]), by combining computer-based training in attention and EF with procedural metacognitive strategies selected for the appropriate developmental period, the training will help to improve these processes as they develop naturally. This should be the criteria when selecting the training strategies. Teaching children to control their own behavior can lead to more durable behavioral changes and less dependency on adult supervision ([@B56]). The student's use of procedural metacognitive strategies, such as selection, monitoring, and control of their learning activities, is crucial for their achievement in all learning situations ([@B91]). This can be justified by the theoretical overlap between EF and procedural metacognition ([@B66]). For this reason, we consider that analyzing strategy-based training is relevant for the increased likelihood of transference and long-term effects. Finally, cognitive training researchers should consider studying strategies that can be applied in attention and EF training at different developmental stages. In this study, we analyzed the compensatory strategies used by participants experiencing difficulties in EF and attention tasks. In addition, we analyzed the developmental factors and cognitive skills that may modulate EF and attention task performance. This is relevant for the future of attention and EF cognitive training design. First, we found a positive correlation between inhibition and vigilance. This result is supported by previous findings suggesting a relation between the two elements ([@B51]; [@B15]; [@B30]; [@B63]; [@B81]). As inhibition is central to EF ([@B20]), and vigilance is central to attention ([@B37]), we believe that the combination of both processes may help to improve more complex subcomponents of attention and EF. The results are consistent with previous findings that connect attention and EF ([@B51]; [@B58]). Regarding the procedural metacognitive strategies used during task performance, our analysis showed that those who obtained lower scores in task performance (either inhibition or vigilance) required more compensatory strategies. Compensatory strategies provide a way for participants to adapt to the training. Specifically, the participants with lower inhibition and vigilance scores in the application required more instructional comprehension as a compensatory strategy. Similarly, those with lower task performance and a higher number of omissions in the DIVISA-R test ([@B71]), which is related to inattention, depended more on the instructional comprehension strategy. As mentioned above, instructional comprehension and self-instruction strategy can help participants to establish a goal, plan and monitor task performance ([@B23]). Moreover, repeating instructions helps to overcome difficulties in working memory ([@B2]). This finding is robust considering the effectiveness that self-instruction has shown in students with difficulties in attention and EF, such as ADHD ([@B36]; [@B35]). For these participants, repeating instructions using self-instruction and goal setting was fundamental. Future strategy-based training designs for attention and EF should consider these findings. One of the objectives of the study was to analyze the influence of age in task performance in order to identify the appropriate age for Nexxo-training. As hypothesized, the older participants obtained better results in inhibition and vigilance tasks; therefore, age moderates task performance. This may be due to neuropsychological changes that occur during child development ([@B24]; [@B13]). In terms of inhibition performance using go/no-go tasks for assessment, it seems that there is an improvement in response inhibition abilities moderated by age ([@B5]; [@B47]), which makes this period relevant. In this regard, our finding is consistent with previous scientific literature. Furthermore, age moderates the use of strategies, as statically negative correlations were found with the total set of compensatory strategies, and the subtypes ("repeat the warning for starting," "comprehension instructions," and "child verbalization"). This finding is consistent with the progressive development of verbal strategies and self-instruction ([@B86]; [@B6]). According to these findings, and, consistent with our results, using this type of training with children up to the age of 8 years old seems ideal. Regarding cognitive skills and task performance, our results shows that higher scores in RIST odd-item-out (fluid intelligence), and lower levels of Omissions in DIVISA (attention test) and in FDT flexibility (cognitive flexibility) predicts better results in vigilance tasks. Recent research shows that working memory, inhibition and shifting, the main components of EF, contribute substantially to general intellectual ability, especially fluid intelligence ([@B11]). Meanwhile, the parietal and frontal areas involved in EF have also been related to fluid intelligence ([@B82]; [@B89]). Consequently, based on this idea, we analyzed the relation between inhibition and vigilance task performance with fluid intelligence. Our results show that fluid intelligence predicts better results in vigilance. Vigilance tasks require attentional control which is related to inhibitory control. We also found that participants with higher levels of performance in vigilance also obtained higher scores in fluid intelligence. Previous findings have suggested a relation between vigilance and intelligence in children at risk of learning disabilities ([@B77]). In this sense, we must add that intelligence benefits vigilance performance. In terms of attention, our results show that the participants with fewer omissions and a lower level of distractibility in neuropsychological tests had better results in vigilance task (after training). As demonstrated in previous studies, omissions and distractibility can be predictors of go/no-go performance ([@B48]). In our view, the fact that lower levels of omissions in the DIVISA-R test is related to better performance in vigilance, is a result which provides validity to the training. Finally, as regards the relation between cognitive flexibility and attention, we consider that cognitive flexibility has a positive influence on vigilance tasks as the instructions change for each game. The transition from one rule (e.g., "tap each time a bear appears on the screen) to another (e.g., "tap when you see the number 5") involves not only an alteration in the type of instructions (target and distractors) but also a change from vigilance tasks to inhibition tasks, as both types of games are played in each session. We hypothesize that individuals with higher cognitive flexibility may better adjust their cognitive resources to these changes. A previous study suggested that cognitive flexibility may become a useful tool for vigilance training strategies, as individual differences in cognitive flexibility predicts better results in vigilance tasks ([@B28]). Another possible explanation refers to the idea of flexibility as a predictor of response speed ([@B19]). Go/no-go tasks involve response speed, i.e., a participant with a low response speed may produce a high number of omissions in the task and, as a result, obtain lower levels of vigilance performance. All these examples demonstrate how cognitive processes are interrelated, and, therefore, how training may have a simultaneous impact on multiple processes. This study has several key strengths. Firstly, it examines a type of strategy-based training in attention and EF functions that provides compensatory strategies adapted to the participant's needs. This is an innovative approach for cognitive training with potential for further research. Secondly, the cognitive training tasks presented in the Nexxo app are based on neuropsychological models ([@B74]; [@B50]). Furthermore, the implemented strategies are based on previous research and have been designed according to the developmental stage at which the training is applied. In this regard, it is important for future strategy-based training designs to consider child developmental factors. In our view, this approach can overcome the limitations of previous cognitive training designs in attention and EF, in terms of generalization and long-term effects ([@B67]). Thirdly, this analysis has helped to clarify the relevance of instructional comprehension and self-instruction as compensatory strategies. This finding should also be taken into consideration for future training designs. This study reveals that child development moderates inhibition and vigilance performance. In addition, this paper demonstrates that there is a relation between fluid intelligence and vigilance. This finding raises the question of whether intelligence can be improved by training vigilance. However, further research is needed in this area. In addition, our paper shows a relation between inhibition and vigilance. Nevertheless, this study also had certain limitations. For example, as the study did not involve groups of older participants, we could not analyze the feasibility of the strategies in different age groups. In addition, due to a technical limitation, we were unable to include processing speed as a variable in our analysis. Therefore, it would be advantageous to include this variable in future training designs. Finally, we focused on Nexxo-training with typically developing children. Further research on Nexxo-training should focus on atypically developing children in terms of attention and EF, such as ADHD. Conclusion {#S5} ========== Nexxo-training is a specific form of strategy-based training that provides not only general procedural metacognitive strategies for the whole group, but also compensatory strategies for individual participants who experience greater difficulties during the training. Considering the proportion of participants who required compensatory strategies at some point in the training period (80.43%), it seems that compensatory strategies are relevant over the course of the training process. Regarding strategy analysis, instructional comprehension and self-instruction (e.g., goal setting and planning) seem to be the most useful strategies for participants with difficulties in inhibitory and vigilance task performance. Finally, developmental factors moderate task performance, while fluid intelligence and cognitive flexibility is related to vigilance performance. Data Availability Statement {#S6} =========================== All datasets generated for this study are included in the manuscript/[Supplementary Files](#SM1){ref-type="supplementary-material"}. Ethics Statement {#S7} ================ In accordance with the Declaration of Helsinki, written informed parental consent was obtained from each participant. This study was approved by the Ethics Committee of the San Carlos Hospital (*n*°186; 15/315-E) in June 2015. Author Contributions {#S8} ==================== TR-P conceived the application. TR-P, EP-H, and JG-M conceived, design, and coordinated the experiment. MQ-G involved in the data analysis. TR-P and MQ-G wrote the manuscript. EP-H reviewed the manuscript. Conflict of Interest {#conf1} ==================== TR-P owns the original idea for the Nexxo application and participated in the design of the Nexxo games. The Nexxo application for iPad is a commercially available app (a free-to-install app with in-app purchases). TR-P is part of the development team for the Nexxo application for iPad. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Marta Rincon Ortega for her collaboration in Nexxo games design and tapp-mobile for software development. We thank Endaze School and Teresa de Berganza Primary School for participating in this study, as well as participants and families enrolled in the study. We thank I+D+i TEA department for material support (SENA and BRIEF questionnaires). We also thank instructors and examiners for their participation: Yolanda Salamanca López, Vivian Randez Rosell, Angee Paola Rodríguez Urrego, Gabriela Alejandra Cajo Pullas, Marcia Sofia Gomes, Tea Habazin, Ana Karen Ortiz Beltrán, Raquel García Gázquez, and Carina Viviana Carrillo. Finally, our sincere thanks to Nathan Florian for his valuable comments and review of the manuscript. Supplementary Material {#S10} ====================== The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fpsyg.2019.02332/full#supplementary-material> ###### Click here for additional data file. [^1]: Edited by: Maria Carmen Usai, University of Genoa, Italy [^2]: Reviewed by: Marie Arsalidou, National Research University Higher School of Economics, Russia; Elisa Bisagno, University of Modena and Reggio Emilia, Italy [^3]: This article was submitted to Developmental Psychology, a section of the journal Frontiers in Psychology

Research for health & the \'Inter-land\' ======================================== The Bamako Call for Action on Research for Health \[[@B1]\] arose from a meeting of Ministers of Health, Education, Science & Technology, Foreign Affairs and International Cooperation, from 59 countries; as well as researchers, policy makers, civil society representatives, journal editors and development agencies. From 17--19th November, 2008, in Bamako, Mali, amid presentations on the conference theme of \"Strengthening Research for Health, Development and Equity\" the call was crafted, having benefited from recent regional \'feeder\' meetings in Algiers, Bangkok, Copenhagen, Rio de Janeiro and Tehran. This Ministerial conference, which occurs once every four years, is agenda setting; on this occasion the agenda being to change the way in which health research is undertaken. IJsselmuiden and Matlin \[[@B2]\] note that the scope of health research is broad, including, for example, biomedical and public health research, research on health policy and systems, environmental health, science and technology, operational research, as well as social science and behavioural research. However, they argue that the range of research needed to protect and promote health and reduce disease is in fact much broader than this: \"the fields of interest span the relationships between health and, among many others, social, economic, political, legal, agricultural and environmental factors\" (p.4). For example, major health gains have been made possible through civil engineering improvements in water quality, sanitation and housing conditions, in addition to medicines and healthcare. As stated in the Bamako call itself: \"The nature of research and innovation for health improvement, especially in the context of the United Nations Millennium Development Goals, is not sufficiently inter-disciplinary and inter-sectoral; there is a need to mobilize all relevant sectors (public, private, civil society) to work together in effective and equitable partnerships to find needed solutions.\" (Recognition Statement 5)\[[@B1]\]. This mammoth revision of how research is conducted in our field could beckon a new age of enlightenment, but one for which we are currently poorly prepared. I argue that there is a need to rethink our approach to research in global and public health and to complement narrow research specialisations with a new cadre of researchers who have expertise concerning the context and process of research, as well as its content, and the interplay of these knowledge domains. Polymaths ========= The Renaissance was an age of enlightenment because it questioned previously narrow understandings of the world. It allowed the activities of questioning and discovery to be respectable, exciting and relevant to the curiosities and the exigencies of people\'s contemporary lives. Luminaries of that age were often distinguished not only by a genius in particular fields of interest, but also by their ability to traverse different fields of specialisation, and to sometimes recognise their interconnections. The \'polymaths\' of Victorian times were also fired by broad curiosities, the delight of discovery and a hunger to better understand how the world worked, and indeed, how they could better work the world. Aristotle was perhaps one of the original polymaths, his work embracing philosophy, logic, biology, astronomy, rhetoric and literary criticism, among others. Benjamin Franklin, as well as being a famous politician, was also a journalist, revolutionary, scientist and inventor; he invented bifocal lenses. A more contemporary polymath might be Umberto Eco, who while writing best selling novels, is an expert on literature, medieval philosophy, and pop culture and also a professor of semiotics. The holding of such broad interests is today still viewed with some awe by the lay person, but rather disparaged by most in the research community. Those with too many research interests are seen as suffering from a sort of fuzzy parallelism; a restless unfocused mind, wandering without much conviction, across what must be a confusing intellectual landscape -- to them anyway. The modern penchant in scientific research is to narrow down interests and expertise, resolutely. Narrow tracts of laser beam breadth enhance the probability that enthusiastic, active and able researchers can claim the coveted \'leading international authority\' status that is so important in producing \'high impact\' research, loading onto university rankings and research incomes. \'Naive Experts\' ================= Commercial, legal and indeed egotistic exigencies, have fuelled the cult of the narrow researcher, and helped to make it a reassuring and satisfying role for many. It is important to acknowledge that this approach to research has also made many valuable contributions, with many benefits for society. However, it is also ironic that the reification of personalised expertise has been accompanied by an apparent depersonalisation of the authority associated with it. As argued in Nagel\'s *The View from Nowhere*\[[@B3]\], that view (uncluttered by personal opinion, context or bias) is sought in the drive for objectivity. That is, a view \'un-socially constructed\' or cluttered by mere human or earthly issues, rather echoing classical views of \'pure knowledge\'. Again, let me stress, our cadre of narrowly focused \'objective\' researchers have an impressive pantheon of achievements. These \'naïve experts\' flourish amidst a view that the world, and the knowledge we now need for it, is too broad, too advanced, too complex, for anyone to really understand, very well, more than just one thing. Doing one thing well, has been magnified even further by the extra brownie points awarded for doing it in just one way -- using a particular research methodology, rather than being \'muddled\' by mixed methods, the results of which may be alarmingly messy to integrate. It is important to stress that the term \'naive experts\', as I use it here, is not meant to be in any way pejorative, but rather descriptive of experts who may lack knowledge -- of the uses or limitations -- of their own knowledge. Research utilisation ==================== The downside to a system that encourages the productions of intentionally naïve experts is their collateral ignorance of other ideas, approaches and possibilities, and most crucially of the utility of their research. Relatively few researchers are engaged with the application, or utilisation, of the knowledge they produce. Indeed, possibly the most intellectually challenging task -- of putting together research findings from diverse perspectives and then deciding what should be done as a result of them -- is left to policy makers and practitioners, who often are poorly prepared to interpret the strengths and weaknesses of different (and sometimes contradictory) research. I believe that researchers have an obligation -- even a moral obligation -- to do more. In some cases, this may already be happening through a broadening of research initiatives, viz multi-disciplinary, inter-disciplinary and trans-disciplinary research. But I believe that we need more broadly minded and broadly skilled researchers to usefully bring such diversity together. What sort of neo-polymaths might we now need? What could we do to enhance the likelihood of research utilisation? One well known approach to policy analysis stresses the importance of distinguishing between content, context and process \[[@B4]\]. Such an approach might also usefully be applied to integrating research about \'what\' (content), with research about \'where\' (context) and \'how\' (process). To take a concrete example, consider the challenges presented by HIV/AIDS. Here *Content Knowledge*could involve an appreciation of critical issues ranging across immunology, stigma and adherence to medication, and the different approaches to researching these. *Context Knowledge*could require understanding how HIV/AIDS is patterned across society, its social epidemiology, especially with regard to gender, disability, age, place, and socioeconomic status. *Process Knowledge*could involve awareness of the systems of application that need to be involved in putting knowledge into action. For instance, providing anti-retroviral medication in Dublin requires working through different systems of delivery than doing so in Durban or New Delhi. Process knowledge is about how to effectively get content knowledge put into practice, in particular contexts. To take another example, and perhaps one that fits in well with the \'health-interland\' described in the Bamako Call, research on health needs to embrace, to a much greater extent, the challenges faced by people with disabilities. People with disabilities have health needs just as do people without disabilities. However, to attain the highest possible standards of health, those researching disability from a health perspective need to interact with and understand other domains -- transport, education and employment, for example. For *Content Knowledge*on prosthetic device development to provide the gains that are possible, it needs to be understood through *Process Knowledge*. For example, how such services are set up -- whether they are targeted through specific NGOs or if there is a well developed interface between community and rehabilitative healthcare services -- may have implications for the provision, servicing and replacement of such devices, and therefore the mobility of their users. Equally, *Context Knowledge*, of say resources and attitudes towards people with disabilities, may play a significant role in the extent to which people with disabilities are included or excluded from mainstream society and other support services that must work together for their well-being \[[@B5]\]. Global health is a \'composite\' field, comprised of biological, clinical and social health sciences, and complemented by other disciplines that are not explicitly \'health related\', such as engineering or political science. Within this cauldron of global health, we do, of course, already have some people who work across conventional intellectual boundaries and practice niches. For instance, Paul Farmer\'s work on HIV/AIDS (*Content*), his socio-political analysis of power relations (*Context*), and his service delivery role in *Partners in Health*(*Process*) are invigoratingly and productively braded together. And there are certainly other people who combine complementary activities and perspectives, but these people tend to emerge individually, we don\'t have an explicit way of producing or encouraging such skills, or encouraging a more integrative orientation in general; and we don\'t have a structure for teaching it. If the Bamako Call for Action on Research for Health is to get traction, then we will need to develop research and research training that more explicitly helps researchers to think through research content, context and process knowledge, and how these can interplay, and contribute to better health. We will also need to examine some of our own \'institutions\' that facilitate a sort of \'broad-minded inertia\': discipline specific research funding streams; high impact journals cultivating a narrow research focus; education programmes that blinker young researchers to the legitimacy of other perspectives. Conclusion ========== While we should not underestimate the challenges of working across traditional boundaries and demarcations \[[@B6]\] nor should we overestimate the value of research that ignores such challenges. We need a reconfiguring of knowledge, not simply a diminution or expansion of it. We need to know more about how things fit together and can be put to good use. We cannot simply hope for broad-minded health researchers to spontaneously spring forth. Research training at postgraduate level should stress the value of *integrative expertise*as well as recognising *depth expertise*. If we can begin to address this challenge then we can do much to promote research utilisation; by making our research more relevant to global health practitioners and policy makers, and to the aspirations in the Bamako Call. Competing interests =================== The author was funded by the EU as a \'European Union Expert Delegate\' at the Global Ministerial Forum on Research for Health, Bamako, 2008. Acknowledgements ================ I am very grateful for the helpful comments made by three anonymous reviewers and an independent reader who have improved this paper.

This Opinion article outlines the relative paucity and emphasizes the need to enhance our knowledge of how subsets of natural killer T (NKT) cells mediate immune mechanisms of elimination of microbial pathogens at sites of inflammation or infection. To date, most studies of how NKT cell subsets migrate upon antigen stimulation have focused on NKT cell activation in the spleen, lymph nodes (LN) and liver ([@B1]). Thus, there currently exists an unmet need to determine the patterns of recirculation and tissue migration of NKT cell subsets and interacting antigen-presenting cells (APCs) that occur at relevant mucosal surfaces in several other organs, including the lung, intestine, and colon. This article proposes and highlights the benefit of *intravital cellular imaging in vivo* of type I and type II NKT cell subsets as an important methodology that may enable the visualization of NKT--APC cellular interactions at mucosal surfaces and enhance the application of this methodology to clinical therapy of antimicrobial immunity. T Cell Recirculation and Migration into Tissues {#S1} =============================================== During an immune response, T cells and B cells traffic to and recirculate between blood and peripheral lymphoid tissues prior to activation by antigen ([@B1]). Chemokines attract T cells to various sites of interaction with antigen-presenting dendritic cells (DCs) in the spleen and LN. After further encounter with antigen, T cells divide and differentiate into effector T cells (Teff) that migrate to different sites of infection to combat and destroy microbial pathogens ([@B2]). Cytokines secreted by Teff also help to clear infectious pathogens from these sites. Interactions between T cells and DCs at various sites of inflammation in LN are crucial for promoting subsequent immunity to microbes ([@B2]). These observations underscore the importance of understanding how T cell recirculation, localization, and interaction *in vivo* in target tissues mediate effective immune responses that either trigger or prevent inflammation and antimicrobial immunity. Type I and Type II NKT Cell Subsets {#S2} =================================== Little is known about the various factors that mediate the recirculation, localization, and interactions of subsets of NKT cells *in vivo* in target tissues and lead to antimicrobial immunity. NKT cells display surface T-cell antigen receptors (TCR) expressed by both conventional T cells and NK cells, such as CD56/161 (humans) and NK1.1 (mice) ([@B3]--[@B5]). NKT cells recognize lipid antigens presented by CD1d MHC class I like molecules ([@B2]--[@B15]) on various APCs, including DCs, macrophages (Mϕ), B cells, thymocytes, adipocytes, and hepatocytes. While the CD1a, CD1b, CD1c, CD1e, and MR1 MHC class I like molecules are also expressed on APCs and can activate various T cell subsets, only analyses of CD1d-mediated responses of type I and type II NKT cell subsets will be presented here. The development of type I NKT cells occurs in the thymus and depends on the activity of several transcription factors including promyelocytic leukemia zinc finger (PLZF), T box transcription factor (T-bet), retinoic acid receptor-related orphan receptor-γt (ROR-γt), and GATA-binding protein 3 (GATA-3) ([@B2], [@B5]). Type I NKT cells respond to α- and β-linked glycolipids. For example, stimulation of type I NKT cells by the α-galactosylceramide (αGalCer) glycolipid agonist induces the secretion of many cytokines that elicit both Th1 \[interferon-γ (IFN-γ)\] and Th2 \[interleukin-4 (IL-4) and IL-13\] responses ([@B2], [@B7]--[@B17]). Type I NKT cells are more prevalent than type II NKT cells in mice than in humans ([@B18]--[@B20]), and comprise about 50% of murine intra-hepatic lymphocytes ([@B21]--[@B23]). The type I NKT cell invariant TCR is encoded mainly by a germline Vα gene (Vα14/Jα18 in mice and Vα24/JαQ in humans), and more diverse non-germline Vβ chain genes (Vβ8.2/7/2 in mice and Vβ11 in humans) ([@B1]--[@B20], [@B24]--[@B26]). The semi-invariant TCR on type I NKT cells preferentially binds to CD1d via its α-chain ([@B3], [@B6], [@B15], [@B25]). Type II NKT cells constitute a minor subset in mice, but are more predominant in humans ([@B18], [@B27]). Most type II NKT cells do not recognize α-linked glycolipids, but rather respond to sulphatide, a self-antigen that occurs naturally on cell membranes in the central nervous system (myelin sheath), pancreas, kidney, and liver. Sulphatide-reactive type II NKT cells may protect from autoimmune diseases by down-regulation of inflammatory responses elicited by type I NKT cells ([@B28], [@B29]). In contrast, non-sulphatide-reactive type II NKT cells may be pathogenic in other diseases, such as ulcerative colitis (UC) ([@B30]). Sulphatide-reactive type II NKT cells express oligoclonal TCRs and express a limited number of Vα and Vβ chains. The antigen specificity of type II NKT cells appears to be conferred by their surface TCR Vβ-chain ([@B31]). CD1d and NKT Cell-Mediated Antimicrobial Immunity {#S3} ================================================= Antimicrobial defense may be mediated by extensive cross-regulation between CD1d, NKT cells, and microbes that function predominantly at mucosal surfaces ([@B32]--[@B34]). The display of microbes at mucosal surfaces, mainly during early postnatal development, controls NKT cell trafficking and function in the intestine, lung, and intestine. Microbial recognition at these sites determines the susceptibility to NKT cell-mediated inflammatory disorders. Conversely, CD1d expression controls the composition of the intestinal microbiota. Whereas microbiota reduce the number and activity of type I NKT cells at mucosal sites, an elevated number and function of type I NKT cells may be stimulated by microbiota in peripheral tissues ([@B32]). Thus, crosstalk between microbiota and type I NKT cells influences mucosal homeostasis and its dysregulation in a bidirectional manner in inflammatory disorders. In human inflammatory bowel disease (IBD) and infectious hepatitis, type II NKT cells are causal to inflammation ([@B10]). In contrast, intestinal inflammation in oxazolone-induced colitis, a mouse model of human UC, is dependent on CD1d and type I NKT cells that express IL-17 and secrete IL-13 ([@B10], [@B35]). Thus, intestinal microbiota influence pathogenic responses in NKT cell-mediated intestinal inflammation. The outcome of these responses depends on the time of microbial exposure, NKT cell subset(s) involved, nature of microbial lipid antigens recognized, and type of APC that presents CD1d-restricted antigens to NKT cells. CD1d-restricted interactions of type I NKT cells with intestinal epithelial cells (IECs) promote IL-10 secretion and mucosal homeostasis, while CD1d-dependent interactions with bone marrow-derived APCs contribute to intestinal inflammation ([@B36]). Further experimentation may reveal whether these various responses result from the expression of different costimulatory molecules by IECs and professional APCs or whether cell-type-specific differences in CD1d trafficking and lipid acquisition contribute to this outcome. The central questions that need to be addressed are: (1) how do specific microbes control mucosal NKT cell abundance and function and determine health vs. disease, (2) what are the pathways of antigen-dependent and cytokine-dependent activation in NKT cells, and (3) do specific alterations in intestinal microbiota (e.g., in patients with IBD) ([@B37]) contribute to intestinal inflammation by the differential homing, proliferation, and activation of NKT cell subsets. Like the intestine, the lung is a site of interaction between commensal microbiota and mucosal NKT cells. Insufficient microbial colonization during neonatal life leads to increased quantities and environmental sensitivity of type I NKT cells in lungs leading to susceptibility to asthma. This notion is supported by the result that exposure to antibiotics during early life but not late life enhances susceptibility to asthma in mice ([@B38]). In addition, elevated numbers of type I NKT cells are found in the lungs of germ-free mice. The latter finding requires the hypermethylation of the *Cxcl16* chemokine gene and increased expression of the CXCL16 chemokine protein, which binds to the CXCR6 cognate chemokine receptor found on NKT cells ([@B39]). These alterations are associated with increased airway resistance, eosinophil infiltration, and proinflammatory cytokine production during ovalbumin (OVA)-induced asthma in mice ([@B39]). Thus, the development, migration, and function of type I NKT cells at mucosal surfaces may be influenced by commensal microbiota ([@B6]). Tracking of T Cells *In vivo* by Intravital Cellular Imaging {#S4} ============================================================ Studies of NKT cell-mediated inflammation at different mucosal surfaces (e.g., intestine, lung, colon) illustrate that increased understanding of the mechanisms of differential recirculation, migration, proliferation, and activation of NKT cells during pathological responses requires the use of a technology that enables the visualization of these NKT cell events in real-time *in vivo*. The technique of two-photon (2P) microscopy coupled with *intravital* imaging enables one to track the location, movement, and interactions of cells ([@B40]--[@B44]). As such, 2P microscopy has improved our knowledge of T cell--DC and T cell--B cell interactions by recording how such cells function in resting tissue and undergo interaction, information exchange, and response to pathogens ([@B40]--[@B43], [@B45]). This methodology has also provided much new information about cellular pathways that arise during disease progression by illustrating the outcome of specific events in real-time ([@B40]--[@B44]). *Intravital imaging* and quantification of cell dynamics *in vivo* requires the use of fluorescently tagged proteins that are expressed transgenically in a cell-type-specific fashion to monitor the migration of single cells from blood vessels to tissues at a maximum tissue depth of 300--400 μm. Initial studies on T cell--APC interactions during the establishment of peripheral tolerance were conducted with conventional CD4^+^ T cells and APCs in the LN and spleen, and showed that the time of contact between CD4^+^ T cells and APCs may vary from long-lived (days) to short-lived (a few hours) ([@B40], [@B43]). This difference in time of T cell--APC contact may influence the relative capacity of an agent administered *in vivo* to treat a given disease and induce (pre-disease) or restore (post-disease) immune tolerance. For example, CTLA-4 and PD-1 inhibitory receptors on Teff or regulatory T (Treg) cells can suppress immune responses by limiting the times of effective interactions of T cells with DCs ([@B44], [@B46], [@B47]). During chronic inflammation, cytokine delivery requires long-term T cell--APC contacts. However, only a relatively small number of cytokine molecules may be secreted at a low antigen concentration ([@B43], [@B44], [@B46], [@B47]). At a high concentration of antigen, the duration of T cell--APC contacts may be sufficiently long to elicit a chronic inflammatory response. Protection against inflammation is more likely to occur at a significantly lower antigen concentration ([@B43]). Further experimentation is required to analyze the effects of antigen concentration, time of cytokine production by CD4^+^ T cells in high vs. low antigen concentration tissue environments, and whether effector cytokines function locally at a particular site or are transported to other distal sites. Nonetheless, the results reported for the tracking and function of conventional CD4^+^ T cells *in vivo* have facilitated analyses of the migration and function of NKT cells *in vivo*. Imaging of NKT Cell Recirculation, Migration, and Activation {#S5} ============================================================ T cell receptor signal strength may determine the cytokine secretion profiles of T cells in a reciprocal manner. That is, the binding of TCRs of type I NKT cells to their antigen ligands can regulate the activity of TCRs on type II NKT cells. In turn, the binding of TCRs of type II NKT cells to their antigen ligands can regulate the activity of TCRs on type I NKT cells. Understanding the basis of how this cross-regulation of NKT cell activation occurs is crucial to develop better strategies to prevent microbial infection ([@B2], [@B8]--[@B12], [@B48]--[@B52]). Such studies require a suitable animal model in which to track NKT cell recirculation and migration *in vivo*. For this reason, heterozygous mice were generated in which the green fluorescent protein (GFP) gene was knocked into a lineage-specific gene enabling certain leukocytes to be fluorescently labeled ([@B53]). In mice that express GFP integrated into the Cxcr6 chemokine receptor gene (Cxcr6gfp/+ mice), type I NKT cells traffic to, and become quite abundant in the liver (20--30% of lymphocytes). However, NKT cell migration within the liver is arrested following interaction with Kupffer cells. The latter interaction occurs within minutes following lipid antigen injection ([@B54]--[@B58]). In addition, both IL-12 and IL-18 proinflammatory cytokines induced following bacterial infection that suppresses type I NKT cell motility in liver sinusoids of Cxcr6gfp/+ mice via a CD1d-independent mechanism. This block in NKT cell movement is evident within 1 h after exposure to the cytokines and precedes NKT cell activation. Further antigen ligation stabilizes an immune synapse formed between NKT cells and interacting APCs. This synapse potentiates LFA-1/ICAM-1 interactions that enable activated type I NKT cells to remain in the liver. Thus, activated type I NKT cells recirculate less than activated conventional CD4^+^ T cells ([@B59]). Identification of the patterns and kinetics of recirculation of type I and type II mouse NKT cells as well as the patterns and kinetics of human type I and type II NKT cells await further study. Future Challenges {#S6} ================= A future goal of studies of human NKT cells is to identify their functional roles in health and disease ([@B1]). Determination of how subsets of human NKT cells migrate and recirculate *in vivo* may advance our understanding of the biology and mechanisms of cellular interaction of different human NKT cells with APCs. Current investigations are being performed in two animal models. First, Cxcr6gfp/+ mice are being used to monitor human NKT cell trafficking, localization, and activation *in vivo* ([@B56]). Second, the kinetics and dynamics of human CD1d (hCD1d)-restricted NKT cell interactions are being analyzed in hCD1d knock-in mice that express hCD1d in place of mCD1d ([@B59]). Subpopulations of mouse type I NKT cells that are similar to human type I NKT cells in phenotype (mouse Vβ8^+^, human Vβ11 homolog^+^, CD4^low^), tissue distribution, and function (anti-tumor activity) are present in hCD1d knock-in mice. The latter mice serve to model how a lipid antigen induces the migration and function of hCD1d-restricted type I NKT cells and type II NKT cells *in vivo* ([@B59]--[@B62]). If type I and type II human NKT cells can be differentially activated or inhibited *in vivo*, this may facilitate the design of new immunotherapeutic protocols in the treatment and prevention of infectious diseases. Additional imaging studies are required to delineate whether, in addition to NKT cells regulation at mucosal surfaces, commensal bacteria also regulate NKT cells at other sites, e.g., the skin where microbiota are in close contact with NKT cells and CD1a-restricted, lipid-reactive T cells ([@B63]--[@B65]). Future work may also establish potential species-specific and antigen-specific effects of microbiota on NKT cells and the roles of viruses and fungi in this process. Finally, it is of major clinical interest to develop therapeutic strategies that may induce changes in the function of type I NKT cells at mucosal surfaces that will promote and/or preserve mucosal homeostasis and antimicrobial immunity. Conflict of Interest Statement {#S7} ============================== 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. [^1]: Edited by: Thierry Mallevaey, University of Toronto, Canada [^2]: Reviewed by: Kristin Hogquist, University of Minnesota, USA; Paul Kubes, University of Calgary, Canada; Irah L. King, McGill University, Canada [^3]: Specialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology

Background {#Sec1} ========== Profound impact of lower extremity fractures {#Sec2} -------------------------------------------- Numerous negative consequences of lower extremity long bone fractures have been documented, including delayed healing, nonunion, malunion, and significantly delayed functional recovery \[[@CR1], [@CR2]\]. Specifically, at 1-year post-injury, most lower extremity long bone fracture patients have not regained their pre-injury function or quality of life \[[@CR3]--[@CR6]\]. Decreasing re-operations, improving fracture healing, and hastening functional recovery remain ongoing priorities for the orthopedic community. To accomplish this, orthopedic surgeons are increasingly using adjuvant medical therapies to complement their surgical interventions. Hypovitaminosis D and adult fracture patients {#Sec3} --------------------------------------------- Several observational studies have reported up to 75% of healthy adult fracture patients (ages 18--50) have serum 25-hydroxyvitamin D (25(OH)D) levels \< 30 ng/mL \[[@CR7], [@CR8]\]. Although these lower serum 25(OH)D levels have yet to be correlated to fracture healing complications, poor fracture outcomes and the high prevalence of hypovitaminosis D have prompted many orthopedic surgeons to routinely prescribe vitamin D supplements to improve fracture healing in healthy non-osteoporotic patients (ages \< 50) \[[@CR9]\]. An emerging practice with no consensus {#Sec4} -------------------------------------- While evidence-based guidelines recommend vitamin D supplements for general bone health and osteoporosis prevention \[[@CR10]\], there is very little data to guide surgeons on the best supplementation strategies or doses to improve fracture healing. Our group recently surveyed 397 orthopedic surgeons and found that more than 29 different dosing regimens of vitamin D were being used to promote fracture healing, with doses ranging from 400 international units (IU) daily to loading doses of 600,000 IU \[[@CR9]\]. This difference in practice patterns suggests that a high level of clinical uncertainty exists for this vitamin D indication. High-dose supplementation {#Sec5} ------------------------- Many orthopedic surgeons prefer high-dose vitamin D supplementation strategies because of the underlying belief that a rapid increase in available vitamin D may be necessary to maximize fracture healing. This rationale is supported by the chronologic steps of bone healing. Lower extremity long bone fractures heal by callus formation, which begins to form within weeks of injury. Experimental studies have implicated vitamin D in this process and demonstrated that supplementation improves fracture healing in animal models \[[@CR11]--[@CR13]\]. Not only do many patients have low 25(OH)D levels, but recent studies have also identified acute drops in levels shortly after a fracture \[[@CR14], [@CR15]\]. Thus, current research provides a mechanistic rationale that supplementation may improve fracture healing. Furthermore, since callus formation is a crucial early healing step, the magnitude of supplementation efficacy may be time dependent, with rapid increases from high doses being beneficial. Problem to be addressed {#Sec6} ----------------------- If high doses of vitamin D supplementation improve fracture healing outcomes in healthy non-osteoporotic patients, then there is a large opportunity to increase its use. Some surgeons have suggested that supplementation should be given to all fracture patients \[[@CR9]\]. While this approach makes intuitive sense, there are several clinical questions that need to be addressed before widespread adoption is promoted or a definitive efficacy randomized controlled trial (RCT) is initiated. These include the following: (1) Does the timing, frequency, or dose of supplements affect fracture healing?, (2) Are serum 25(OH)D levels associated with fracture healing?, and (3) Does the response to supplementation differ based on a patients' serum 25(OH)D status? If these questions are not addressed, then there may be a missed opportunity (e.g., ineffective dose, duration, or patient selection) to optimize outcomes in adult fracture patients. Progression to a definitive randomized controlled trial {#Sec7} ------------------------------------------------------- Using a phase II exploratory design, we will test three common dosing strategies of vitamin D~3~ for early surrogate treatment response. Given the paucity of literature on the efficacy of vitamin D supplementation and fracture healing, a phase II exploratory trial is the necessary first step in determining the response of vitamin D and the optimal dosing in a fracture patient population. We will use our study results to design a future definitive RCT that will determine if a high dose of vitamin D~3~ provides a greater response than a common low dose (600 IU) or placebo to reduce re-operations in healthy adult fracture patients (ages 18--50). Selecting which high dose to be used in the future trial is the overarching objective of this exploratory study. Through the results of our long-term definitive trial, we will be poised to have an immediate global impact on the care of fracture patients. Vita-Shock exploratory trial {#Sec8} ---------------------------- We propose a 96-patient, 4-arm blinded exploratory trial that will compare two high-dose regimens (loading dose and daily dose), a low-dose vitamin D~3~ supplementation, and a placebo for fracture healing in non-osteoporotic patients (ages 18--50). The impetus for our study is fueled by our team's previous research demonstrating the poor outcomes of tibia and femur fractures and the lack of evidence-based guidance for the dose, frequency, or duration of vitamin D supplements to improve fracture healing. Our proposed study will test the central hypothesis that vitamin D~3~ dose and timing of administration is critical for improving fracture healing at 3 months. Study aims and objectives {#Sec9} ========================= Primary feasibility objective {#Sec10} ----------------------------- The primary aim is to assess the feasibility of vitamin D~3~ supplementation on fracture healing at 3 months. Fracture healing will be assessed as follows: (1) clinical fracture healing will be measured using the Function IndeX for Trauma (FIX-IT) \[[@CR16]\], (2) radiographic fracture healing will be measured using the Radiographic Union Score for Tibial fractures (RUST) \[[@CR17]--[@CR20]\], and (3) biological fracture healing will be measured using serum levels of cross-linked C-terminal telopeptides of type I collagen (CTX) and amino-terminal procollagen propeptides of collagen type I (PINP) \[[@CR21]\]. Secondary feasibility objectives {#Sec11} -------------------------------- The main secondary aim is to determine if 25(OH)D serum levels are associated with fracture healing at 3 months. The other secondary objective is to confirm study protocol feasibility for a larger definitive RCT to determine the optimal vitamin D~3~ dosing regimen to reduce re-operations for fracture healing complications in healthy adult patients. Feasibility outcomes include rate of participant enrolment (24 months to enroll 96 participants), adherence with the daily and loading dose vitamin D supplementation (at least 80% compliance), compliance with blood draws (at least 80% compliance), proportion of participants with complete follow-up at 3 months and 12 months post-fracture (90% follow-up at 3 months and 80% at 12 months post-fracture), and level of data quality (95% complete data for completed visits). Hypotheses for the primary and secondary feasibility objectives {#Sec12} --------------------------------------------------------------- ### Primary feasibility objective {#Sec13} Lower extremity shaft fractures heal via callus formation and secondary bone healing. This seminal process begins within a few weeks of injury and vitamin D metabolites have been extensively implicated in this stage of healing. During these early weeks, circulating vitamin D levels are most likely to be critical to bone healing; therefore, we hypothesize the following: *High doses (loading or daily) will increase healing compared to low daily dose*. Using high doses will rapidly increase the circulating vitamin D available during fracture callus formation.*High loading dose increases healing compared to high daily dose.* Loading doses will overcome medication adherence issues and increase circulating vitamin D even more rapidly than daily doses.*Low daily dose will increase healing compared to placebo*. While the low daily dose is not expected to increase circulating vitamin D as rapidly as the high-dose strategies, this comparison will determine if rapid serum increases are necessary to improve fracture healing. ### Secondary feasibility objective {#Sec14} Based on experimental data and the role of vitamin D on bone metabolism, a correlation between circulating vitamin D levels and fracture healing is expected \[[@CR11]--[@CR15]\]; however, the potential efficacy of various supplementation strategies may be dependent on the patient's baseline vitamin D status or other related changes. For example, it is known that the dose response of supplementation varies depending on the patient's serum 25(OH)D levels, with larger increases seen in patients with serum levels \< 20 ng/ml. Methods {#Sec15} ======= Study setting {#Sec16} ------------- The Vita-Shock trial will be conducted at the R Adams Cowley Shock Trauma Center (STC) in Baltimore, Maryland, USA, that treats femoral or tibial shaft fractures in young adults. The Vita-Shock trial was registered at [ClinicalTrials.gov](http://clinicaltrials.gov) (identifier NCT02786498) on June 1, 2016 (<https://clinicaltrials.gov/ct2/show/NCT02786498>). This protocol paper adheres to the SPIRIT checklist (Additional file [1](#MOESM1){ref-type="media"}) as a guide for reporting. Inclusion criteria {#Sec17} ------------------ Patients who meet all the following criteria will be included in the study: Adult men or women aged 18--50 yearsClosed or low-grade open (Gustilo type I or II) tibial or femoral shaft fracture \[[@CR22]\]Fracture treated with a reamed, locked, intramedullary nailAcute fracture (enrolled within 7 days of injury)Provision of informed consent Fifty years was selected as the upper age limit to minimize potential confounding with post-menopausal endocrine changes that affect bone metabolism. For the purposes of the study, femoral shaft fractures will be defined as any injury in which the majority of fracture line is distal to the lesser trochanter and proximal to the distal metaphyseal flare of the femoral condyles (Fig. [1](#Fig1){ref-type="fig"}). Intertochanteric extension or distal articular extension is permitted. Similarly, a tibial shaft fracture will be defined as an injury with a primary fracture line between the proximal meta-diaphyseal flare to the distal metaphyseal region ending one joint width proximal to the tibial plafond (Fig. [2](#Fig2){ref-type="fig"}). Intra-articular extension is permitted. Fig. 1Femur fracture Fig. 2Tibia fracture Exclusion criteria {#Sec18} ------------------ Patients who meet any one or more of the following criteria will be excluded from the study: Osteoporosis (a participant was deemed to have osteoporosis if there was prior medical evidence in their medical records or whether the patient indicated that they had this condition. We will not use bone scans to diagnose osteoporosis as part of the study protocol.)Stress fracturesElevated serum calcium (\> 10.5 mg/dL)Atypical femur fractures as defined by American Society for Bone and Mineral Research criteria \[[@CR23]\]Pathological fractures secondary to neoplasm or other bone lesionPatients with known or likely undiagnosed disorders of bone metabolism such as Paget's disease, osteomalacia, osteopetrosis, and osteogenesis imperfectaPatients with hyperhomocysteinemiaPatients with an allergy to vitamin D or another contraindication to being prescribed vitamin DPatients currently taking an over the counter multivitamin that contains vitamin D and are unable or unwilling to discontinue its use for this studyPatients who will likely have problems, in the judgment of the investigators, with maintaining follow-upPregnancyPatients who are incarceratedPatients who are not expected to survive their injuriesOther lower extremity injuries that prevent bilateral full weight-bearing by 6 weeks post-fracture. Patients with multiple injuries or multiple tibial and femoral shaft fractures will be eligible for inclusion; however, only the most severe eligible fracture will be included (as determined by the treating surgeon using the grade of soft tissue injury using the Tscherne classification system for closed fractures \[[@CR24]\] and the Gustilo classification system for open fractures) \[[@CR22]\]. Recruitment strategy and patient screening {#Sec19} ------------------------------------------ All patients presenting to participating surgeons between the ages of 18 to 50 years with a tibial or femoral shaft fracture will be screened. Potentially eligible patients will be approached to participate in the trial. All screened patients will be classified as included or excluded. Allocation of patients to study groups {#Sec20} -------------------------------------- Each participant will be randomized to one of four treatment groups: (1) 150,000 IU loading dose vitamin D~3~ plus daily dose placebo, (2) loading dose placebo plus 4000 IU vitamin D~3~ per day, (3) loading dose placebo plus 600 IU vitamin D~3~ per day, or (4) loading dose placebo plus daily dose placebo. The daily vitamin D~3~ supplements/placebo will be provided in a blinded manner. The daily treatment will commence within 1 week of injury and will be taken for 3 months. The loading dose vitamin D~3~ supplements/placebo will be given within 1 week of injury and at 6 weeks (± 2 weeks) post-injury. Allocation to the four study groups will be concealed using a centralized 24-h computerized randomization system that will allow internet-based allocation. The treatment allocation will be stratified on the following prognostic factors to ensure balance between the intervention groups: fracture type (closed vs. open) and long bone fracture (tibia vs. femur). Vitamin D~3~ (cholecalciferol) treatment groups {#Sec21} ----------------------------------------------- ### Blinded administration {#Sec22} The loading dose of 150,000 IU will consist of three 50,000 IU capsules of vitamin D~3~. The loading dose placebo will consist of three capsules that are identical to the 50,000 IU capsules with no active ingredient. The loading dose vitamin D~3~ supplements/placebo will be given within 1 week of injury and at 6 weeks (± 2 weeks) post-injury while in hospital or at the outpatient fracture clinic. The daily vitamin D~3~ supplements/placebo will be provided in a blinded manner and the daily treatment will commence within 1 week of injury. The daily doses (4000 IU, 600 IU, and placebo) will be identical and will be comprised of one capsule. Patients will be given a bottle of either active vitamin D~3~ or placebo capsules and will be instructed to take one capsule daily for 3 months. The placebo capsules will have no active ingredients and will be identical to the vitamin D capsules. To measure supplementation adherence, participants will be asked to bring their bottles to their follow-up visits. At the 3-month visit, participants will return their bottle to the clinical research coordinator. If the participant does not return the bottle, the clinical research coordinator will provide them with an envelope to return it via mail. All doses of vitamin D and placebo will be obtained from Bio-Tech Pharmacal, Inc. (Fayetteville, AK). The unblinding protocol can be found in Fig. [3](#Fig3){ref-type="fig"}. Following the completion of the study, participants may be unblinded their treatment group upon request. Fig. 3Unblinding of personnel for emergency medical management ### Vitamin D~3~ dose rationale {#Sec23} The doses selected are based on biologic rationale, current practice patterns, and existing guidelines. The goal of the high dose arms is to rapidly increase circulating vitamin D and serum 25(OH)D during the early callus fracture healing periods. Conversely, while the low daily dose is not expected to increase circulating vitamin D as quickly as the high-dose strategies, this treatment arm will determine if rapid serum increases are necessary to improve fracture healing. Finally, the placebo control arm is needed to demonstrate the relative potential efficacy of each treatment arm and is also necessary to represent current practice at most trauma centers in North America. High loading dose. One hundred fifty thousand international units D~3~ loading doses can be administered easily with three 50,000 IU D~3~ pills. We expect this dose to increase circulating vitamin D levels the fastest. While we acknowledge that many non-orthopedic clinicians may prefer more frequent large doses, such as 50,000 IU weekly, our loading dose strategy has been chosen to correspond with the standard post-operative clinical follow-up schedule. This is important for generalizability and is likely to overcome potential supplementation adherence issues within the adult fracture population that is often predominantly lower socioeconomic patients. This high loading dose is also in the mid-range of other previous large loading doses used safely in fracture patients and is similar to the total cumulative 3-month dose of our high daily dose group.High daily dose. Four thousand international units D~3~ represents an alternative high dose strategy and it corresponds to the tolerable upper daily intake level suggested by the Institute of Medicine (IOM) \[[@CR25]\]. While this is the IOM's upper limit, the Endocrine Society has recommended adults can safely take up to 10,000 IU per day \[[@CR26]\], further suggesting that our 4000 IU dose should be well-tolerated.Low daily dose. Six hundred international units D~3~ is a common dose and approved indication for maintaining general bone health. Six hundred international units is also the IOM's Recommended Dietary Allowance for all individuals ages 1--70 years \[[@CR25]\]. This represents our most conservative supplementation strategy, but its use is common among surgeons prescribing vitamin D and previous studies have shown its efficacy for increasing serum 25(OH)D levels.Placebo. Finally, we are including a placebo group because it is important to include placebo-controlled comparisons to our active supplements during this exploratory phase of research. Not only does placebo reflect our usual clinical practice of no supplementation, this exploratory trial will also define our rationale and selection of the control group for the definitive trial. If there are no preliminary response differences between low-dose supplementation and placebo, then the low-dose supplement could be used as the control group in the definitive trial. This would obviate potential criticisms for performing a definitive placebo-controlled trial in a population with a high prevalence of hypovitaminosis D. ### Storage and administration {#Sec24} As per the standard operating procedures at the STC, the study supplements/placebo will be stored at room temperature in accordance with the manufacturer's recommendations. The research or pharmacy personnel will maintain an inventory and temperature log to ensure the integrity of the supplements. Study supplementation will begin within 1 week of injury, and it is expected that the research personnel will provide the supplementation to the participant upon discharge from the hospital. ### Potential adverse events associated with vitamin D {#Sec25} A recent systematic review comprehensively examined the effectiveness and safety of vitamin D supplementation among all ages of adult fracture patients \[[@CR25]\]. The majority of research has been performed in elderly fracture populations; however, the safety of a wide range of doses is well established. Studies with doses of 4000 IU daily and loading doses from 50,000 IU for up to 7 days or single loading doses up to 500,000 IU have been used without complication \[[@CR27]\]. Since vitamin D regulates parathyroid hormone (PTH) and serum calcium levels, it is theoretically possible that vitamin D supplementation could lead to hypercalcemia. Of the 1088 patients included in the systematic review, four cases of hypercalcemia were reported (0.4%) \[[@CR28]\]. Furthermore, there have been no cases of hypercalcemia in several high loading dose clinical trials. Regardless, we will monitor serum calcium levels at enrolment, 6 weeks, and 3 months post-fracture, and clinical signs of hypercalcemia will be sought at all clinical encounters. If hypercalcemia is identified, participants will be instructed to stop their vitamin D supplementation immediately and the hypercalcemia will be treated as indicated. Finally, we will monitor for increased falls among the study participants. While we do not expect to observe this adverse event in our 18 to 50 year-old adult population, a recent study of 200 elderly fracture patients found that a 60,000 IU monthly loading dose and 24,000 IU monthly loading dose plus 300 μg of calcifediol were associated with increased falls compared to the control group \[[@CR29]\]. This single study contradicts several other high loading dose clinical trials, and these concerns have not been borne out of the healthy adult fracture population. This may be a result of the fact that supplementation in the non-osteoporotic fracture population has not been extensively studied (highlighting the need for the proposed research) or because these concerns regarding the risk of falls do not apply to healthy adults without osteoporosis. ### Concomitant calcium supplementation {#Sec26} In addition, although calcium supplementation is often recommended concomitantly with vitamin D for osteoporosis prevention, for our non-osteoporotic study population the necessity of calcium supplementation is controversial and will not be provided because of the increased risk of kidney stones, hypercalcemia, and potential confounding. This rationale has also been outlined by other researchers performing RCTs involving vitamin D supplementation. Surgical technique and post-operative rehabilitation {#Sec27} ---------------------------------------------------- ### Surgical technique {#Sec28} The study protocol will not dictate the surgical technique. Based on the study's eligibility criteria, all participants must receive a reamed, locked, intramedullary nail for their tibial or femoral shaft fracture. The number and orientation of locking screws is at the discretion of the treating surgeon, as there have been no studies that demonstrate clinical superiority of any locking screw strategy. Any concomitant fracture lines that extend into the adjacent articular areas may be treated with additional fixation as indicated. ### Post-operative rehabilitation {#Sec29} Full weight-bearing as tolerated is recommended for all isolated tibial and femoral shaft fractures. In the presence of additional lower extremity fractures, intra-articular extension, or other concomitant soft tissue injuries, participants may be restricted to protected weight-bearing (partial or no weight) for up to 6 weeks post-fracture. If additional contralateral injuries are present, both limbs must be eligible for full weight-bearing by 6 weeks post-fracture. Primary and secondary outcome measures {#Sec30} -------------------------------------- ### Primary outcome {#Sec31} Fracture healing will be assessed as follows: (1) clinical fracture healing will be measured using FIX-IT, (2) radiographic fracture healing will be measured using the RUST, and (3) biological fracture healing will be measured using serum levels of CTX and PINP. #### Clinical healing {#FPar1} FIX-IT is a standardized measure of weight-bearing and pain in patients with lower extremity fractures, specifically tibia and femur fractures \[[@CR16]\]. Preliminary validation of the FIX-IT has demonstrated high inter-rater agreement and moderate correlation with the physical scores of the Short Form-36 \[[@CR16]\]. It has been used in other studies to assess clinical fracture healing. #### Radiographic healing {#FPar2} The RUST score assesses the presence of bridging callus or a persistent fracture line on each of four cortices \[[@CR17]--[@CR20]\]. This score has been previously validated and found to have greater inter-rater reliability when compared with surgeons' general impression of the cortical bridging \[[@CR17]--[@CR20]\]. RUST has been widely used to assess radiographic fracture healing \[[@CR17]--[@CR20]\]. An orthopedic surgeon who is independent of the study will review the images and assign a RUST score. #### Biological healing {#FPar3} CTX is a bone-resorption marker and previous research has found that it rises 1 week after fracture of the tibial shaft and remains elevated throughout fracture healing \[[@CR21]\]. PINP is a bone-formation marker and prior research has found that it is highest at 12 weeks after fractures of the tibial shaft and proximal femur \[[@CR30]\]. The primary time point for assessing fracture healing will be at 3 months post-injury. This time point was selected because it coincides within the standard clinical follow-up schedule, and because it has the greatest potential to detect differences in short-term fracture healing. Additionally, radiographic fracture healing is a surrogate predictive of re-operations related to fracture healing complications. More specifically, the presence of early radiographic callus (\< 4 months) has shown to be the strongest predictor of reoperation, with one study reporting 99% accuracy and an area under the curve of 0.995 (*p* \< 0.0001) \[[@CR31]\]. Data from our institution confirmed these findings with the 3-month RUST score being the most powerful predictor of nonunion surgery for tibia fractures \[[@CR32]\]. While we expect the 1-year fracture union rate to be approximately 95% for the femur fractures \[[@CR30]\] and 75% for the tibia fractures (unpublished data from the SPRINT trial) \[[@CR33]\], improved early fracture healing is biologically plausible. The median time to fracture union for tibia fractures is 4 months; therefore, many patients are still experiencing morbidity from their injury at the 3-month visit and decreasing the time to union would be an important patient benefit. ### Secondary outcomes {#Sec32} The main secondary outcome for the definitive trial will be assessed by measuring 25(OH)D serum levels. Serum levels will be collected in a blinded manner. Correlations will be assessed between participants' 25(OH)D levels at enrolment, changes in 25(OH)D levels from enrolment to 3 months, and 25(OH)D levels at 3 months and fracture healing as described above. The other secondary outcomes will include assessing supplementation adherence between daily and loading doses, confirming participant safety as measured by adverse events and serum levels of calcium and PTH, and assessing protocol adherence (e.g., completion of outcome assessment and participant follow-up). *Adherence with vitamin D supplementation* will be assessed by participant self-report, by counting the tablets for the daily doses at each follow-up, and by direct observation for the loading doses. *Participant safety* will be assessed by adverse events, defined as any symptom, sign, illness, or experience that develops or worsens in severity during the course of this study. Within the adverse events collected, fracture healing complications will be identified and will include nonunion (defined as failure of the fracture to progress towards healing for 2 consecutive months and at least 6 months post-fracture), delayed union (defined as a failure of progression of fracture healing beyond the expected median healing time of 4 months with pain at the fracture site), hardware failure (defined as a broken or bent nail or locking screw) \[[@CR34]\], wound healing problems (previously published criteria by Anglen \[[@CR34]\]), and infection (superficial and deep as defined by Centers for Disease Control and Prevention criteria). Wound healing problems and infection are a part of the composite fracture healing complication outcome because previous animal and infectious disease clinical research has suggested that vitamin D can improve wound healing and reduce infections \[[@CR35]--[@CR38]\]. In addition to adverse events, serum levels of calcium and PTH will be monitored and we will record results of the participants' pre-operative metabolic profile. These data will be used to understand the baseline metabolic health of the participants and will be used as needed in the event of suspected adverse events. *Participant adherence with the protocol* will be assessed by monitoring the completion of outcome measures, including clinic assessments (FIX-IT), radiographs (RUST), and blood work (CTX, PINP, 25(OH)D, calcium, and PTH), documentation of adverse events and re-operations, and completion of follow-up to 12 months. Research personnel conducting the outcome assessments will be blinded to the allocation. Data collection and participant follow-up {#Sec33} ----------------------------------------- Upon providing informed consent, baseline demographics will be collected from the patient and from their medical chart (Table [1](#Tab1){ref-type="table"}). This includes demographic, medical history, pre-operative blood work-up details (e.g., kidney and liver function tests, calcium, phosphate, and albumin), injury details, fracture characteristics, details on the surgical management of their fracture, and rehabilitation details. Participants will have blood drawn within the fracture clinic that will be analyzed for calcium levels and for CTX, PINP, 25(OH)D, and PTH. Post-operative x-rays will be taken as per standard of care. Table 1Schedule of eventsAssessmentVisit 1: screening and baselineVisit 2: 6 weeksVisit 3: 3 monthsVisit 4: 6 monthsVisit 5: 9 monthsVisit 6: 12 monthsScreening●Serum calcium analysis●\*●●Informed consent●Randomization●Collection of baseline data (demographic, serum metabolic panel, fracture, and surgical data)●Nutritional/placebo supplementation\*\*●●●Assessment of clinical fracture healing (FIX-IT)●●●●●X-rays of tibia or femur●●●●●●Assessment of radiographic fracture healing (RUST)●●●●●Serum bone marker analysis (CTX and PINP)●●●Assessment of adherence to supplementation●●Laboratory serum 25(OH)D analysis●●●Assessment for adverse events●●●●●Serum PTH level analysis●●●Assessment of fracture healing complications●●●●●\*To be assessed as eligibility criteria\*\*Must occur within 1 week of fracture Feasibility of the study will be assessed over 12 months post-fracture. Participants will be followed at standard clinical visit intervals for 12 months post-injury including 6 weeks, 3 months, 6 months, 9 months, and 12 months. The schedule of events (Table [1](#Tab1){ref-type="table"}) details the requirements and procedures for each visit. Participants will have blood drawn within the fracture clinic that will be analyzed for calcium, CTX, PINP, 25(OH)D, and PTH serum levels at 6 weeks and 3 months. Post-operative x-rays will be taken as per standard of care at each follow-up visit. Participants will be assessed clinically for FIX-IT at each visit. All study outcomes (as defined above) will be documented on the case report forms (CRFs) at each follow-up visit. A 12-month follow-up was selected because it is a standard follow-up period for patients with tibial and femoral shaft fractures and it is a commonly used follow-up period for similar fracture trials \[[@CR33], [@CR39]\]. In addition, it is a commonly referenced time period for fracture healing complications requiring reoperation and will further inform decisions surrounding the larger, definitive RCT. ### Analysis of blood samples {#Sec34} Serum calcium testing will be performed by the hospital laboratory and will be part of the unblinded medical chart for patient safety. The remainder of serum samples (PTH, 25(OH)D, PINP, CTX) will be analyzed in a blinded manner at the end of the study. Laboratory personnel at the University of Maryland's Muscle Research Laboratory will process the samples from STC for storage in the − 80 °C freezer. Upon completion of all blood work for the study, the serum samples will be transferred to the Institute for Clinical and Translational Research Clinical Research Unit Core Laboratory to be analyzed as a single batch to eliminate inter-batch assay variability. The results of the analyses will be sent to the Center for Evidence-Based Orthopaedics (CEO) to be added to the study database and included within the final data analysis. The treating surgeon will remain blinded to these results. Participants may request the results of their blood analysis at the end of the study. ### Analysis of radiographs {#Sec35} The radiographs of participants recruited at STC will be stored in the STC Picture Archiving and Communication System and then sent to the CEO for the review of RUST by an independent practicing orthopedic surgeon. Participant retention {#Sec36} --------------------- Once a participant is enrolled in the trial, every reasonable effort will be made to follow the participant for the entire duration of the study period. The expected follow-up rate for this study is greater than 90% based on similar fracture trials \[[@CR33], [@CR39]--[@CR41]\]. To maximize participant retention, all possible attempts should be made to collect as much data as possible and to reduce loss to follow-up. We have implemented procedures to improve participant retention (Fig. [4](#Fig4){ref-type="fig"}) \[[@CR42]\]. Fig. 4Retention strategies We will only deem participants lost to follow-up after all exhaustive measures have been taken to locate the participant. Participants should not be deemed lost to follow-up until the 12-month visit is due and all attempts to contact the participant have been exhausted. We will not remove participants from the study if the study protocol was not adhered to (e.g., participant received wrong treatment arm, early discontinuation of supplements, occurrence of protocol deviations, missed follow-up visits). We will document the reasons for participant withdrawal from the trial (e.g., withdrawal of consent or lost to follow-up). Statistical plan {#Sec37} ================ Sample size determination {#Sec38} ------------------------- The trial will use a phase II randomized screening design to facilitate non-definitive comparisons of three vitamin D~3~ dosing regimens. Using the principles outlined by Rubinstein et al., the statistical parameters have been carefully chosen to ensure a reasonable sample size for our definitive trial and meaningful results \[[@CR43]\]. Consistent with previous recommendations, an *α* and *β* of 0.20 was chosen with a target mean difference of 17--20%, depending on the fracture healing measure. There will be no adjustments for multiple testing given the exploratory nature of the study design. Based on the original instrument development and validation in tibia and femur fracture patients, it is expected that the low dose and control groups will have a mean 3-month FIX-IT score of 8 (standard deviation (SD) 3) \[[@CR16]\]. Assuming the high dose groups will achieve a mean 2-point increase (17% mean difference), 21 patients are required in each group. The same sample size requirements will be applied for comparisons using the RUST instrument based on similar assumptions and recent literature (2-point mean difference, 8 vs. 6, SD 3) \[[@CR17]--[@CR20]\]. Clinically important changes in the PINP and CTX markers are unknown; however, in a previous study of tibia fracture healing, Veitch et al. observed concentrations of both bone turnover markers approximately 100% greater than baseline values \[[@CR44]\]. Given the large changes observed in these bone turnover markers, the same criteria will be applied for identifying a potentially clinically beneficial regimen and remain powered to detect a mean difference of 20% (SD 30%). Finally, the sample size will be increased to account for a 10% loss to follow-up, for a total enrolment of 24 patients per allocation group (96 total). Statistical methods {#Sec39} ------------------- All outcome analyses will be exploratory and adhere to the intention-to-treat principle. Per-protocol sensitivity analyses will also be conducted. Our statistical analysis plan will provide additional details on the analyses and will be finalized prior to conducting the final analyses. ### Specific aim {#Sec40} Each measure of fracture healing will be described with its mean and SD. For our primary analysis, comparisons for the three hypotheses will be made using an independent *t-* test and significance set at *α* = 0.20 (Table [2](#Tab2){ref-type="table"}). Hypothesis 1 compares high-dose supplementation versus low dose. To test this hypothesis, we will combine the two high-dose groups (loading and daily) for a 2:1 comparison against the low daily dose group. All other comparisons will be 1:1 based on the treatment groups outlined. Table 2Primary outcome analysisObjectiveHypothesisFracture healing outcomeMethod of analysisTo determine the response of vitamin D~3~ dose on fracture healing at 3 months*High doses of supplementation (loading or daily) will increase healing compared to low daily dose*. Using high doses will rapidly increase the circulating vitamin D available during fracture callus formation.1. FIX-IT (clinical)Patients in the high loading dose and high daily dose groups will be combined for a 2:1 comparison against low daily dose group using an independent *t*-test (alpha = 0.20).\*2. RUST (radiographic)3. PINP (biologic)4. CTX (biologic)To determine the response of vitamin D~3~ frequency on fracture healing at 3 months*High loading dose increases healing compared to high daily dose.* Loading doses will overcome medication adherence issues and increase circulating vitamin D even more rapidly than daily doses.1. FIX-IT (clinical)Comparisons between the high loading dose and high daily dose groups will be made using an independent t-test (alpha = 0.20).\*2. RUST (radiographic)3. PINP (biologic)4. CTX (biologic)To determine the response of low amounts of vitamin D~3~ supplementation on fracture healing at 3 months*Low daily dose will increase healing compared to placebo*. While the low daily dose is not expected to increase circulating vitamin D as rapidly as the high dose strategies, this comparison will determine if rapid serum increases are necessary to improve fracture healing.1. FIX-IT (clinical)Comparisons between the low daily dose and placebo groups will be made using an independent *t*-test (alpha = 0.20).\*2. RUST (radiographic)3. PINP (biologic)4. CTX (biologic)\*Using a phase II screening trial approach, comparisons are non-definitive and an increased alpha level has been adopted ### Secondary aims {#Sec41} To test the hypotheses of the main secondary aim, adjusted regression models will be used to explore associations between 3-month fracture healing and three assessments of serum 25(OH)D levels: enrolment, 3 months, and change in levels between enrolment and 3 months. Significance will be set at *α* = 0.20. Additional descriptive analyses will be performed for serum 25(OH)D at each time point (Table [3](#Tab3){ref-type="table"}). Table 3Secondary outcomes analysisObjectiveHypothesisOutcomeMethod of analysisMain secondary outcome To determine if 25(OH)D serum levels are associated with fracture healing at 3 monthsThere will be an association between fracture healing and:1. FIX-IT (Clinical)Associations will be quantified using univariate analysis (alpha = 0.20).\*.2. RUST (Radiographic)1) Patients' enrolment serum 25(OH)D3. PINP (Biologic)4. CTX (Biologic)2) Their change in 25(OH)D from enrolment to 3 months3) Their 25(OH)D level at 3 monthsOther secondary outcomes Supplementation adherenceDaily vitamin D~3~ adherence will be \< 80% and loading dose vitamin D~3~ adherence will be \> 95%.Self-reportSummary statistics of means and confidence interval.Count of pills Participant safetyAdverse events will be rare across all 4 treatment groups.Adverse eventSummary statistics of proportions.Re-operations for a composite of fracture healing complications will follow the same 3 hypotheses as fracture healing.Re-operations for a composite of fracture healing complicationsSummary statistics of proportions.Levels of serum calcium will be similar across the 4 treatment groups. Levels of serum calcium will be within normal reference ranges.Serum calciumSummary statistics of means and confidence interval.Levels of serum PTH will be similar across the 4 treatment groups. Levels of serum PTH will be within normal reference ranges.Serum PTHSummary statistics of means and confidence interval. Protocol adherenceProtocol adherence will be acceptable.Complete follow-up assessments including x-rays and bloodworkSummary statistics of proportions.\*Using a phase II screening trial approach, comparisons are non-definitive and an increased alpha level has been adopted All other secondary outcomes will be presented using point estimates and appropriate measures of variance to describe supplementation adherence, participant safety, and key aspects of participant compliance with the protocol (Table [3](#Tab3){ref-type="table"}). Supplement adherence will be summarized using means and 95% confidence intervals (CIs) for participant self-reporting and the mean cumulative dose taken at 3 months. The incidence of adverse events and re-operations for fracture healing complications in each group will be described with counts and proportions. Serum levels of calcium and PTH will be summarized using means and 95% CIs. Participant compliance with the protocol will be summarized descriptively with counts and proportions. ### Per-protocol sensitivity analyses {#Sec42} The specific aim and the relevant other secondary outcome analyses will be repeated following as-treated analyses. These sensitivity analyses will be completed after the above outcome analyses have been completed and once unblinding has occurred. Per protocol will be defined as participants who received both loading doses of vitamin D and participants who did not miss 20 or more daily doses of vitamin D. Therefore, participants who missed a loading dose of vitamin D and participants who missed 20 or more daily doses of vitamin D will not be included in the as-treated sensitivity analyses. Data management {#Sec43} =============== The CRFs will be the primary data collection tool for the study. All data requested on the CRF must be recorded. All data will be entered into the trial database (McMaster University) and double verified. Data and safety committee {#Sec44} ========================= An orthopedic surgeon at the University of Maryland will monitor patient safety for the Vita-Shock study. The surgeon will be responsible for reviewing adverse events, enrolment numbers, and medical compliance. All adverse events, both serious and non-serious, will be reported through safety reports that will be sent to the surgeon on an annual basis for review. Reports will contain the following information: Brief narrative introduction that describes the status of the study, progress or findings to-date, issues, and the procedures that produced the report (e.g., data obtained by a specific date).Administrative tables that describe study status.Aggregate tables of adverse events and serious adverse events.Listings of serious adverse events. Adverse event reporting and definitions {#Sec45} --------------------------------------- ### Adverse event {#Sec46} An adverse event is any symptom, sign, illness, or experience that develops or worsens in severity during the course of this study. ### Serious adverse event {#Sec47} Adverse events are classified as serious or non-serious. A serious adverse event is any adverse event that is any of the following: FatalLife threateningRequires or prolongs hospital stayResults in persistent or significant disability or incapacityA congenital anomaly or birth defectAn important medical event All serious adverse events must be recorded and promptly submitted to the University of Maryland Institutional Review Board (IRB), as well as be reported to the Methods Centre immediately. ### Unanticipated problems resulting in risk to participant or others {#Sec48} Any incident, experience, or outcome that meets all of the following criteria: Unexpected in nature, severity, or frequency (e.g., not described in study-related documents such as the ethics-approved protocol or consent form).Related or possibility related to participation in the research (i.e., possibly related means there is reasonable possibility that the incident experience or outcome may have been caused by the procedures involved in the research).Suggests that the research places participants or others at greater risk of harm (including physical, psychological, economic, or social harm). All unanticipated problems resulting in risk to participants or others must be recorded and promptly submitted to the University of Maryland IRB, as well as be reported to the Methods Centre immediately. ### Adverse drug reactions {#Sec49} An adverse drug reaction is an injury caused by taking a medication. All adverse drug reactions that are considered both serious and unexpected are to be reported to the US Food and Drug Administration. Dissemination {#Sec50} ============= Results from the study will be disseminated through a publication in an academic journal and through presentations at relevant orthopedic conferences regardless of whether or not there are significant findings. Every attempt will be made to ensure that the amount of time between completion of data collection and release of study findings is minimized. Supplementary information ========================= {#Sec51} **Additional file 1.** SPIRIT 2013 Checklist. 25(OH)D : 25-Hydroxyvitamin D CEO : Center for Evidence-Based Orthopaedics CI : Confidence interval CRF : Case report form CTX : C-Terminal telopeptides of type I collagen FIX-IT : Function IndeX for Trauma IRB : Institutional review board IU : International units PINP : Amino-terminal procollagen propeptides of collagen type I PTH : Parathyroid hormone RCT : Randomized controlled trial REB : Research ethics board RUST : Radiographic union score for tibial fractures SD : Standard deviation STC : R Adams Cowley Shock Trauma Center Vita-Shock : A Blinded Exploratory randomized controlled trial to determine optimal vitamin D3 supplementation strategies for acute fracture healing **Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information ========================= **Supplementary information** accompanies this paper at 10.1186/s40814-019-0524-4. Vita-Shock Investigators Writing Committee: Sheila Sprague, Sofia Bzovsky, Daniel Connelly, Lehana Thabane, Jonathan D. Adachi, and Gerard P. Slobogean Principal Investigators: Gerard P. Slobogean, University of Maryland, Baltimore and Sheila Sprague, McMaster University Co-Investigators: Jonathan D. Adachi, Mohit Bhandari, Lehana Thabane (McMaster University) and Michael F. Holick (Boston University) McMaster University Methods Centre: Sofia Bzovsky (Project Manager), Nicole Simunovic (Grants Management), Kim Madden (Unblinded Project Manager, Data Management), Heather Dwyer (Unblinded Project Manager, Data Management), Taryn Scott (Unblinded Project Manager, Data Management), and Diane Heels-Ansdell (Statistical Analysis) R Adams Cowley Shock Trauma Center, University of Maryland: Daniel Connelly, Joshua Rudnicki, Zachary Hannan, Andrew N. Pollak, Robert V. O'Toole, Christopher LeBrun, Jason W. Nascone, Marcus F. Sciadini, Yasmin Degani, Raymond Pensy, Theodore Manson, W. Andrew Eglseder Jr., Lucas Marchand, Andrea Howe, Dimitrius Marinos, Daniel Mascarenhas, George Reahl, Katherine Ordonio, Marckenley Isaac, Ugochukwu Udogwu, Mitchell Baker, Alexandra Mulliken, and Haley Demyanovich All authors listed under the Acknowledgments section reviewed the manuscript and provided critical input regarding its intellectual content. All authors read and approved the final manuscript. Supported by research grants from the Orthopaedic Trauma Association and McMaster Surgical Associates. The funding bodies had no role in the design of the study and collection of data and will have no role in the analysis and interpretation of data and in writing the manuscript. All data generated in this study will be analyzed upon completion of participant follow-up and will be published in a journal article in the future. This protocol, the consent form template, and the CRFs have been reviewed and approved by the University of Maryland IRB and the McMaster University REB prior to commencement of the study. In addition, an Investigation New Drug Application was approved by the Food and Drug Administration. Any amendments to the study protocol which may affect the conduct of the study, or the potential safety of or benefits to participants (e.g., changes to the study objectives, study design, sample size, or study procedures) will require a formal amendment to the protocol. Any protocol amendments will be approved by the principal investigators and will require approval by the University of Maryland IRB and the McMaster University REB. Administrative changes (e.g., minor corrections or clarifications that have no effect on the way the study is conducted) will not need to undergo a formal amendment process. Not applicable The authors declare that they have no competing interests.

Rico‐Guevara A, Mickley J. Bring your own camera to the trap: An inexpensive, versatile, and portable triggering system tested on wild hummingbirds. Ecol Evol. 2017;7:4592--4598. <https://doi.org/10.1002/ece3.3040> 1. INTRODUCTION {#ece33040-sec-0001} =============== Studying animals in their natural habitat is advantageous and preferable to working with them in captivity for a variety of research questions. Natural habitats allow for the study of behavior in situ, offering opportunities to measure energetic performance of free‐living animals, and opening doors to understanding intra and interspecific interactions. Researchers face three main challenges while working with wild animals: (1) neutralizing the observer effect (Baker & McGuffin, [2007](#ece33040-bib-0005){ref-type="ref"}; Wade, Zalucki, & Franzmann, [2005](#ece33040-bib-0037){ref-type="ref"}), (2) dealing with long waiting times (review in Cutler & Swann, [1999](#ece33040-bib-0009){ref-type="ref"}), and (3) compensating for human sensory limitations (Weale, [1961](#ece33040-bib-0038){ref-type="ref"}). These challenges are solved with camera traps (reviews in Cutler & Swann, [1999](#ece33040-bib-0009){ref-type="ref"}; O\'Connell, Nichols, & Karanth, [2011](#ece33040-bib-0020){ref-type="ref"}; Rowcliffe & Carbone, [2008](#ece33040-bib-0029){ref-type="ref"}), which are used to detect animals in a given area (Karanth, [1995](#ece33040-bib-0015){ref-type="ref"}; Silveira, Jácomo, & Diniz‐Filho, [2003](#ece33040-bib-0031){ref-type="ref"}), and to study behavior (Bischof, Ali, Kabir, Hameed, & Nawaz, [2014](#ece33040-bib-0006){ref-type="ref"}; Gula, Theuerkauf, Rouys, & Legault, [2010](#ece33040-bib-0011){ref-type="ref"}; Ohashi, D\'Souza, & Thomson, [2010](#ece33040-bib-0021){ref-type="ref"}). Nevertheless, commercial camera traps are effective with relatively few taxa and can be prohibitively expensive (Meek & Pittet, [2012](#ece33040-bib-0018){ref-type="ref"}). Our goal was to design an alternative with the flexibility to study a larger variety of taxa at the lowest cost possible. We developed a system with many advantages over available camera traps: (1) it functions mechanically and can be coupled with cameras that do not support remote triggering, (2) it can incorporate most sensors, which allows for other modes of detection besides movement (e.g., light, color, sound), (3) it offers versatility to position multiple sensors separately from the camera, adapting to the subject of interest, (4) it is inexpensive enough to be affordable in field projects requiring multiple camera traps, (5) as it is not married to any particular camera, it can be updated when a given technology becomes obsolete, (6) it is powered by standard AA batteries for long durations, facilitating recharging, easy replacement, and accessibility in remote locations, (7) it is weatherproof, light, and portable, allowing for the deployment of several units with few personnel, and (8) the triggering and sensor systems are easy to customize in the field to adapt to changing conditions or objectives. Most camera traps have been designed to capture large animals using passive infrared motion sensors (PIR) (Meek & Pittet, [2012](#ece33040-bib-0018){ref-type="ref"}; Rovero, Zimmermann, Berzi, & Meek, [2013](#ece33040-bib-0028){ref-type="ref"}; Welbourne, Claridge, Paull, & Lambert, [2016](#ece33040-bib-0040){ref-type="ref"}), although some applications have been aimed at small mammals (Pearson, [1960](#ece33040-bib-0022){ref-type="ref"}; Soininen & Jensvoll, [2015](#ece33040-bib-0032){ref-type="ref"}; Villette, Krebs, Jung, & Boonstra, [2016](#ece33040-bib-0036){ref-type="ref"}), birds (Bolton, Butcher, Sharpe, Stevens, & Fisher, [2007](#ece33040-bib-0007){ref-type="ref"}; Kross & Nelson, [2011](#ece33040-bib-0016){ref-type="ref"}), and arthropods (review in Steen & Ski, [2014](#ece33040-bib-0034){ref-type="ref"}). PIR sensors detect a change in surface temperatures, such as when an animal with a different surface temperature than the background enters the scene (Welbourne et al., [2016](#ece33040-bib-0040){ref-type="ref"}). Despite broad use, PIR sensors present various problems such as false negatives when the subject\'s surface temperature differs minimally from the background (Welbourne et al., [2016](#ece33040-bib-0040){ref-type="ref"}), or false positives from vegetation blowing in the wind (Rovero et al., [2013](#ece33040-bib-0028){ref-type="ref"}; Welbourne et al., [2016](#ece33040-bib-0040){ref-type="ref"}). Innovations for improving detection of smaller (or poikilothermic) taxa include active infrared sensors (AIR) that trigger when an infrared beam is crossed (Hernandez, Rollins, & Cantu, [1997](#ece33040-bib-0013){ref-type="ref"}; Rovero et al., [2013](#ece33040-bib-0028){ref-type="ref"}; Swann, Hass, Dalton, & Wolf, [2004](#ece33040-bib-0035){ref-type="ref"}), cameras with video motion detection that trigger when there is movement in the selected field of view (Bolton et al., [2007](#ece33040-bib-0007){ref-type="ref"}; Kross & Nelson, [2011](#ece33040-bib-0016){ref-type="ref"}), and multiple sensors to improve detectability (Meek & Pittet, [2012](#ece33040-bib-0018){ref-type="ref"}). Unlike PIR, the former two innovations are not affected by surface temperature of the background or subject. In addition to sensor limitations, available camera traps generally lack specialized features such as tele‐macro and high‐speed video. External sensors and triggering systems (e.g., Trailmaster^®^, Cognisys^®^) provide control over the sensors used, and allow the use of some specialized cameras (Brooks, [1996](#ece33040-bib-0008){ref-type="ref"}; Hernandez et al., [1997](#ece33040-bib-0013){ref-type="ref"}; Kucera & Barrett, [1993](#ece33040-bib-0017){ref-type="ref"}), but all require cameras that can be remotely triggered. A review of the market found no suitable camera trap for capturing high‐speed video of hummingbird floral visits: limitations included a combination of slow trigger speed (latency to start recording 0.5 s, cf. Meek & Pittet, [2012](#ece33040-bib-0018){ref-type="ref"}), low video frame rate (\<60 frames/s), and no remote triggering. Our system overcomes these limitations by pairing with nearly any camera or sensor setup, allowing a researcher to choose optimal camera and sensor configurations separately to match their organism and application. We tested the system by tailoring it specifically to hummingbirds, but as it is presented here, it can be coupled to any kind of sensor and camera in order to study a wide variety of subjects (e.g., AIR sensors and night‐vision cameras to study nocturnal poikilotherms). We present a test of this novel system using hummingbirds (Figure [1](#ece33040-fig-0001){ref-type="fig"}). These small, superb fliers visit flowers quickly without perching--difficult subjects for camera traps to detect and record. We have studied hummingbirds drinking nectar from artificial feeders (Rico‐Guevara & Rubega, [2011](#ece33040-bib-0026){ref-type="ref"}) and have developed predictions from biomechanical principles (Rico‐Guevara, Fan, & Rubega, [2015](#ece33040-bib-0025){ref-type="ref"}) that necessitate testing in the wild. Hummingbirds may visit a flower at intervals from 10 minutes to a few hours (Araujo & Sazima, [2003](#ece33040-bib-0004){ref-type="ref"}; Rodrigues & Rodrigues, [2014](#ece33040-bib-0027){ref-type="ref"}), so camera trapping becomes imperative to collect data without researcher‐intensive monitoring. A camera trap that can capture hummingbirds visiting wild flowers requires: (1) detection of small animals, (2) high‐speed video, and (3) tele‐macro functionality (close‐up videos from a distance). {ref-type="supplementary-material"}](ECE3-7-4592-g001){#ece33040-fig-0001} We aimed to quantify a hummingbird\'s net energy gain during a floral visit, for which we needed to measure wing beat frequency to estimate energy expenditure in addition to energy acquired from the nectar (c.f. Anderson, [1991](#ece33040-bib-0003){ref-type="ref"}). A hummingbird may completely deplete a flower in less than a second; capturing this process requires fast triggering. With nectar licking rates up to 20 Hz (Rico‐Guevara, [2014](#ece33040-bib-0024){ref-type="ref"}) and wingbeat frequencies usually around 20--50 Hz (Altshuler & Dudley, [2003](#ece33040-bib-0001){ref-type="ref"}; Hedrick, Tobalske, Ros, Warrick, & Biewener, [2012](#ece33040-bib-0012){ref-type="ref"}), high‐speed video (\>200 frames/s) is required to count the number of licks and wing beats during a single visit to a flower, information of primary importance to understand their energetics and consequent decision‐making behaviors in nature. 2. MATERIALS AND METHODS {#ece33040-sec-0002} ======================== 2.1. Sensor selection and testing {#ece33040-sec-0003} --------------------------------- We choose to use PIR motion sensors over the alternatives listed above because they did not need to be in close proximity to the flower, were cheap and easy to deploy, and successfully detected hummingbirds. We assessed their ability to detect hummingbirds by filming each sensor connected to a light‐emitting diode, at different distances (measured with a laser range finder: Simmons 600) at hummingbird feeders and flowers at the Finca Colibrí Gorriazul, a private field station near Fusagasugá, Colombia. PIR sensors successfully detected over 15 species of hummingbird, varying in size and behavior, at both feeders and flowers. Individuals of one of the smallest species (3.5--4 g), *Chaetocercus mulsant* (Table [S3](#ece33040-sup-0003){ref-type="supplementary-material"}) were reliably detected at distances of 50 cm, and individuals of one of the largest species (6--9 g), *Colibri coruscans* (Table [S3](#ece33040-sup-0003){ref-type="supplementary-material"}) were reliably detected at distances of 100 cm. 2.2. Triggering mechanism {#ece33040-sec-0004} ------------------------- Our triggering mechanism consisted of two PIR sensors connected to a triggering circuit (Figures [2](#ece33040-fig-0002){ref-type="fig"}, [3](#ece33040-fig-0003){ref-type="fig"}, [S2](#ece33040-sup-0003){ref-type="supplementary-material"}). When either of the PIR sensors activated, the triggering circuit briefly turned on a mechanical actuator, which manually pushed the shutter button of the camera (Figure [2](#ece33040-fig-0002){ref-type="fig"}). The PIR sensors' retrigger delay was set to 45 s to prevent retriggering before the camera was ready to record again (see [supplemental methods](#ece33040-sup-0003){ref-type="supplementary-material"}); this delay may be unnecessary depending on the study subject and camera selected. Our application also generated a wireless signal to a control box (Fig. [S1](#ece33040-sup-0003){ref-type="supplementary-material"}) some distance away so that a researcher was notified when any traps had triggered to allow for nectar measurements (see below). PIR sensors generally have a range of \<7 m for larger‐bodied animals, although with smaller‐bodied animals, such as hummingbirds, their effective range is \<1 m. Using multiple external sensors, we were able to optimally position them with respect to the camera, decreasing the likelihood of false negatives. For example, if background vegetation movement was triggering a sensor, we could reposition that sensor (pointing it away from the piece of moving vegetation) without compromising detectability because of the redundancy achieved by having more than one sensor. Furthermore, our standalone sensors (Figure [3](#ece33040-fig-0003){ref-type="fig"}) included an option to adjust sensitivity, allowing for fine‐tuning by optimizing the tradeoff between increased sensitivity and false positives. Our final costs per triggering mechanism were \<\$50 (Table [S2](#ece33040-sup-0003){ref-type="supplementary-material"}) plus the cost of the camera (Table [S1](#ece33040-sup-0003){ref-type="supplementary-material"}, we used pre‐owned cameras of \~\$200); more inexpensive than other proposed homemade systems (that generally cost \$500--\$1,500---Pierce & Pobprasert, [2007](#ece33040-bib-0023){ref-type="ref"}; Gula et al., [2010](#ece33040-bib-0011){ref-type="ref"}; Steen, [2014](#ece33040-bib-0033){ref-type="ref"}) or commercial solutions (that can cost upwards of \$1,000--5,000---Meek & Pittet, [2012](#ece33040-bib-0018){ref-type="ref"}; Rovero et al., [2013](#ece33040-bib-0028){ref-type="ref"}). Triggering mechanisms weighed \~500 g including batteries and the two sensors, much smaller and lighter than many of the alternatives. Additionally, a number of cost‐saving innovations accompany our system and are broadly applicable to any ecological research using homemade electronics or sensors (see [supplemental methods](#ece33040-sup-0003){ref-type="supplementary-material"}). {ref-type="supplementary-material"} as well as additional photos and diagrams (Figure [S1](#ece33040-sup-0003){ref-type="supplementary-material"}, [S2](#ece33040-sup-0003){ref-type="supplementary-material"}, supplementary circuit diagrams)](ECE3-7-4592-g002){#ece33040-fig-0002} {ref-type="fig"}b). (c) Lateral view of the trigger box where the weatherproof ports for the sensor phone cord (labeled in red) and the actuator connections (on the right side) are visible. (d) Frontal view of the trigger box in which the power switch (labeled in orange) and the connections for the power wires of the actuator (in blue) can be observed](ECE3-7-4592-g003){#ece33040-fig-0003} 2.3. Camera selection and specifications {#ece33040-sec-0005} ---------------------------------------- More than two frames per wing beat cycle are convenient to estimate hummingbird wing beat frequency (cf. Altshuler & Dudley, [2003](#ece33040-bib-0001){ref-type="ref"}). Consequently, we needed cameras with high‐speed video and a prerecord mode (recording a brief amount of video before the shutter is pressed) to compensate for the camera\'s shutter lag. We compared available high‐speed video cameras to balance affordability of a multicamera setup with the required features, which we include in the Table [S1](#ece33040-sup-0003){ref-type="supplementary-material"}. We opted to use consumer‐grade high‐speed video cameras (cf. Steen, [2014](#ece33040-bib-0033){ref-type="ref"}), and after experimenting with different models with our triggering system (first four rows in Table [S1](#ece33040-sup-0003){ref-type="supplementary-material"}, recording length section in Supplement), we chose the Casio EX‐FH20/5, which has already been used for biological research (e.g., Ryerson & Schwenk, [2011](#ece33040-bib-0030){ref-type="ref"}). These cameras featured video recording at 210 fps and 480 × 360‐pixel resolution, along with a prerecord mode. We mounted the cameras on light tripods (with triggering system attached) and shielded them with reflective‐layered foam covers, offering rain and sun protection. Cameras were powered externally by two 4xAA battery packs wired in parallel and plugged into the camera\'s AC adapter port. 2.4. System tests {#ece33040-sec-0006} ----------------- We studied hummingbird feeding at Peña del Aserradero Natural Reserve (cloud forest \~2,400 m.a.s.l.) in the Northern Andes of Colombia. We tested the cameras during 3 days (pilot fieldwork), then stopped filming to review the videos and make adjustments (see Section "[3](#ece33040-sec-0007){ref-type="sec"}"), and finished with seven more days of filming. We deployed camera traps (Figures [4](#ece33040-fig-0004){ref-type="fig"}, [S3](#ece33040-sup-0003){ref-type="supplementary-material"}, [S4](#ece33040-sup-0003){ref-type="supplementary-material"}) at focal flowers (May--June 2015), experiencing copious rain, cold (lowest temperatures under 5°C), and intense sun (peak temperatures above 30°C). We collected data simultaneously from four high‐speed cameras situated at focal flowers in different feeding territories. To assess the reliability of the system by documenting missed visits or false positives, backup cameras at each site continuously filmed both the focal flowers and the camera traps at 30 fps (Video [S1](#ece33040-sup-0001){ref-type="supplementary-material"}). We camouflaged all the cameras and systems at the field site (Video [S1](#ece33040-sup-0001){ref-type="supplementary-material"}, Fig. [S3](#ece33040-sup-0003){ref-type="supplementary-material"}), with two researchers alternating waiting for the signals at the base camp and measuring nectar volume and concentrations of the flowers adjacent to the focal ones. {#ece33040-fig-0004} To study energetics, we needed to obtain an estimate of the nectar energy available to a hummingbird at the time of the visit. Therefore, at each camera trap, we bagged a flower next to the focal flower, and immediately after a bird visit, we emulated a "visit" to the bagged flower, and measured nectar volume and concentration. Our triggering system only started recordings at the beginning of a visit; therefore, recordings were stopped manually while researchers measured nectar. We reached each camera location within 1---2 min and did not record hummingbirds re‐visiting the focal flower during these intervals. As triggering systems sent a wireless signal to a base camp when activated, we were able to monitor all four traps simultaneously and immediately emulate flower visits. From the videos, we measured licking rate, bill insertion distance, handling time, amount of nectar collected, nectar properties, and aerodynamic parameters (e.g., wingbeats/s). These allowed us to obtain extraction efficiency (μl/s), energy content in the nectar consumed (cal/μl), and net energy gain (by subtracting the costs of hovering). 3. RESULTS {#ece33040-sec-0007} ========== We manually reviewed the pilot fieldwork videos from the backup cameras at 3× speed and compared the number of visits with those captured by our systems. There were 35 hummingbird visits to the focal flowers (e.g., Video [S2](#ece33040-sup-0002){ref-type="supplementary-material"}), and the cameras were triggered 60 times; of these 60, 34 were recordings of actual visits, and 26 were videos of the focal flower without a visiting hummingbird (false positives). One hummingbird visit did not trigger our systems (a false negative) (Table [1](#ece33040-tbl-0001){ref-type="table-wrap"}). This false negative occurred when a hummingbird arrived during the 45 s trigger delay immediately following a false positive triggered by wind. False positives occurred in only one location and only during the afternoons; studying the backup videos we conjectured that all were caused by strong wind moving vegetation. This location was particularly exposed and windy in the afternoons compared to other locations. Review of backup videos showed that, despite the camouflage, the hummingbirds inspected the cameras and sensors. Nevertheless, after an initial inspection (Video [S1](#ece33040-sup-0001){ref-type="supplementary-material"}), all hummingbirds visited the focal flower and did not inspect them the second and third days. Actuator motion and sound were minimal and occurred away from the flower, provoking no observable behavioral changes in the hummingbirds. Following pilot fieldwork (Figure [4](#ece33040-fig-0004){ref-type="fig"}), we performed a series of fixes that minimized the false positives through trial and error at the problematic windy location. We greatly reduced the false positives by repositioning the sensors (away from the piece of vegetation previously triggering them) and decreasing their sensitivity (to ignore background vegetation movement detection), minimizing triggering by vegetation moving the wind. In addition, we enhanced the quality of the data from the videos collected through the triggering systems by improving the zoom and framing (to capture both hummingbird hovering and feeding), and accounted for lighting changes throughout the day (avoiding dark recordings). We also discarded videos from the first day to minimize observer effects. ###### Performance of the systems. The number of correctly captured visits, false positives, and false negatives are shown for both our initial pilot fieldwork, and after adjusting locations and PIR sensitivity to minimize false positives. Percentages in parentheses are shown relative to total triggers or total visits, denoting rates of true positives, false positives, and false negatives Initial pilot After adjustment ---------- --------------- ------------------ ---- ----------- -------- ----- Visit 34 (57%) 1 (3%) 35 107 (87%) 0 (0%) 107 No visit 26 (43%) --- 26 16 (13%) --- 16 Total 60 1 61 123 0 123 John Wiley & Sons, Ltd In the extended fieldwork, we documented 107 floral visits by hummingbirds in high‐speed video: There were no false negatives, and we only recorded 16 false positives (Table [1](#ece33040-tbl-0001){ref-type="table-wrap"}). We collected data on visits to eight plant species by 11 species of hummingbirds (Table [S3](#ece33040-sup-0003){ref-type="supplementary-material"}). The lack of false negatives is a testament to the usefulness of multiple external PIR sensors for capturing hummingbirds, and our final false positive rate of \<15% is trivial in comparison to the benefits of our system. It took less than a minute to review one of the false‐positive videos (for a total of about 15 min in the extended fieldwork phase), but about 180 hr to visually review the videos from the continuously filming backup cameras at 3× speed for the same fieldwork phase. By using macro, backlit‐filming techniques (cf. Rico‐Guevara, [2014](#ece33040-bib-0024){ref-type="ref"}), we visualized and measured the amount of nectar inside flowers, and tracked the bill and tongue inside the corolla. Through this combination of automated macro, high‐speed, and backlit videography, we were able to observe what was previously unobservable--wild hummingbirds depleting nectar inside flowers. Our triggering system drew 15 mA of current, with a one‐second 350 mA pulse when triggered. We were able to run each trigger on one set of batteries (8xAA) for the entire study (\~100 hr). Battery life depends somewhat on the rate of triggering, but 140--160 hr is reasonable for 2,500 mAh AA batteries and greatly exceeds that of the cameras, even when using external battery packs. The external battery packs for the cameras were changed every 12--15 hr of monitoring and never fully drained. However, their run time was considerably less than that of the triggering mechanisms. Therefore, battery life of the entire system is generally dependent on the chosen camera, not the triggering mechanism. 4. DISCUSSION {#ece33040-sec-0008} ============= Filming animal behaviors in their natural environment, while minimizing observer disruption is costly and time‐consuming and camera traps help solve this problem. However, the flexibility of available camera traps is limited, and no options exist for filming high‐speed video of small‐bodied animals such as hummingbirds. We solved this logistical challenge by splitting the camera trap into two parts: the camera and the triggering mechanism. We designed a system that could mechanically trigger specialized cameras and receive input from multiple external sensors positioned separately from the camera, all while being cheap, portable, weatherproof, battery‐efficient, and easy to upgrade. We were able to independently pick the ideal camera and sensor configuration for our application, allowing more control in picking critical camera features such as video frame rate and prerecord mode, and more flexibility with designing sensor configurations to optimize sensitivity. Our system simply presses the camera\'s shutter button; therefore, cameras can be upgraded and recoupled. The system can also be adapted for cameras with remote triggering by closing the camera\'s remote trigger switch instead of operating an actuator. While our application is unique in using high‐speed video camera traps, the main novelty of our design is the decoupling of camera and triggering system, increasing camera trap flexibility. While we used high‐speed video cameras, the system could be adapted to other specialized cameras such as starlight or thermal cameras to study nocturnal animals, including cameras that researchers already own or those with future technological advances. While we used PIR sensors to detect movement, the design is not limited to PIR, and other sensors for light, color, or sound could be employed to trigger the camera instead of or in combination with PIR sensors. Alternatives to our approach include several technologies already used for studying small animals. Camera traps triggered by AIR sensors (Hernandez et al., [1997](#ece33040-bib-0013){ref-type="ref"}) and mini‐DVR video recorders or cameras with video motion detection that start recording when motion in the video is detected (e.g., auto‐record mode on JVC GC‐PX100 or surveillance software such as Scene Analyzer™, i‐PRO SmartHD™, iSpy) are appropriate (Bolton et al., [2007](#ece33040-bib-0007){ref-type="ref"}; Kross & Nelson, [2011](#ece33040-bib-0016){ref-type="ref"}). So are computer vision algorithms (e.g., Anandan, Bergen, Hanna, & Hingorani, [1993](#ece33040-bib-0002){ref-type="ref"}; Joshi & Thakore, [2012](#ece33040-bib-0014){ref-type="ref"}; Nordlund & Uhlin, [1996](#ece33040-bib-0019){ref-type="ref"}; Zeljkovic, [2013](#ece33040-bib-0041){ref-type="ref"}) that have recently been applied to biological studies to filter video for animal activity after recording (e.g., Dell et al., [2014](#ece33040-bib-0010){ref-type="ref"}; Weinstein, [2014](#ece33040-bib-0039){ref-type="ref"}). However, none of these systems currently support a wide array of cameras, including any with high‐speed video. Most cost in excess of \$500 and many use heavy 12 V batteries. We found the auto‐record mode of many video motion detection solutions was too slow to capture the start of a visit by hummingbirds. Computer vision algorithms require continuous prerecorded high‐speed video and acquiring this under field conditions faces significant drawbacks such as short camera battery life and storage problems (a 32 GB memory card lasts 2 hr). Therefore, our solution allows for cost‐effective camera trapping with more functionality than previously possible. One limitation of our system was the high maintenance level of the cameras we used. Cameras needed to be protected with rainproof covers, were turned off at night, and were not deployed for long periods of time. One advantage of commercial camera traps is that they are completely weatherproof and designed for deployments of weeks or months. These features are not useful when filming hummingbirds, due to high turnover rates of inflorescences. Nevertheless, if researchers require longer filming periods, we recommend weatherproof cameras (e.g., GoPro^®^), that can maintain standby mode for long durations. 5. CONCLUSION {#ece33040-sec-0009} ============= We are unaware of a recent camera trap application for ecological research in which the triggering system was separated from the camera itself. This approach leads fewer design compromises and has the potential to minimize the limitations of many extant camera traps (Meek & Pittet, [2012](#ece33040-bib-0018){ref-type="ref"}; Rovero et al., [2013](#ece33040-bib-0028){ref-type="ref"}). In our application, we were able to use a specialized camera with much improved video features from standard camera traps, situate the camera separately from the triggering sensors, modify the sensors, and add wireless capabilities; all at a much lower cost. Our system used simple integrated circuits, although the Arduino^®^ platform could provide future innovations in camera trapping above and beyond those of our system because of its immense flexibility. Arduinos (and similar microcontrollers) have the capability to set video recording duration, trigger flashes, and weigh the input of multiple sensors in complex ways. They can also collect and store ancillary data with sensors for ambient temperature, humidity, and many other aspects of the environment, while being cheap and power efficient. CONFLICT OF INTEREST {#ece33040-sec-0012} ==================== None declared. AUTHOR CONTRIBUTIONS {#ece33040-sec-0013} ==================== AG conceived the camera trap idea. JM built the traps and the control box. AG and JM performed initial tests, and AG conducted field tests in Colombia and analyzed videos. AG and JM co‐wrote the manuscript. Both authors contributed to all drafts and gave final approval for publication. Supporting information ====================== ######   ###### Click here for additional data file. ######   ###### Click here for additional data file. ######   ###### Click here for additional data file. ######   ###### Click here for additional data file. We are deeply indebted to Kristiina Hurme, Margaret Rubega, the UConn Electrical Engineering Department and Austin Warner, Daniel Lees, Jacob Moulton, and Rajeev Bansal for assisting in the early stages of this project. We thank Jesse Joy, Briana Lechkun, Sebastian Aragon, Catalina Peña, and Miguel Ángel Muñoz for field assistance, and Kelsey O\'Connor and Jessica Attalah for help organizing data and measuring videos. Carl Schlichting, Timothy Moore, and two anonymous reviewers kindly read the manuscript and provided valuable comments. We thank Diego Sustaita, Jessica Lodwick, Kevin Burgio, Holly Brown, and the UConn Ornithological Research Group for feedback. A. R‐G. thanks the Miller Institute for funding. This work was supported by the UConn EEB Department, and NSF IOS‐DDIG 1311443. The authors declare no conflict of interest. [^1]: The authors contributed equally to this paper.

**Citation:** Iliyasu Z, Galadanci HS, Oladimeji AI, Babashani M, Gajida AU, Aliyu MH. Predictors of safer conception practices among HIV-infected women in Northern Nigeria. *Int J Health Policy Manag*. 2019;8(8):480--487. doi:10.15171/ijhpm.2019.27 Introduction {#s1} ============ The HIV/AIDS pandemic has challenged human development, especially in high burden low-resource settings.^[@R1]-[@R4]^ In 2016, women constituted nearly half (17.8 million) of the world's 36.7 million people living with HIV.^[@R5]^ Of the 1.8 million new HIV acquisitions that year, 160 000 were in children. In Nigeria, an estimated 37 000 infants acquired HIV infection vertically in the same year, one of the highest globally.^[@R5]^ In sub-Saharan Africa, the virus is predominantly transmitted heterosexually. Childbearing among HIV-infected couples carries substantial risk of HIV transmission not only to the uninfected partner, but to the fetus as well. In addition, even among seroconcordant couples, there is the risk of acquiring a different strain through secondary transmission. Therefore, in the early phase of the epidemic, HIV-infected couples were not only discouraged from procreation, but offered termination of pregnancy to avert vertical transmission.^[@R6]^ However, these all changed with the discovery of reduced transmission risk through timed unprotected sexual intercourse, antiretroviral therapy (ART) pre-exposure prophylaxis (PrEP), artificial insemination and male medical circumcision.^[@R7]^ Recent additions include: sperm washing, intrauterine insemination, in-vitro fertilization and intra-cytoplasmic sperm injection.^[@R8]^ However, these methods are not 100% effective nor are they within reach of most affected couples in low-resource settings. Paradoxically, this is where fertility demand is high.^[@R9]^ It is therefore, likely that HIV-infected couples in these settings are conceiving naturally, despite well-documented risks.^[@R10]^ With increasing access to antiretrovirals and prevention of mother to child transmission services, the proportion of HIV-infected women in seroconcordant and serodiscordant partnerships who desire to have children has increased from about 26% to 69% in different regions.^[@R11]^ Recent studies of pregnant women on highly active antiretroviral treatment suggest that pregnancy does not speed up the progression of HIV disease.^[@R12],[@R13]^ Despite these promising data, women living with HIV still face significant social and health system challenges when contemplating parenthood, including stigma and resistance from some healthcare providers and the general public.^[@R14]^ The current reach of affordable, timely and stigma-free safer conception methods is restricted. Although higher rates of use of safer conception methods have been reported in recent demonstration projects among HIV-positive women,^[@R15]^ the proportion of women practicing safer conception ranges from almost none in South East Asia to 7% in parts of sub-Saharan Africa.^[@R16]^ With the expected increase in patient enrolment on ART following the adoption of "test and treat" policy across the globe, even less time is likely to be available for discussions about reproductive plans and safer conception, particularly in low-resource settings.^[@R17],[@R18]^ Very little is known about risk perception, fertility desires and intentions, safer conception practices and their predictors among HIV-infected women in northern Nigeria, the region with the highest total fertility rate in the country.^[@R19]^ The objective of this study is to assess the fertility desires and intentions, risk perceptions and predictors of ever use of at least one safer conception method among HIV-infected women attending a teaching hospital in northern Nigeria. Methods {#s2} ======= Study Setting and Population {#s2-0-1} ---------------------------- The study was conducted at the S. S. Wali ART Centre, Aminu Kano Teaching Hospital (AKTH), Kano, Nigeria. AKTH is a 550 bed government-owned tertiary health institution serving a catchment population of over 13 million.^[@R20]^ The inhabitants are mainly Hausa-Fulani. The ART clinic operates daily, and does not charge for clinical examinations, laboratory tests and antiretroviral drugs. Patient support groups, counseling, testing and home-based services are also available. HIV infection is diagnosed using a sequential HIV rapid screening algorithm based on the national guidelines.^[@R21]^ All patients undergo pre-and post-test counseling.^[@R22]^ Included in the study were consenting women age 18-49 years, diagnosed with HIV (≥6 months earlier) attending AKTH. Persons who withheld consent or were too sick to be interviewed were excluded. Study Design and Sampling {#s2-0-2} ------------------------- We employed a cross-sectional study design. Sample size *n* was obtained using Fisher formula \[n=z^2^p(1-p)/d^2^=, where z = standard normal variate (1.96), p = proportion of women aware of (≥1) safer conception method from a previous study (53%)^[@R23]^ and d = tolerable error of 5%\]. The sample size obtained (n = 384) was then increased by 10% to account for anticipated non-response (384/0.90 = 427), giving a final sample size of 427. Systematic sampling was used to recruit participants as they arrived at the ART center. Using average clinic attendance and estimated sample size, a sampling interval was computed. On arrival, the client was registered, given a sequential serial number, and allocated a consulting room. While waiting, clients were informed about the research. The first respondent was determined by picking a random number between 1 and the sampling interval. Subsequent respondents were identified by adding the sampling interval to the preceding respondent's serial number. Women whose serial number tallied with the sampling process were individually invited into a separate room after the clinic consultation and informed consent obtained. Participant names were not recorded on the questionnaire and all other identifying information were kept confidential. Non-participation had no effect on subsequent care. Referral for professional counselling was available, if required. Only participants who provided informed consent were interviewed in private. Study Instrument and Data Collection {#s2-0-3} ------------------------------------ A pre-tested structured interviewer-administered questionnaire with five sections was adapted from a previous study.^[@R24]^ This previous tool provided the concepts, but the questions used in the present study were adapted for our population and re-validated. The first section inquired about socio-demographic characteristics. The second section elicited detailed obstetric history, HIV diagnosis, partner serostatus, disclosure and treatment history. Section 3 assessed transmission risk perception, fertility desires and intentions, while section four focused on awareness and practice of safer conception methods. Fertility desire was assessed using the question "Do you wish to have any/more children?" with 3 options, 'Yes,' 'No,' and 'Undecided,' whereas fertility intention was elicited using the question "Do you plan to have another baby within the next 3 years?" with 'Yes' and 'No' options. Awareness of safer conception methods was determined with the question "Are you aware of any safer conception method for HIV-positive couples?" This question was followed by "If yes, describe the method (s) you are aware of." The interviewer was instructed not to read out the options, but to compare the description by the respondent with a list of options provided, and select all responses mentioned. Risk perception was assessed based on the response to questions on risk of infecting a partner during conception and the chances of serodiscordant and seroconcordant couples bearing an uninfected child. Other questions included risk of HIV transmission during pregnancy, childbirth and breastfeeding and whether or not HIV transmission via these routes can be prevented. Theoretical Framework and Measurements {#s2-0-4} -------------------------------------- Based on Miller's Traits-Desires-Intentions-Behavior framework for understanding childbearing motivations,^[@R25]^ fertility desire was assessed by asking "Do you wish to have any (more) children?" with dichotomous response options "Yes" coded as 1 or "No" coded as 0. If the respondent answered "Yes," they were asked the total number of children they desire to have. The response was recorded as a discrete number. Further, to assess fertility intention, respondents were asked, "Are you planning to have any (more) children in the next 3 years?" ("Yes" coded as 1 or "No" coded as 0). Practice of safer conception was classified as 'Yes' coded as 1, if the respondent ever practiced at least one method of safer conception, specifically one, a combination or all of the following: used antiretroviral drugs while trying to conceive, timed unprotected intercourse, timed intercourse with PrEP for HIV-negative partner, sperm washing with intrauterine insemination and artificial intra-vaginal insemination. Data Analysis {#s2-0-5} ------------- Data were analyzed using SPSS version 22.^[@R26]^ Continuous data were summarized using mean ± SD or median and range. Categorical data were presented as frequencies and percentages. At bivariate level, Pearson's chi-square or Fisher's exact test was used to assess significance of associations at *P *\<* *.05. Risk perception was dichotomized into high and low/no risk perception based on the responses as follows: (1) Respondents who indicated there is no risk of infecting a partner during conception and no transmission risk in utero, at delivery and through breastfeeding were considered to perceive no risk; (2) Participants who were of the view that transmission was possible at least during one of these times, but was not preventable, were characterized as having low risk perception; (3) Respondents who felt that a transmission risk exists at all of these times, but that transmission is preventable, were considered as having high risk perception. A logistic regression model was developed with variables that had *P *\<* *.10 at bivariate level or those that were considered conceptually important, irrespective of their significance at that level (age group, marital status, occupation, number of children, risk perception, fertility desire and couple contraceptive use, partner's HIV status). The dependent variable was 'ever practiced at least one safer conception method,' classified as 'Yes' (coded as 1), if the respondent has ever practiced any, a combination or all of the following safer conception methods: used ART drugs while trying to conceive, timed intercourse with PrEP for HIV-negative partner, and artificial intra-vaginal insemination. Adjusted odds ratios (AORs) and associated 95% CI were used to compute effect estimates. A secondary analysis of the same outcome was conducted only on women who desired children. In the second model, all the previous independent variables were included in the model, except fertility desire. Results {#s3} ======= Sociodemographic Characteristics {#s3-0-1} -------------------------------- Of 427 eligible women, 328 completed the interviews, a response rate of 76.8%. Non-response was mainly related to earlier commitments. Respondents' mean age (±SD) was 33.0 (±7.5) years. Over half of respondents were Hausa/Fulani (52.7%) and Muslims (60.7%). More than a quarter of respondents (29.0%) were teachers. Over two-thirds (71.7%) of respondents had at least secondary education, but nearly a fifth (19.2%) had no for­mal education ([Table 1](#T1){ref-type="table"}). ###### Socio-Demographic and Obstetric Characteristics of HIV-Infected Women, Kano, Nigeria (N = 328) --------------------- ------------- **Characteristics** **No. (%)** Age group \<30 104 (31.7) 30-39 139 (42.4) ≥40 85 (25.9) Ethnicity Hausa/Fulani 173 (52.7) Yoruba 53 (16.2) Igbo 43 (13.1) Others 59 (18.0) Religion Islam 199 (60.7) Christianity 129 (39.3) Education No formal 63 (19.2) Primary 30 (9.1) Secondary 81 (24.7) Post-secondary 154 (47.0) Marital status Single 69 (21.0) Married 195 (59.5) Divorced 27 (8.2) Widowed 37 (11.3) Occupation Homemaker 47 (14.3) Teaching 95 (29.0) Trading 81 (24.7) Civil servant 66 (20.1) Others 39 (11.9) Parity 0 72 (22.0) 1 50 (15.2) 2-4 143 (43.6) ≥5 63 (19.2) --------------------- ------------- Majority of the respondents (59.5%) were married. The median parity of respondents was 2.0 (range, 0 to 10). Of the 259 ever married respondents, 167 (50.9%) had disclosed to their partners. Almost half (n = 122, 47.1%) of the ever married women had seroconcordant partners. The rest were either serodiscordant (n = 62, 23.9%) or partner status was unknown (n = 75, 29.0%). Risk Perception and Fertility Desires and Intentions {#s3-0-2} ---------------------------------------------------- [Table 2](#T2){ref-type="table"} presents risk perception, fertility desires and intentions. Over two-thirds of the women (68.9%) were aware of transmission risk during pregnancy (69.5%), delivery (75.3%) and breastfeeding (78.9%) in the absence of interventions. Over two-thirds (68.9%) of the participants were aware of the possibility of bearing HIV-free children without infecting their partners. Overall, (n = 122, 37.2%) respondents perceived the risk of HIV transmission to partner and foetus/infant as low or non-existent while (n = 206, 62.8%) perceived it as high. ###### Risk Perception and Fertility Intention Among HIV-Infected Women, Kano, Nigeria, 2016 (N = 328 unless otherwise stated) ------------------------------------------------------------------------------- ------------- ------------- ------------------ **Yes**\ **No**\ **Do not Know**\ **No. (%)** **No. (%)** **No. (%)** **Risk Perception** Possibility of serodiscordant couple conceiving without infecting the partner 226 (68.9) 33 (10.1) 69 (21.0) Likelihood of serodiscordant couple bearing HIV-negative baby 243 (74.1) 21 (6.4) 64 (19.5) Possibility of seroconcordant couple bearing HIV-negative baby 225 (68.6) 43 (13.1) 60 (18.3) Risk of HIV transmission in utero 228 (69.5) 50 (15.2) 50 (15.2) Risk of HIV transmission during delivery 247 (75.3) 31 (9.5) 50 (15.2) Risk of HIV transmission through breastfeeding 262 (78.9) 24 (7.3) 42 (12.8) Possible to prevent mother to child transmission of HIV? 206 (62.8) 122 (37.2) \- **Fertility Desires and Intentions** Wants to have more children 150 (45.7) 147 (44.8) 31 (9.5) Total number of (biological) children desired ‏- \- \- None 22 (6.7) \- \- 1 16 (4.9) \- \- 2-4 143 (43.6) \- \- ≥5 147 (44.8) \- \- Intents to have a child/another child in the next 3 years (n = 150) 134 (89.3) 16 (10.7) \- Discussed fertility plans with healthcare provider (n = 134) 79 (59.0) 55 (41.0) \- Healthcare provider supportive of fertility plans (n = 79) 75 (94.9) 4 (5.1) \- Discussed fertility plans with husband/partner (n = 150) 118 (78.7) 32 (21.3) \- Husband/partner supportive of fertility plans (n = 118) 108 (91.5) 10 (8.5) \- Current contraceptive use 67 (20.4) 261 (79.6) \- Partner\'s current contraceptive use 91 (27.7) 104 (31.7) 133 (40.6) ------------------------------------------------------------------------------- ------------- ------------- ------------------ More than three-fourths of respondents who desire children had discussed their plans with their partners (78.7%). Most partners (91.5%) and healthcare workers (94.9%) were supportive. At the time of the study, 1 in 5 respondents (n = 67, 20.4%) and more than 1 in 4 partners (n = 91, 27.7%) were using contraceptives. In over half (n = 110, 56.4%) of the 195 currently married respondents, at least one partner was using a modern contraceptive method. Awareness and Practice of Safer Conception Methods {#s3-0-3} -------------------------------------------------- [Table 3](#T3){ref-type="table"} presents awareness and ever-practice of safer conception methods. Almost two-thirds of women (n = 213, 64.9%) were aware of at least one method of safer conception. The most common methods of safer conception listed included ART (62.8%), timed unprotected intercourse (29.3%) and timed intercourse with PrEP for HIV-negative partner (23.2%). Sperm washing and artificial insemination were mentioned by less than 1 in 10 respondents. The proportions that ever practiced the listed methods of safer conception were 23.2%, 15.9%, 9.8%, 4.3%, and 6.7%, respectively. ###### Awareness and Ever Practice of Safer Conception Methods Among HIV-Infected Women, Kano, Nigeria, 2016 ---------------------------------------------------------------------------------- ------------- ------------- **Yes**\ **No**\ **No. (%)** **No. (%)** Awareness of safer conception methods (n = 328) Aware of at least one safer conception method for HIV-positive couples (n = 328) 213 (64.9) 115 (35.1) Use of ART drugs 206 (62.8) 122 (37.2) Timed unprotected intercourse 96 (29.3) 232 (70.7) Timed intercourse with PrEP for HIV-negative partner 76 (23.2) 252 (76.8) Sperm washing with intra uterine insemination 40 (12.2) 288 (87.8) Artificial intra-vaginal insemination 34 (10.4) 294 (89.6) Safer conception methods ever used (n = 328) ART drugs 76 (23.2) 252 (76.8) Timed unprotected intercourse 52 (15.9) 276 (84.1) Timed intercourse with PrEP 32 (9.8) 296 (90.2) Sperm washing with intrauterine insemination 14 (4.3) 314 (95.7) Artificial intra-vaginal insemination 22 (6.7) 306 (93.3) ---------------------------------------------------------------------------------- ------------- ------------- Abbreviations: ART, antiretroviral therapy; PrEP, pre-exposure prophylaxis. Predictors of Safer Conception Practice {#s3-0-4} --------------------------------------- [Table 4](#T4){ref-type="table"} presents the factors associated with practice of safer conception. At bivariate level, safer conception practice was associated with respondent's age, ethnicity, religion, education, occupation, number of children, couple contraceptive use and risk perception (*P* \< .05). At multivariate level, including all respondents, marital status, occupation, number of children, couple contraceptive use, husband/partner serostatus and risk perception remained statistically significant predictors of safer conception practice. Nearly half of the HIV-infected women (n = 150, 45.7%) interviewed wanted more children. The median number of children desired was 4 (range, 1 to 12). Most respondents (n = 134, 89.3%) intend to have a child within 3 years. ###### Logistic Regression Model for Predictors of Safer Conception Practice Among HIV-Infected Women, Kano, Nigeria, 2016 (N = 328) ------------------------------ ------------------------------------- -------------------------------------- ------------------------------- ------------------- ------------------- **Characteristics** **Ever Practiced Safer Conception** **Never Practiced Safer Conception** **Crude Odds Ratio (95% CI)** **AOR (95% CI)** ***P*** **Value** Age group \<30 36 (34.6) 68 (65.4) Ref 30-39 50 (36.0) 89 (64.0) 1.08 (0.73-1.92) 0.64 (0.41-1.41) .38 ≥40 44 (51.8) 41 (48.2) 2.08 (1.25-3.72) 1.39 (0.71-2.86) .43 Marital status Single 14 (20.3) 55 (79.7) Ref Married 93 (47.7) 102 (52.3) 3.46 (1.91-6.92) 1.50 (1.10-3.55) .014^b^ Divorced 17 (63.0) 10 (37.0) 6.71 (2.64-17.81) 1.73 (0.48-5.34) .66 Widowed 6 (16.2) 31 (83.8) 0.84 (0.33-2.31) 0.21 (0.07-0.78) .031^b^ Occupation Trading 41 (50.6) 40 (49.4) Ref Teacher 30 (31.6) 65 (68.4) 0.45 (0.24-0.83) 0.33 (0.15-0.72) .034^b^ Civil servant 33 (50.0) 33 (50.0) 0.98 (0.51-0.77) 0.37 (0.16-0.86) .027^b^ Others 26 (30.2) 60 (69.8) 0.42 (0.22-0.79) 0.44 (0.19-1.01) .12 No. of children 0 5 (6.9) 67 (93.1) Ref 1 29 (58.0) 21 (42.0) 18.50 (6.36-53.90) 26.2 (7.80-87.80) .011^b^ 2-4 68 (47.6) 75 (52.5) 14.10 (4.62-31.90) 12.1 (3.70-39.80) .037^b^ ≥5 28 (44.4) 35 (55.6) 10.70 (3.81-30.20) 5.60 (1.44-21.90) .023^b^ Husband\'s serostatus^a^ Serodiscordant 24 (38.7) 38 (61.3) Ref Seroconcordant 71 (58.2) 51 (41.8) 2.24 (1.24-7.46) 1.51 (1.13-4.64) .012^b^ Serostatus unknown 21 (28.0) 54 (72) 0.72 (0.17-0.93) 0.53 (0.22-0.83) .024^b^ Couple contraceptive use^a^ Yes 63 (61.2) 40 (38.8) 1.80 (1.47-7.59) 1.62 (1.16-5.83) .032^b^ No 53 (34.0) 103 (66.0) Ref Fertility desire No 60 (40.8) 87 (59.2) Ref Yes 62 (41.3) 88 (58.7) 1.03 (0.78-3.64) 0.68 (0.12-2.65) .67 Undecided 8 (25.8) 23 (74.2) 2.8 (0.53-7.45) 1.42 (0.25-5.63) .53 Transmission risk perception Low/no risk 36 (29.5) 86 (70.5) Ref High risk 94 (45.6) 112 (54.4) 2.61 (1.14-5.76) 2.14 (1.18-3.90) .014^b^ ------------------------------ ------------------------------------- -------------------------------------- ------------------------------- ------------------- ------------------- Abbreviations: AOR, adjusted odds ratio; Ref, reference group. ^a^Only for ever married respondents. ^b^Significant at *P* \< .05. Specifically, after adjusting for other variables, married respondents had 50% increased chance of practicing safer conception relative to single women. Similarly, the likelihood of use of safer conception were 67% and 63% lower among teachers and civil servants, respectively, compared with traders. Further, women with one child had a much higher chance of using safer conception relative to nulliparous women. Seroconcordant couples were 51% more likely to have practiced safer conception relative to serodiscordant couples. Similarly, women whose partner was of unknown serostatus were nearly half (47%) as likely to have practiced safer conception compared to serodiscordant couples. Couples using modern contraception were 62% more likely to practice safer conception compared to those who did not. Finally, respondents who perceived the risk of HIV transmission to infants and partners as high during conception had more than two-fold odds of practicing safer conception compared to those who felt the risk was low or non-existent. Parity, couple contraceptive use, husband's serostatus and transmission risk perception remained significant predictors of safer conception practice on secondary analysis limited to couples that wanted to have babies (n = 150, [Table 4](#T4){ref-type="table"}). Discussion {#s4} ========== Two-thirds of the HIV-infected women in this study were aware of the prospects of conceiving without infecting their partner or fetus. A similar proportion knew at least one method of safer conception. The most popular methods were ART, timed unprotected intercourse and a combination of the 2 methods. Nearly half of the respondents wanted more children within 3 years. Less than a quarter used contraceptives and an even lower proportion practiced safer conception. The practice of safer conception was associated with marital status, maternal occupation, the number of living children, husband's serostatus, contraceptive use, and transmission risk perception. The proportion of HIV-infected women who wanted more children was lower than the figure (75.8%) reported from Birnin Kudu in northern Nigeria.^[@R27]^ However, it was similar to the findings among HIV-infected women in Ethiopia (45.5%),^[@R28]^ but higher than among their counterparts in Uganda (35%).^[@R29]^ A multi-country study in 10 sub-Saharan African countries reported that fertility preferences and contraceptive behaviors of HIV-positive women were relatively similar across countries, where HIV-positive women were less likely to want more children and their partners more likely to use male condoms than HIV-negative women.^[@R32]^ Our figure was also lower than the proportions of HIV-infected women who were desirous of more children in the United States (50%) and the United Kingdom (75%).^[@R33],[@R34]^ These differences could be related to variations in study population demographics, methods and currency of the studies, and cultural preferences. The proportion of respondents aware of timed unprotected intercourse and artificial insemination as methods of safer conception were lower than that reported among their contemporaries in Uganda (51% and 53%, respectively).^[@R29]^ Further, the proportion of women aware of sperm washing was low in both studies (12.2% versus 15.0%), respectively.^[@R35]^ A qualitative study from Uganda also reported that while several clients had heard about safer conception methods, especially timed unprotected intercourse, only a few fully understood it. It was not surprising, therefore that, the couples felt that they were taking a gamble.^[@R36]^ Similarly, in Kenya, HIV-positive women mentioned safer conception methods including sperm washing, but could not provide details.^[@R37]^ The proportion of women who reported using the various methods of safer conception in the present study was higher than the baseline rates reported among their counterparts in Uganda, where 12% and 2% used timed unprotected intercourse and PrEP, respectively, while none had used manual self-insemination and sperm washing. Even after 24 months follow up, only 9% reported the use of PrEP, 3 male partners used manual self-insemination, while none used sperm washing.^[@R29]^ The situation was also similar among HIV-positive women in a study in South Africa.^[@R30]^ However, another South African study reported higher uptake of safer conception methods.^[@R31]^ This disparity could reflect the availability and familiarity with these methods among patients and healthcare providers. Considering the high premium accorded childbearing in sub-Saharan Africa, it is important to make low-technology, affordable safer conception methods accessible to couples that desire to procreate, satisfying the desire to bear children while reducing to a minimum the probability of mother-to-child transmission of HIV.^[@R17]^ The contraceptive prevalence among HIV-infected women was higher than that reported from Birnin Kudu, northern Nigeria (10%),^[@R27]^ and among the general populace in Nigeria (6.3%).^[@R38]^ Although, there was near universal support for the reproductive plans of HIV-infected women among partners and healthcare providers in our study, this was not the case elsewhere. For instance, more than 40% of women in Kenya reported being advised, primarily by healthcare providers and family members, to abstain from sex and terminate the pregnancy.^[@R39]^ Like others, women living with HIV are entitled to information and services that would guarantee safer sexual and reproductive choices.^[@R40]^ There is a need to integrate counselling on contraception, safer sex and reproduction for HIV positive couples into ART programs. Our finding of an association between marital status, occupation and number of living children with safer conception practice is similar to reports from Uganda. In the Uganda study, however, additional factors such as perceived willingness to use safer conception methods, knowledge of respondent's HIV status, HIV-seropositivity and equitable decision-making powers in relationships correlated with participants' awareness and attitude towards safer conception methods.^[@R35]^ The role of marital status is not surprising, as pre-marital childbearing is abhorred in this setting. Therefore, it is unlikely that single HIV-infected women would have attempted to conceive, a likely reason for their limited knowledge of such methods. This group needs to be targeted with information before commencing procreation. Similarly, occupation, being a proxy for educational status is likely to influence health literacy and act as a cue for adopting safer practices. Women in skilled professions are likely to be better-educated and more likely to seek information and practice safer conception.^[@R41]^ However, the apparent paradox where use of safer conception was similar between civil servants and traders raises the possible role of affordability, as some safer conception methods could be expensive. As residents of the commercial center of northern Nigeria, traders in Kano tend to be wealthier than persons in other occupations. All HIV-positive women should nevertheless have access to such services to avoid transmission. Finally, children of HIV-positive women are more likely to survive if the mother adopted safer conception methods, hence the correlation between number of living children and ever practice of safer conception in this study. Logistic model includes the following variables: age group, marital status, occupation, number of children, husband's serostatus, couple contraceptive use, fertility desire and transmission risk perception. It is not surprising that couple contraceptive use and risk perception were significantly associated with safer conception practice in our study. Both contraception use and the ability to perceive high transmission risk are indicative of a degree of level of knowledge of health-related issues. Couples who use contraception are likely to have had encounters with the health system and thereby become exposed to health education messages, which might include information on safer conception. The predictive role of husband's serostatus on safer conception practice is also not unique. For instance, a qualitative study in Uganda demonstrated the importance of partner serostatus and couple communication dynamics on the uptake of safer conception methods.^[@R42]^ This finding highlights the influence of the male partner's serostatus on the appropriateness of safer conception options and the dominant role of men in family decisions, especially in patriarchal settings. Our study findings underscore the importance of counselling HIV-affected couples on mutual disclosure, harmonization of fertility intentions and the adoption of safer conception methods that enable the achievement of couples' reproductive goals at minimal risk to both parents and their offspring. Our study has limitations. First, the study setting and participants may not be representative of the general population of women in northern Nigeria. Our respondents were a group of better-educated women, accessing ART services at a tertiary referral centre. They are likely to be more informed about safer conception, and to have higher contraceptive prevalence and lower fertility desires and intentions compared to their rural counterparts. Safer conception and family planning counselling and services are likely to be provided at such centres. Second, although individual interviews were conducted in private by trained interviewers from the same culture, social desirability bias cannot be ruled out. Responses could also differ in community-based surveys. In conclusion, our findings highlight the mismatch between fertility desires/intentions, risk perception and safer conception practice in a low-resource setting. These findings are inimical to the goal of eliminating mother-to-child HIV transmission worldwide. Rather than being prescriptive, healthcare providers should pro-actively discuss fertility desires and intentions, the associated risks and safer conception options with their clients. This is often not the case in the study area and in South Africa where such conversations occur when client-initiated.^[@R43]^ It has been suggested that before any safer conception intervention, it is important, to the extent feasible, to ensure that the clients' clinical state is favourable to a lower likelihood of transmission, specifically low viral load, high CD4+ cell count, and no AIDS-defining symptoms. Both partners should also be free of other sexually transmitted infections or should be receiving treatment, and should preferably be in a stable relationship.^[@R44]^ Where possible, fertility screening is also recommended, including semen analysis for HIV-infected men and the spinnbarkeit test for the woman to detect ovulation. Interventions should also be sensitive to the vulnerability of HIV-infected women, their unique psychosocial disposition and the considerable pressure they may face from male partners to get pregnant, even if they do not wish to.^[@R17]^ The prevention of unintended pregnancy is itself a major service component of prevention of mother-to-child transmission programs. Efforts should therefore be made to incorporate safer conception education in family planning programs, especially among women with HIV infection. Ethical issues {#s5} ============== Ethical approval for this study was obtained from the AKTH Ethics Committee, approval number NHREC/21/08/2008/AKTH/EC/1576. Competing interests {#s6} =================== Authors declare that they have no competing interests. Authors' contributions {#s7} ====================== ZI, HSG, and AIO conceived and designed the study. AIO, AUG, and MB collected the data while ZI, MHA, and HSG analysed the data and prepared the draft manuscript. All authors reviewed and approved of the final manuscript. Authors' affiliations {#s8} ===================== ^1^Department of Community Medicine, Bayero University, Kano, Nigeria. ^2^Centre for Infectious Diseases Research, Bayero University, Kano, Nigeria. ^3^Department of Obstetrics and Gynecology, Bayero University, Kano, Nigeria. ^4^Department of Medicine, Bayero University, Kano, Nigeria. ^5^Department of Health Policy & Vanderbilt Institute for Global Health, Vanderbilt University Medical Center, Nashville, TN, USA. Key messages {#s9} ============ Implications for policy makers {#key1} ------------------------------ - Healthcare providers should pro-actively discuss fertility desires and intentions, and associated risks and safer conception options with their clients. - Partner HIV testing and disclosure should be encouraged. - Policies should prioritize building healthcare provider capacity to enhance safer conception counseling and service delivery. - Couple contraceptive counseling and services should be provided as a component of programs for prevention of mother-to-child HIV transmission. Implications for public {#key2} ----------------------- This paper provides recommendations for policy-makers and other stakeholders that would address the findings of high fertility desires and intentions, moderate risk perception and low uptake of safer conception practices among HIV-infected women in Kano, northern Nigeria. Well-designed and sustainable approaches to facilitating safer conception choices among such women will enhance the elimination of mother-to-child HIV transmission in Nigeria, the country with the highest burden of vertical HIV transmission in the world.

Under conditions of excessive caloric intake, adipose tissue (AT) undergoes structural and functional rearrangement characterized by hypertrophy and hyperplasia of adipocytes; insufficient neovascularization; aberrant fibrogenesis; and migration and activation of macrophages, natural killers, and lymphocytes \[[@B1], [@B2]\]. Thus, the overproduction of proinflammatory adipokines, such as tumor necrosis factor (TNF)-*α*, interleukin (IL)-1*β*, IL-6, and IL-8 \[[@B3]\], along with reduced AT capability of storing free fatty acids and the resultant aberrant efflux of free fatty acids into the circulation, lead to insulin resistance and its related diseases \[[@B1], [@B4][@B5]--[@B6]\]. Furthermore, we recently demonstrated that AT dysfunction is a determinant of worse metabolic profile and higher cardiovascular risk in patients with type 2 diabetes \[[@B7]\]. Wnt1-inducible signaling pathway protein 1 (WISP1, or Cyr61/CTGF/NOV) is an extracellular matrix--associated protein belonging to the Cyr61/CTGF/NOV family, which includes matricellular proteins operating at the border between cells and extracellular matrix and exerts regulatory actions on several cellular responses. Thus, WISP1 is involved in a broad spectrum of biological functions and pathological processes \[[@B8][@B9][@B10][@B11][@B12][@B13][@B14][@B15]--[@B16]\]; is mainly expressed during organ development and under diseased conditions, such as fibrosis \[[@B11][@B12][@B13][@B14]--[@B15]\], cancer \[[@B9], [@B10], [@B13]\] , and inflammatory diseases \[[@B16]\]; and has recently been proposed as a novel adipokine \[[@B17]\]. WISP1 is widely expressed in visceral (VAT) and subcutaneous (SAT) human AT, is released by fully differentiated human adipocytes, and stimulates cytokine responses in AT-associated macrophages \[[@B17]\]. Circulating WISP1 concentration correlates with its expression in AT, which therefore represents a major source of this adipokine in humans \[[@B17]\]. Among the many cell types on which it exerts proliferative effects, WISP1 has induced proliferation of mesenchymal stem cells and, thus, AT expansion, in experimental models of visceral obesity \[[@B18]\]. Although an association between WISP1 expression in AT inflammation and insulin resistance has been described in persons with normal glucose tolerance \[[@B17]\], no evidence on its role in AT inflammation in conditions of impaired glucose regulation has been produced so far. Increased circulating WISP1 levels were recently reported in a study among women with gestational diabetes; this study identified greater body mass index (BMI) and insulin resistance during pregnancy as determinants of increased WISP1 \[[@B19]\]; however, as far as we know, no study has evaluated circulating WISP1 levels in patients with type 2 diabetes. Therefore, the aim of this study was to investigate the role of plasma WISP1 levels in identifying patterns of AT inflammation in patients with type 2 diabetes. 1. Methods {#s6} ========== A. Population {#s7} ------------- For this study, we recruited 97 consecutive patients referred to our diabetes outpatient clinics for metabolic evaluations. Of these, 71 had a diagnosis of type 2 diabetes, with or without obesity \[n = 35, 26 men and nine women, mean age ± standard deviation (SD), 51.9 ± 9.2 years; n = 36, 21 men and 15 women, mean age ± SD, 50.4 ± 9.7 years, respectively\], and 26 were obese without diabetes (nine men and 17 women, mean age ± SD, 42.4 ± 9.8 years). Furthermore, a population of 21 normal-weight nondiabetic individuals was recruited as a control group (14 men and seven women, mean age ± SD, 52.3 ± 12.4 years). To be eligible, participants had to fulfil the following inclusion criteria: men or women aged 25--70 years; white ethnicity; no history of excessive alcohol intake (average daily consumption of alcohol \>30 g/d in men and \>20 g/d in women); and absence of hepatitis B surface antigen and antibodies to hepatitis C virus, cirrhosis or other chronic liver diseases, malignancies, autoimmune diseases, and treatment with corticosteroids and/or other agents affecting the immune system. The study protocol was reviewed and approved by the Ethics Committee of this hospital, and the study was conducted in conformance with the Helsinki Declaration. All patients provided written consent before the study. B. Laboratory Determinations {#s8} ---------------------------- The study population underwent clinical workup, including medical history collection; physical examination with measurement of BMI (kg/m^2^), waist circumference (cm), and systemic blood pressure \[systolic and diastolic (mmHg)\]; and fasting blood sampling to assess total cholesterol (mg/dL), high-density lipoprotein cholesterol (mg/dL), triglycerides (mg/dL), aspartate aminotransferase (IU/L), alanine aminotransferase (IU/L), blood urea nitrogen (mg/dL), creatinine (mg/dL), and C-reactive protein (CRP; mg/dL) by standard laboratory methods. The glyco-metabolic profile was evaluated by measuring fasting blood glucose (FBG; mg/dL), insulin (IU/L; PANTEC srl, Italy) and glycosylated hemoglobin (% - mmol/L). The homeostasis model assessments of insulin resistance and insulin secretion were calculated as described elsewhere \[[@B20]\]. Diabetes mellitus was diagnosed according to American Diabetes Association 2009 criteria \[[@B21]\]. Among circulating markers of AT inflammation, we measured the concentrations of IL-8, IL-6, and TNF-*α* (pg/mL; Multiplex, BioRad Laboratories, Hercules, CA) and adiponectin (μg/mL; enzyme-linked immunosorbent assay; Tema Ricerca srl, Italy) on sera frozen immediately after sampling and stored at −25°C for a few days. WISP1 concentration was detected in plasma samples by enzyme-linked immunosorbent assay commercial kits (ng/mL; RayBiotech Inc., Norcross, GA). C. MRI {#s9} ------ Data on hepatic fat fraction (%) and abdominal SAT and VAT area (cm^2^), obtained by MRI (1.5-T magnet; Magnetom Avanto, Siemens Medical Systems, Erlangen, Germany), were available for 67 patients with type 2 diabetes enrolled for the Eudract 2011-003010-17 study, as described elsewhere \[[@B22]\]. Briefly, nonalcoholic fatty liver disease (NAFLD) was diagnosed in patients with hepatic fat fraction of ≥5.5% \[[@B23]\]; VAT and SAT were quantified by acquiring a three-dimensional gradient echo T1-weighted volumetric interpolated breath-hold examination sequence on an axial plane modified by Dixon \[repetition time, 4.7 msec; echo time, 2.3 msec; flip angle, 10°; matrix, 256 ×192 mm; section thickness, 5 mm (reconstructed 2.5 mm); intersection gap, 0\] and analyzing the fat-only data sets with specific software (Slice-O-Matic; Tomovision Inc., Montreal, QC, Canada) \[[@B24], [@B25]\]. Data were calculated from AT area at L1--L2, L2--L3, L3--L4, and L4--L5 levels; free-form regions of interest and manual threshold were used to select fat tissue within VAT and SAT slides. A single radiologist, blinded to the clinical characteristics of study participants, conducted an exploratory evaluation of VAT quality by detecting the presence of inhomogeneous areas, likely referable to AT inflammation, in the context of visceral compartment (between L1 and L3). In physiologic conditions, AT signal intensity at MRI is homogenous and markedly brighter than that observed in surrounding tissues and organs on both T2-weighted and T1-weighted sequences. Inflammation is generally characterized by the presence of edema, which represents the main feature of phlogosis and appears as an area with slightly higher signal intensity, in comparison with normal tissue, at T2-weighted sequences with fat saturation. Therefore, VAT nonhomogeneity has been defined as the presence of areas characterized by slightly lower signal intensity on T1-weighted sequences and slightly higher signal intensity on T2-weighted fat-saturated sequences at MRI. D. Statistical Analysis {#s10} ----------------------- All analyses were performed with SPSS statistical package, version 23 (IBM, Armonk, NY). Values are reported as mean ± SD for continuous variables and as a percentage for categorical variables. Non--normally distributed variables underwent log-transformation before the analyses. Comparisons between two groups were performed by Student *t* test for independent samples and by Pearson *χ*^2^ for categorical variables. Analysis of variance analysis with Bonferroni adjustment was performed for comparisons between multiple groups. Comparisons between subgroups of individuals with or without impaired VAT signal intensity, categorized as 0 = absence and 1 = presence of VAT inhomogeneity at MRI, were performed by the nonparametric Mann--Whitney test for continuous variables and Fisher test for categorical variables. Bivariate correlation analyses were calculated by Pearson and Spearman rank correlations or by an age- and sex-adjusted partial correlation test. A two-tailed *P* value \< 0.05 was considered to indicate a statistically significant difference, with a 95% confidence interval. 2. Results {#s11} ========== In the total study population (n = 118), the mean plasma WISP1 concentration was 460.5 ± 1540.4 ng/mL; this value linearly increased throughout different classes of obesity (Kruskal--Wallis test; *P* \< 0.001) ([Fig. 1](#F1){ref-type="fig"}). WISP1 levels did not differ significantly between participants with and without type 2 diabetes (432.4 ± 1521 ng/ml vs 535 ± 1639.5 ng/mL; *P* = 0.29); no specific association between WISP1 levels and diagnosis of diabetes was found even after stratification of the diabetic population for presence of obesity ([Table 1](#T1){ref-type="table"}). {#F1} ###### **Clinical and Biochemical Characteristics of Study Population According to Presence of Type 2 Diabetes and Obesity** **Parameter** **NonOb-T2D** **(n = 36)** **Ob-T2D (n = 35)** **Ob-NonT2D (n = 26)** **NonOb-NonT2D (n = 21)** ***P* Value** --------------------------- ---------------------------- --------------------- ------------------------ --------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------- Age (y) 50.4 ± 9.7 51.9 ± 9.2 42.4 ± 9.8 52.3 ± 12.4 0.007[*^a^*](#t1n1){ref-type="table-fn"}; 0.003[*^b^*](#t1n2){ref-type="table-fn"}; 0.002[*^c^*](#t1n3){ref-type="table-fn"} Sex (male/female) 21/15 26/9 9/17 14/7 0.02[*^d^*](#t1n4){ref-type="table-fn"} BMI (kg/m^2^) 26.6 ± 1.8 34.2 ± 4.4 40.7 ± 6.1 24.4 ± 2.9 \<0.001[*^a^*](#t1n1){ref-type="table-fn"}^,^[*^b^*](#t1n2){ref-type="table-fn"}^,^[*^c^*](#t1n3){ref-type="table-fn"}^,^[*^e^*](#t1n5){ref-type="table-fn"} Waist circumference (cm) 96 ± 6.9 112.8 ± 12.4 121.2 ± 14 85.8 ± 14.8 \<0.001[*^a^*](#t1n1){ref-type="table-fn"}^,^[*^b^*](#t1n2){ref-type="table-fn"}^,^[*^c^*](#t1n3){ref-type="table-fn"}^,^[*^e^*](#t1n5){ref-type="table-fn"} T2D duration (y) 7.6 ± 7.6 6.2 ± 5.1 --- --- NS SBP (mmHg) 124 ± 13.8 135.1 ± 16.9 128.1 ± 19.3 123.1 ± 9.1 0.03[*^e^*](#t1n5){ref-type="table-fn"}; 0.02[*^f^*](#t1n6){ref-type="table-fn"} DBP (mmHg) 79 ± 8 86.7 ± 19.6 81.2 ± 7.8 78.3 ± 5.9 NS FBG (mg/dL) 131.7 ± 37.8 130 ± 38.2 93.6 ± 11.8 98.2 ± 16.9 0.01[*^e^*](#t1n5){ref-type="table-fn"}^,^[*^g^*](#t1n7){ref-type="table-fn"}; \<0.001[*^c^*](#t1n3){ref-type="table-fn"}^,^[*^b^*](#t1n2){ref-type="table-fn"} HbA1c (%-mmol/mol) 6.3 ± 0.5 6.8 ± 1.3 5.3 ± 0.5 --- 0.001[*^c^*](#t1n3){ref-type="table-fn"}^,^[*^b^*](#t1n2){ref-type="table-fn"} Total cholesterol (mg/dL) 178.1 ± 36.9 175.8 ± 38 191.4 ± 21.1 197.6 ± 26.9 NS HDL cholesterol (mg/dL) 49.1 ± 13.3 50.3 ± 15.9 49.9 ± 19.2 51.2 ± 10.9 NS Triglycerides (mg/dL) 134.6 ± 70.8 138 ± 51.5 133.1 ± 37.5 114 ± 75.3 NS AST (IU/L) 23.5 ± 13.6 24.8 ± 10.7 23.7 ± 10.5 18.1 ± 4.6 NS ALT (IU/L) 31.9 ± 24.9 33.9 ± 16.7 29.8 ± 16.7 23.6 ± 12.4 NS Uric acid (mg/dL) 5.6 ± 1.3 5.8 ± 0.9 5.6 ± 1.4 5.2 ± 1.04 NS Blood creatinine (mg/dL) 0.97 ± 0.3 1.06 ± 0.2 1 ± 0.1 1.05 ± 0.2 NS FBI 10.7 ± 5.5 13.6 ± 5.1 14.6 ± 11.9 --- NS HOMA-IR 3.4 ± 1.7 4.2 ± 1.8 3.5 ± 3.4 --- NS HOMA*β* % 63.8 ± 64.7 105.7 ± 71.2 173.2 ± 132 --- 0.03[*^f^*](#t1n6){ref-type="table-fn"}; \<0.001[*^c^*](#t1n3){ref-type="table-fn"} IL-8 (pg/mL) 69.8 ± 109.2 79.4 ± 130.4 21.1 ± 30.8 22.9 ± 29.6 NS WISP-1 (ng/mL) 249.4 ± 546.3 610.4 ± 2067 725 ± 2039.2 241 ± 695.7 NS Unless otherwise noted, values are mean ± SD. Bonferroni-adjusted analysis of variance analysis. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; DBP, diastolic blood pressure; HbA1c, hemoglobin A1c; HOMA*β*%, homeostasis model assessment of insulin secretion; HOMA-IR, homeostasis model assessment of insulin resistance; nonOb-nonT2D, nonobese nondiabetic patients; nonOb-T2D, nonobese diabetic patients; Ob-nonT2D, obese nondiabetic patients; ObT2D, obese diabetic patients; SBP, systolic blood pressure; T2D, type 2 diabetes. Ob-nonT2D vs nonOb-nonT2D. Ob-nonT2D vs Ob-T2D. NonOb-T2D vs Ob-nonT2D. *χ*^2^ Pearson test between groups. Ob-T2D vs nonOb-nonT2D. NonOb-T2D vs Ob-T2D. NonOb-T2D vs nonOb-nonT2D. Among all clinical and metabolic determinants, bivariate correlation analyses showed that WISP1 levels were strongly associated with higher IL-8 concentration (*r* = 0.49; *P* \< 0.0001; Supplemental Data 1) and increased BMI (*r* = 0.23; *P* = 0.016); in contrast, no relationship was found with age (*r* = −0.05; *P* = 0.6), sex (*r* = 0.13; *P* = 0.2), systemic blood pressure \[systolic: *r* = −0.08 (*P* = 0.4); diastolic: *r* = −0.13 (*P* = 0.2)\], or metabolic control \[FBG: *r* = −0.06 (*P* = 0.56); total cholesterol: *r* = 0.13 (*P* = 0.17); high-density lipoprotein cholesterol: *r* = 0.07 (*P* = 0.44); low-density lipoprotein cholesterol: *r* = 0.12 (*P* = 0.2); triglycerides: *r* = 0.08 (*P* = 0.4)\]. Circulating IL-6 was detectable in 60% of study participants and TNF-*α* in 45% of study participants \[median (25th--75th percentile) IL-6: 1.6 (0--3.9) pg/mL; TNF-*α*: 0.39 (0--3.93) pg/mL\]. No significant differences were found between patients with and without type 2 diabetes \[IL-6: patients with diabetes, 1.5 (0--3.36) pg/mL; patients without diabetes: 1.97 (0--4.9) pg/mL; TNF-*α*: patients with diabetes: 0.68 (0--3.38) pg/mL; patients without diabetes: 0.22 (0--6.8 pg/mL); *P* = not significant\]. Interestingly, patients with increased plasma IL-6 and TNF-*α* levels (*i.e.*, those in the highest quartile of both circulating cytokines) showed significantly higher WISP1 levels (*r* = 0.24; *P* = 0.02) and a trend toward greater IL-8 concentration (*r* = 0.20; *P* = 0.054). In patients with type 2 diabetes, higher WISP1 levels were specifically associated with the presence of VAT---rather than SAT---fat distribution, as expressed by greater VAT area and VAT/SAT ratio, and lower adiponectin levels, whereas no correlation with total body adiposity (*i.e.,* BMI, waist circumference) and NAFLD was observed ([Table 2](#T2){ref-type="table"}). ###### **Plasma WISP-1 Levels in Patients With Type 2 Diabetes: Bivariate Correlation Analyses** **Parameter** **Correlation Coefficient**[*^a^*](#t2n1){ref-type="table-fn"} ***P* Value** --------------------- ---------------------------------------------------------------- ----------------------------------------- Sex −0.15 0.23[*^b^*](#t2n2){ref-type="table-fn"} Age −0.15 0.22 T2D duration −0.17 0.17 BMI 0.04 0.77 Waist circumference −0.006 0.95 SBP −0.05 0.69 DBP −0.24 0.06 FBG 0.01 0.92 HOMA-IR −0.009 0.94 HOMA-*β*% −0.11 0.41 HbA1c −0.09 0.49 IL-8 0.71 \<0.0001 Adiponectin −0.25 0.04 VAT area 0.56 0.005 SAT area −0.37 0.01 VAT/SAT ratio 0.26 0.03 NAFLD (yes/no) 0.10 0.39[*^b^*](#t2n2){ref-type="table-fn"} Abbreviations: DBP, diastolic blood pressure; HbA1c, hemoglobin A1c; HOMA*β*%, homeostasis model assessment of insulin secretion; HOMA-IR, homeostasis model assessment of insulin resistance; SBP, systolic blood pressure; T2D, type 2 diabetes. Pearson correlation coefficients are shown. Spearman correlation coefficient, with WISP-1 as a continuous variable. The multivariate linear regression analysis performed in the entire study population demonstrated that circulating IL-8 concentration was the main determinant of increased plasma WISP1 levels, independent of sex, age, IL-6, TNF-*α*, diabetes, and total adiposity (*R*^2^ = 0.58; *P* \< 0.001), as shown in [Table 3](#T3){ref-type="table"}. ###### **Multivariate Linear Regression Analysis** **Parameter** **Unstandardized Coefficients** **Standardized Coefficient: *β*** ***t*** ***P* Value** --------------------- --------------------------------- ----------------------------------- --------- --------------- ---------- Constant 722.6 884.4 0.87 0.39 IL-8 9.65 1.005 0.75 9.6 \<0.0001 IL-6 −3.95 18.7 −0.14 −0.21 0.83 TNF-*α* 2.32 19.45 0.08 0.12 0.90 BMI 40.93 41.38 0.14 0.99 0.33 Waist circumference −14.91 13.57 0.16 −1.1 0.27 Age −10.34 10.25 −0.07 −1.00 0.32 Sex (M/F) 36.5 248.01 0.01 0.15 0.88 T2D (yes/no) −189.65 254.68 −0.06 −0.74 0.46 *R* = 0.762; *R^2^* = 0.58; corrected *R^2^* = 0.539; SD error = 931.4877. Plasma WISP1 concentration is the dependent variable. Abbreviations: T2D, type 2 diabetes. Inhomogeneous areas referable to VAT inflammation were detected in nine out of 67 diabetic patients (13.4%; four without obesity and five with obesity) who underwent MRI ([Fig. 2](#F2){ref-type="fig"}; MRI images from all the patients with VAT inhomogeneity are shown in Supplemental Data 2). In all these patients, spots with impaired MRI signal, represented by lower intensity on T1-weighted and higher intensity on T2-weighted images in comparison with the surrounding AT (suggestive of edema and inflammation), were localized near the mesenteric root and jejunum vessels. Of note, none of these individuals had a history of abdomen surgery, inflammatory bowel diseases, gastrointestinal infection, or malignancy. Patients with qualitative VAT alterations were all men, had a higher prevalence of insulin therapy, and exhibited peculiar systemic inflammatory profile (characterized by greater WISP1, IL-8, and CRP levels) in comparison with diabetic individuals with homogeneous VAT at MRI ([Table 4](#T4){ref-type="table"}). {#F2} ###### **Inflammatory Profile and Body Fat Distribution in Patients With Type 2 Diabetes With or Without Impaired VAT Homogeneity** **Parameter** **Impaired VAT (n = 9)** **Normal VAT (n = 58)** ***P* Value** -------------------------- -------------------------- ------------------------- ------------------------------------------ Age (y) 55.7 ± 8 59.2 ± 9.7 0.21 Gender (M/F) 9/0 38/20 0.035[*^a^*](#t4n1){ref-type="table-fn"} BMI (kg/m^2^) 32.4 ± 5.6 29.5 ± 4 0.17 Waist circumference (cm) 112.9 ± 20.2 102 ± 10.5 0.20 T2D duration (y) 6.7 ± 4.9 7.1 ± 6.8 0.89 WISP-1 (ng/mL) 1319.9 ± 1480 297 ± 1545 \<0.0001 IL-8 (pg/mL) 159.6 ± 119.7 60.1 ± 116.5 0.006 CRP (mg/dL) 7 ± 5.4 2.8 ± 3.8 0.009 Adiponectin (μg/mL) 4.6 ± 1.7 6.7 ± 3.5 0.08 VAT area (cm^2^) 236.1 ± 83.8 183.1 ± 67.8 0.14 SAT area (cm^2^) 246.3 ± 163.5 238.1 ± 115.2 0.9 VAT/SAT 1.1 ± 0.4 0.96 ± 0.6 0.19[*^a^*](#t4n1){ref-type="table-fn"} Prevalence of NAFLD (%) 55 56 0.99[*^a^*](#t4n1){ref-type="table-fn"} Obesity (%) 55.6 50 0.75[*^a^*](#t4n1){ref-type="table-fn"} Insulin therapy (%) 44.4 12 0.038[*^a^*](#t4n1){ref-type="table-fn"} Mann--Whitney independent sample *U* test unless otherwise noted. Abbreviations: T2D, type 2 diabetes. Fisher test. The association between systemic inflammation and impaired VAT homogeneity persisted after adjustment for sex, age, insulin treatment, and abdominal adiposity, as expressed by total VAT and SAT area \[WISP1: *r* = 0.27 (*P* = 0.03); IL-8: *r* = 0.34 (*P* = 0.009); CRP: *r* = 0.37 (*P* = 0.004); partial correlation analyses\]. Notably, the area under the receiver-operating characteristic curve for WISP1 for identifying the presence of VAT inhomogeneity was 0.87 (*P* \< 0.001; CI: 0.734--1.00); plasma WISP1 levels \>130.7 ng/mL were capable of predicting VAT inflammation at MRI with a sensitivity of 78% and a specificity of 83% (Supplemental Data 3). In contrast, no correlation was found between impaired VAT intensity and indexes of insulin-resistance/secretion, obesity, or glycemic control (data not shown). 3. Discussion {#s12} ============= This study demonstrated that circulating WISP1 levels are increased in obese persons and directly related to visceral adiposity, independent of glycemic status or insulin resistance. Furthermore, higher WISP1 is strongly associated with increased IL-8, reduced adiponectin, and impaired VAT homogeneity at MRI. This study investigated WISP1 in the presence of type 2 diabetes, its potential implication in AT dysfunction, and its related conditions. Recently, Murahovschi *et al.* \[[@B17]\] conducted an elegant systematic investigation of *WISP1* gene expression in human SAT and VAT samples from mostly glucose-tolerant participants with a broad spectrum of body weight. These authors detected high WISP1 expression in AT---mostly in the visceral compartment---and found a negative association with insulin sensitivity and circulating adiponectin levels, whereas WISP1 expression directly correlated with visceral fat content at MRI. These findings suggested a role of this adipokine as a potential marker of visceral fat accumulation and insulin resistance \[[@B17]\]. In agreement with our results obtained in patients with type 2 diabetes, Murahovschi *et al.* did not find any association between WISP1 expression and the presence of NAFLD. In our study, WISP1 concentration did not differ significantly between diabetic and nondiabetic participants. Bivariate correlation analyses performed in all study participants showed that plasma levels of this adipokine were not associated with FBG or lipid profile and, in the presence of type 2 diabetes, did not correlate with glycemic control or duration of diabetes. On the other hand, IL-8 was the main determinant of increased WISP1 concentration in diabetic and nondiabetic patients. Because IL-8 is a well-known mediator of AT inflammation \[[@B3]\], overall our results may suggest that increased WISP1 levels do not portray conditions such as overweight or diabetes *per se* but rather are a systemic marker of impaired AT homeostasis and function. Studies conducted in other pathological conditions (*i.e.*, lung idiopathic primary fibrosis \[[@B11], [@B12]\] and cardiac fibrosis \[[@B15], [@B16]\]) demonstrated that WISP1 displays an active role in determining tissue fibrosis and remodeling. In particular, increased WISP1 levels were detected in fibroblasts from idiopathic primary fibrosis \[[@B11]\]; in these cells, proinflammatory and profibrotic cytokines, such as TNF-*α* and TGF-*β*1, induced WISP1 expression through a nuclear factor *κ*B--dependent mechanism; WISP1, in turn, promoted IL-6--mediated fibrosis processes \[[@B12]\]. In addition, WISP1 and WNT pathways took part in processes leading to cardiac fibrosis and myocardial remodeling \[[@B14]\] and mediated angiotensin-induced cardiomyocyte hypertrophy and fibrotic damage \[[@B15]\]. Therefore, it is plausible to speculate that the increase in WISP1, in the presence of AT inflammation, may induce AT remodeling and aberrant fibrogenesis, which is responsible, in turn, for AT loss of function, insulin resistance, and its consequences \[[@B26][@B27]--[@B28]\]. This hypothesis, ascribing to WISP1 a causal role in AT dysfunction, is strongly corroborated by data from Murahovschi *et al.* \[[@B17]\] showing that in human macrophages and mesenchymal stem cell--derived adipocytes, 24-hour incubation with WISP1 induces significant and dose-dependent increase of IL-6, TNF-*α*, IL-1*β*, and IL-10 messenger RNA expression and protein level in medium \[[@B17]\]. In our study, we explored the possibility of identifying areas of impaired VAT homogeneity at MRI, which could reflect AT inflammation, by qualitative assessment of AT intensity. We found inhomogeneous VAT in a subgroup of patients with type 2 diabetes characterized by a peculiar systemic proinflammatory profile; notably, circulating WISP1 levels showed a great predictive power for VAT inhomogeneity in our population. Previous experimental studies demonstrated that standard routine MRI sequences can depict brown AT, characterized by intense metabolic activity and increased cellularity, *in vivo* \[[@B29], [@B30]\]. Similarly, data from the Multi-Ethnic Study of Atherosclerosis showed that the presence of low VAT radiodensity at computed tomography was independently associated with a worse systemic inflammatory profile and higher prevalence of metabolic syndrome in a large US population \[[@B31]\]. The current study investigated the possible role of standard MRI, a radiation-free alternative to computed tomography, in identifying AT inhomogeneity likely referable to local inflammation. This approach could represent a complementary tool for risk stratification in patients at increased cardiovascular risk and warrants further investigation in larger populations and in the context of trials specifically designed for this purpose. Our study explored WISP1 in the presence of type 2 diabetes and assessed the association between circulating WISP1 levels and markers of AT inflammation. In our study population, WISP1 assessment showed low sensitivity and high SDs, which might represent a limitation for using this molecule as a routine marker for screening AT inflammation. Besides this consideration, increased WISP1 levels have been shown to specifically identify a peculiar phenotype of patients characterized by the presence of visceral fat distribution and worse circulating proinflammatory profile. Furthermore, 93% of patients with plasma WISP1 levels \>0 displayed high IL-8 levels, and the association between the circulating concentration of these two molecules persisted after adjustment for possible confounders. Although we are aware that specific conditions, such as tissue inflammation, may benefit from a biopsy-proven diagnosis, our population did not meet any clinical criteria for performing an invasive procedure, such as AT biopsy. Nevertheless, in our study, AT dysfunction has been estimated by dosing highly accurate circulating biomarkers and exploring qualitative aspects at MRI. Furthermore, our population underwent detailed metabolic phenotyping, allowing us to investigate a broad spectrum of potential correlates of increased WISP1 levels in plasma. Thus, this composite cluster of systemic and imaging data related to AT dysfunction may prove to be useful in identifying dysmetabolic conditions and in the follow-up of targeted treatments. Finally, we acknowledge that the cross-sectional design of our study does not allow us to establish with certainty a causal nexus between higher WISP1 and IL-8 levels or between VAT inhomogeneity and worse circulating inflammatory profile. Therefore, further studies with longitudinal design are warranted for fully understanding the pathophysiologic processes behind our observations and the possible clinical implications. In summary, circulating WISP1 levels are increased in obese persons and directly related to adiposity, independent of glycemic status or insulin resistance; moreover, they are strongly associated with increased plasma IL-8 and signal abnormalities of VAT. The overall data add insights to the mechanisms underlying metabolic alterations and may open a scenario for innovative therapeutic approaches to diabetes prevention and care. Abbreviations: ATadipose tissueBMIbody mass indexCRPC-reactive proteinFBGfasting blood glucoseIL-8interleukin-8MRImagnetic resonance imagingNAFLDnonalcoholic fatty liver diseaseSATsubcutaneous adipose tissueSDstandard deviationTNFtumor necrosis factorVATvisceral adipose tissueWISP1Wnt1-inducible signaling pathway protein-1. Author contributions: I.B., M.G.B., and M.G.C. designed the study. D.C., F.A.C., L.B., F.L., and M.D.B. coordinated the study, oversaw patient recruitment and data collection, and finalized the dataset. F.A.C., F.M., A.P., and R.D.G. performed laboratory experiments. I.B. and M.G.B. conducted the statistical analyses. M.D.M. performed the MRI and analyzed the data set. I.B. and M.G.C. drafted the paper, which was reviewed by all authors. All authors read and approved the final manuscript. This study has been carried out with the contribution of the Italian Society of Diabetology (SID) Borse di studio Diabete Ricerca-MSD 2014 (I.B.) and was funded by research grants from the Sapienza University Ateneo Scientific Research (M.G.C., I.B.). Disclosure Summary: The author reports no conflicts of interest in this work

Due to ethical restrictions imposed by the Ethics Committee of Parma Province as well as legal restrictions imposed by Italian legislation, data cannot be made publicly available. All relevant data is available upon request to the corresponding author. Introduction {#sec005} ============ Gait speed, or *walking speed*, measured at the individual's usual pace has been reported to be a relevant clinical marker of health, well-being and functional status of older population \[[@pone.0153583.ref001]\]. Epidemiological studies addressing the reliability and validity of gait speed assessment in this age-group indicate that this parameter is an independent predictor of a wide range of poor clinical outcomes in older persons, including falls \[[@pone.0153583.ref002]\], hospitalization/institutionalization \[[@pone.0153583.ref003]\], disability \[[@pone.0153583.ref004]\] and mortality \[[@pone.0153583.ref005]\]. In particular, the 4-meter gait speed test is one of the most widely used assessment tools in the clinical geriatric settings \[[@pone.0153583.ref006]\]. This measurement, performed by a stopwatch, is simple, quick, reproducible, inexpensive, feasible, and can even be assessed by non-professional trained staff. Recent recommendations from the *Mobility Working Group* have identified the timed 4-meter usual gait speed as the main tool to diagnose dismobility, a condition characterized by poor mobility and defined by gait speed slower than 0.6 m/s \[[@pone.0153583.ref007]--[@pone.0153583.ref008]\]. However, the current standard methodology of assessment of the walking time during the test by a stopwatch can be biased by high inter- and intra-operator variability \[[@pone.0153583.ref009],[@pone.0153583.ref010]\]. These limitations might persist even after an intensive training for the staff devoted to its use. Over the past two decades, the increasing need of accurate and objective techniques in the assessment of physical activity led to the technological development of inexpensive, miniature accelerometer sensors \[[@pone.0153583.ref011],[@pone.0153583.ref012]\]. The quality of the information provided by these sensors is potentially more reliable and valid and may theoretically give better quantitative measures of gait in older individuals, identifying deteriorating gait and dismobility \[[@pone.0153583.ref013]\]. However, studies investigating the correlation between the assessment of gait speed, when conducted with a traditional stopwatch and by using accelerometers, are missing. Scarce information is also available on the relationship between the results obtained with these two assessment methods and other objective tests of physical performance \[[@pone.0153583.ref014]\]. In a recent population-based cohort study carried out in older individuals without activities of daily living (ADL) disability, a good correlation between manually-measured 4-meter gait speed and Short Physical Performance Battery (SPPB) was found, but instrumental measurements with accelerometers were not performed \[[@pone.0153583.ref015]\]. Given the clinical and research relevance of slow gait speed, we sought to investigate the correlation between its manual (using a stopwatch, 4-MM) and technological (using an accelerometer, 4-MA) assessment in older individuals. The study was also aimed at evaluating the correlation of 4-MM and 4-MA with other measures of physical performance, namely hand grip strength and the 6-minute walking test (6MWT). Materials and Methods {#sec006} ===================== Design, Participants and Ethical Statement {#sec007} ------------------------------------------ This community-based observational study is an ancillary project of the MED&SANO study, an epidemiological study conducted in a representative sample of non-disabled older persons living in the Medesano geographic area (Emilia Romagna Region, Italy). The current analysis used cross-sectional data from the baseline comprehensive geriatric assessment conducted between November 2012 and April 2013 as part of the Provide multicenter European study, whose aims and design are detailed elsewhere \[[@pone.0153583.ref016]\]. A total of 455 non-disabled older community-dwellers aged 65 years or older were eligible for study enrolment after a screening evaluation by general practitioners of the Medesano area, according to the absence of physical disability. Among them, 205 were randomly selected to undergo a second-level specialist evaluation including comprehensive geriatric assessment. All subjects with overt disability (Barthel index score \<65), with severe chronic diseases or cancer were excluded from the study at this second step (n = 8). For the present analysis, 172 participants (69 men and 103 women) with complete data for the measures of interest were considered. Thus, 33 subjects were excluded since they did not complete the battery of physical tests included in this study. The study protocol was approved by the Ethics Committee of Parma Province. All participants were informed about the study procedures, purposes, and known risks, and all gave their written informed consent. Physical Performance and Muscle Strength Measures {#sec008} ------------------------------------------------- The battery of physical evaluation tests included the assessment of gait speed by using a stopwatch (4-MM) and an accelerometer (4-MA), SPPB, 6MWT and hand grip strength. For each participant, all measures were collected during the same day. SPPB was assessed first, followed, respectively, by 6MWT, 4-meter gait speed and handgrip strength. A period of 2--3 minutes of rest was granted between one test and another. ### Gait Speed Assessment {#sec009} The manual and instrumental assessment of gait speed were performed during the same 4-meter walk. A straight clearly marked course was used. The manual measurement was made by a trained operator using a stopwatch (4-MM). A tri-axial accelerometer was instead used for the instrumental assessment (4-MA) \[[@pone.0153583.ref017]\]. Instructions to walk at usual pace from a still standing position behind the starting line were provided to participants. Timing started at the first foot movement and ended when a foot completely crossed the finish line. Evaluations were conducted in a standardized way by five trained investigators. Canes and walkers were allowed if the subject normally used this equipment in his/her daily life. The inertial triaxial sensor (Free4Act®, LorAn-Engineering, Bologna, Italy) consists of a small case of 78x48x20 mm weighting 48 g only, easy to use, requires no specialized equipment, does not interfere with regular walking, and could be used to analyze walking in clinical practice. The accelerometer, placed on a semi-elastic belt covering the L4-L5 inter-vertebral space, transmitted the data to a PC via Bluetooth. The sensitive axes of the sensing unit were automatically aligned along the anatomical vertical, medio-lateral, and antero-posterior axis. Short Physical Performance Battery (SPPB) {#sec010} ----------------------------------------- Lower extremity performance was evaluated using the SPPB, a strong predictor of physical disability in older adults \[[@pone.0153583.ref018]\]. The SPPB includes three timed subtests: the chair stand test, the usual gait speed test, and the balance test. The timed results of the subtests were converted to an ordinal scale ranging from 0 (worst performance) to 12 (best performance) according to predefined and previously published cut-points. ### 6-minute Walking Test (6MWT) {#sec011} The 6MWT was performed indoors, using a 30 meter-long walking course with a hard surface. The length of the corridor was marked every 3 meter and cones marked the turnaround points \[[@pone.0153583.ref019]\]. A starting line, defining the beginning and the end of each 60-meter lap, was marked on the floor using a brightly colored tape. Participants were invited at walking as far as possible over the circuit for a period of 6 minutes. The test evaluates the global and integrated responses of all the systems involved during exercise, including the pulmonary and cardiovascular systems, systemic circulation, peripheral circulation, blood, neuromuscular units, and muscle metabolism \[[@pone.0153583.ref019]\]. ### Handgrip Strength {#sec012} Isometric hand grip strength was measured using a handheld dynamometer (Jamar Plus Digital Hand Dynamometer). The device measured strength in kilograms, with a precision of 0.1 kg. Participants were asked to perform the task twice with each hand. The average of the best result obtained with each hand was used for the present analyses. ### Other Measures {#sec013} Weight and height, objectively measured and employing a standard protocol, were used to calculate the body mass index as kg/m^2^. The Barthel Index was assessed for measuring the participants\' functional ability. Cognitive performance was assessed using the Mini-Mental State Examination (MMSE). Depressive symptoms were assessed by the 15-item Geriatric Depression Scale (GDS-15), which is a widely used screening instrument for depressive symptoms in the elderly. The GDS-15 detects changes in depressive symptoms after a major negative life event \[[@pone.0153583.ref020]\]. Nutritional status was determined using the Mini Nutritional Assessment--Short Form (MNA-SF), which is a reliable and practical screening test validated in all geriatric settings \[[@pone.0153583.ref021]\]. Information on drug use was collected through the expert evaluation of clinicians as routine approach. The prevalence of specific medical conditions was established using standardized criteria that combine information from self-reported history, medical records, and a clinical medical examination. Diagnostic algorithms were modified versions of those adopted in the Women's Health and Aging Study \[[@pone.0153583.ref022]\]. Statistical Analysis {#sec014} -------------------- Categorical variables were expressed in numbers and percentages, and continuous variables were reported according to gender as means (and standard deviations, SD) for normally distributed parameters or as median and interquartile range (IQR) for those non-normally distributed. Since reference values for some muscle function and gait parameters in non-disabled older individuals are different between males and females \[[@pone.0153583.ref023]\], a gender-specific analysis was carried out. Scatterplots of data were built to examine the correlation between 4-MM and 4-MA. Then, unadjusted Pearson correlations were calculated, as appropriate. Correlations of 4-MM and 4-MA with hand grip strength and 6MWT were also separately assessed in men and women using the same tests. 4-MA and 4-MM were categorized by using the cut-off point for dismobility syndrome of 0.6 m/sec \[[@pone.0153583.ref007],[@pone.0153583.ref008]\]. The concordance between 4-MM and 4-MA in diagnosing dismobility syndrome was then assessed. Statistical significance was defined as p≤0.05. SAS 8.2 statistical package (SAS Institute, Inc., Cary, NC, USA) was used for all analyses. Results {#sec015} ======= Characteristics of the study population (n = 172, 69 M, 103 F) are summarized in [Table 1](#pone.0153583.t001){ref-type="table"}. The mean age of participants was 80.7 (SD 4.8) years. 10.1371/journal.pone.0153583.t001 ###### Sociodemographic and Clinical Characteristics of Study Sample of the MED&SANO study. {#pone.0153583.t001g} Men(n = 69) Women(n = 103) ------------------------ -------------- ---------------- Age (years) 79.03 ± 4.87 78.18 ± 5.64 Barthel Index 97.78 ± 5.45 96.65 ± 6.47 Handgrip strength (kg) 39.42 ± 8.57 23.85 ± 5.01 Total SPPB (score) 9.74 ± 1.14 8.97 ± 1.65 4-MM (m/s) 0.91 ± 0.26 0.78 ± 0.22 6MWT (m/s) 0.88 ± 0.22 0.86 ± 0.21 4-MA (m/s) 1.06 ± 0.17 0.89 ± 0.18 BMI (kg/m^2^) 26.56 ± 3.18 26.24 ± 3.80 MMSE score 26.13 ± 3.17 26.81 ± 3.55 GDS Short Form (score) 3 \[1--5\] 3 \[2--6\] MNA Short Form (score) 13.14 ± 1.47 12.45 ± 2.07 Drug use (n) 3 \[2--5\] 4 \[3--6\] \*Data are presented as number of cases (percentage), mean ± SD or median and interquartile range. \*\*SPPB; Short Physical Performance Battery; 6MWT: Six Minute Walking Test; 4-MA: 4-m gait speed accelerometer; 4-MM: 4-m gait speed manual; BMI: Body Mass Index; MMSE: Mini Mental State Examination Score; GDS: Geriatric Depression Scale; MNA: Mini Nutritional Assessment. Scatterplots of correlations between 4-MM and 4-MA in men and women are shown in Figs [1](#pone.0153583.g001){ref-type="fig"} and [2](#pone.0153583.g002){ref-type="fig"}, respectively. In both genders the correlations were statistically significant (β±SE men 0.75±0.14, p\<0.001, women 0.77±0.08, p\<0.001). Figs [1](#pone.0153583.g001){ref-type="fig"} and [2](#pone.0153583.g002){ref-type="fig"} also show the categorization of subjects as having or not having dismobility syndrome according to the cut-off value of 0.6 m/s. {#pone.0153583.g001} {#pone.0153583.g002} Calculation of unadjusted Pearson correlations confirmed that 4-MM was significantly and positively correlated with 4-MA in both genders, as highlighted in [Table 2](#pone.0153583.t002){ref-type="table"} (r = 0.62, p\<0.001 in men; r = 0.73, p\<0.001 in women). Among males, the number of subjects categorized as having dismobility syndrome (gait speed \<0.6 m/s) was 6 (9%) according to 4-MM, and 19 (28%) according to 4-MA. Among females, 22 subjects (21%) had dismobility syndrome according to 4-MM and 30 (29%) according to 4-MA. [Table 3](#pone.0153583.t003){ref-type="table"} shows the categorization of participants according to the different measurement techniques and using 0.6 m/s as gait speed cut-off in men and women, respectively. In 13/69 males (19%) and 24/103 females (23%) the two methods of gait speed assessment disagreed for the presence of dismobility syndrome. 10.1371/journal.pone.0153583.t002 ###### Unadjusted coefficient correlation investigating the relationship between 4-MM, 4-MA and objectives measures of physical performance and functional capacity. {#pone.0153583.t002g} Men(N = 69) Women(N = 103) ----------------------- ------------- ---------------- ------ ---------- ------ ---------- ------ ---------- **Handgrip strength** 0.51 \< .0001 0.40 0.005 0.38 0.0001 0.29 0.001 **4-MM** \- \- 0.62 \< .0001 \- \- 0.73 \< .0001 **6-MWT** 0.59 \< .0001 0.50 0.0004 0.49 \< .0001 0.22 0.048 **4-MA** 0.62 \< .0001 \- \- 0.73 \< .0001 \- \- 4-MM: 4-meter walking speed measured manually by stopwatch; 4-MA: 4-meter walking speed measured instrumentally by accelerometer; 6MWT: 6-minute walking test. 10.1371/journal.pone.0153583.t003 ###### Categorization of male (n = 69) and female participants (n = 103) by using gait speed cut-off 0.6 m/s according to the two different methods of gait speed assessment (4-MM, manual assessment; 4-MA accelerometer assessment). 4-MM showed a poor sensitivity to detect dismobility syndrome (32% for males and 47% for females). {#pone.0153583.t003g} ------------------------- ------------------------- ------------------- **Males (N = 69)** **4-MA4-MM** \<0.6 m/s (dismobility) ≥0.6 m/s (normal) \<0.6 m/s (dismobility) 6 (8.7%) 0 ≥0.6 m/s (normal) 13 (18.8%) 50 (72.5%) **Females (N = 103)** **4-MA4-MM** \<0.6 m/s (dismobility) ≥0.6 m/s (normal) \<0.6 m/s (dismobility) 14 (13.6%) 8 (7.8%) ≥0.6 m/s (normal) 16 (15.5%) 65 (63.1%) ------------------------- ------------------------- ------------------- The correlations of 4-MM and 4-MA with other measures of functional performance are also shown in [Table 2](#pone.0153583.t002){ref-type="table"}. A significant positive correlation with 6MWT was found for both 4-MM and 4-MA in men and women (4-MM men r = 0.59, p\<0.001; women r = 0.49, p\<0.001; 4-MA men r = 0.50, p = 0.0004; women r = 0.22, p = 0.048). 4-MA showed a significant positive correlation with handgrip strength (r = 0.40, p = 0.005 in men; r = 0.29, p = 0.01 in women), as also 4-MM (r = 0.51, p\<0.001 in men; r = 0.38, p = 0.0001 in women). Discussion {#sec016} ========== In a cohort of community-dwelling older individuals, we found a significant correlation between the assessment of gait speed using a manual (i.e., stopwatch) and technological (i.e., accelerometer) technique. However, our results suggest that the concordance of two tests is less strong than anticipated and might be suboptimal in the classification of single subjects. This is the first study investigating the correlation between these two assessment modalities of gait speed. There is a wide range of methods available for assessing physical function in both research and clinical practice \[[@pone.0153583.ref024]\]. The final choice on the best measurement should take into account the inter-rater and test-retest reliability, accuracy, feasibility and costs. Namely, 4-meter gait speed test was shown to be extremely robust. Moreover, its design makes it particularly suitable for use in routine clinical and research activities. These characteristics led to its integration in the SPPB, the most used objective tool for the assessment of lower extremity functioning in older persons \[[@pone.0153583.ref024]\]. The present study shows that both 4-MM and 4-MA were correlated to other tests of physical performance like 6MWT and hand grip strength. The correlation found between the 4-MM and the 4-MA highlights that further studies are needed to investigate the role of accelerometer-measured gait speed assessment. Accurate, standardized and reproducible techniques of measurement are in fact needed, privileging those techniques less affected by methodological issues, and inter- and intra-operator variability that may represent a relevant bias \[[@pone.0153583.ref009],[@pone.0153583.ref010]\]. Four-meter gait speed depends on lower limb muscle function and is a good predictor of mobility impairment and adverse outcomes \[[@pone.0153583.ref008],[@pone.0153583.ref025],[@pone.0153583.ref026]\]. Thus, it is important to use appropriate, reliable and accurate techniques to measure it during the comprehensive geriatric assessment, in order to correctly diagnose dismobility syndrome. In fact, a 4-meter walking speed lower than 0.6 m/s is nowadays widely recognized as a diagnostic criterion for this syndrome \[[@pone.0153583.ref007]\]. We acknowledge that this cut-off point was developed using 4-MM and not based on 4-MA. Our data clearly show that a large number of cases were categorized differently according to the method of assessment. By hypothesizing 4-MA as gold standard, 4-MM showed a poor sensitivity to detect dismobility syndrome (32% for males and 47% for females, [Table 3](#pone.0153583.t003){ref-type="table"}). As such, the way gait speed is measured may have strong clinical implications. The use of an accelerometer may represent a promising alternative to manual assessment, theoretically giving more "objective" results. However, the method of wearing the device in 4-MA might influence the gait information obtained from this instrument. In addition, further longitudinal studies should investigate the correlation of 4-MA with functional outcomes, particularly in those subjects categorized differently according to the two techniques. Moreover, these results reinforce the concept that there are some gender-related differences in gait speed, that should be considered when interpreting results of functional tests during a comprehensive geriatric assessment. 4-MM remains the easiest and commonest way to assess physical performance \[[@pone.0153583.ref006]\]. Nevertheless, when using this technique, a certain degree of variability, potentially influencing its results, should always be taken into account. The routine application of 4-MA may help to overcome these limitations in both clinical and research settings where a high degree of concordance is needed to detect dismobility syndrome,. The problem of insufficient accuracy of manual measurements of walking speed is well known in medical literature \[[@pone.0153583.ref009],[@pone.0153583.ref010],[@pone.0153583.ref027]\]. Some studies addressed the importance of an initial specific training of the staff to reduce the potential inaccuracy of evaluation \[[@pone.0153583.ref009],[@pone.0153583.ref010]\]. Others used a photocell-based method (with devices mounted at the starting and finishing lines) associated with a rigid protocol of administration. As compared to manual methods of assessment, gait-accelerometers provide a wider spectrum of additional information that can be clinically relevant for exploring the risk of functional declines in older subjects (e.g., variability of movements, balance, static and dynamic acceleration). Accelerometers are also characterized by a portable and low cost acquisition system. In addition, this testing is not restricted to a laboratory environment since this instrument easily allows the assessment of usual gait speed in all the settings \[[@pone.0153583.ref026]\]. Several investigations evaluating measures of gait in subjects with different neurological conditions with pathological gait impairment, compared to healthy subjects, have already suggested that a triaxial accelerometer is a good practical tool for capturing altered ambulation \[[@pone.0153583.ref028]\]. Our study has some limitations. The relatively small sample size and the cross-sectional nature of the study do not allow definitive conclusions. The capacity of 4-MA to predict functional outcomes in older community-dwellers is in fact still poorly investigated. The studies available in the literature, and performed with accelerometers, did not use the same position to place the device. In our study, the accelerometer was placed on a semi-elastic belt covering the L4-L5 intervertebral space. Thus, our results cannot be completely comparable with other studies using different positions \[[@pone.0153583.ref029]\]. A standardized position of wearing the device is needed in the future. We acknowledge that process of data extraction could have represented another limiting factor. However, in our study, the accelerometer directly transmitted the data to a PC via Bluetooth. The sensitive axes of the sensing unit were automatically aligned along the anatomical vertical, medio-lateral, and antero-posterior axis. Finally, gold standard measurements of gait speed assessment, such as instrumented walkways and stereo photogrammetry \[[@pone.0153583.ref030]\], were not used in the present study. Despite these limitations this is the first study having assessed the correlation of 4-MM with 4-MA. The association between these two techniques of gait speed assessment should be better investigated in larger populations, with longitudinal design, including also subjects with known dismobility syndrome. On this regard longitudinal studies comparing the two tests are needed to validate 4-MA as a gold standard. Meanwhile, 4-MM remains the easiest approach to be used in the clinical practice. The implementation of technological methods of gait speed assessment in second-level geriatric care services or for clinical research purposes should however be considered although based on our findings it becomes hard to make recommendations on the better usefulness of automatic than manual measurement. Conclusions {#sec017} =========== In a population of elderly community-dwellers without known dismobility syndrome, we found a significant correlation between the assessment of gait speed using manual and technological techniques. The concordance of gait speed manually or instrumentally assessed is not optimal and manual measures might lead to misclassification of a substantial number of subjects. However, there is need of future longitudinal studies comparing the two techniques, using standardized procedures and taking quantitative gait analysis as gold standard reference, before to recommend the usefulness of accelerometers in the comprehensive geriatric assessment. We are grateful to the participants for the time and energy they spent while taking part in this Study. We are thankful to the energy and commitment of all research staff members of the School of Geriatrics (Drs Marco Mantovani, Valeria Buttò, Eleonora Sutti, Giuliana Bondi) and Physiatry (Drs Laura Galuppo, Federica Petraglia) of the University of Parma. [^1]: **Competing Interests:**The present study was unconditionally funded by Nutricia Research, Nutricia Advanced Medical Nutrition, as part of the Provide multicenter European study. The funder had no role in study design, data collection, analyses, decision to publish and preparation of the manuscript. The study was performed by independent researchers that had no personal financial conflict of interest with the funding source. None of the authors was employed by the funding source at the moment of the execution of the study. None of the authors received financial compensation or grants by the funding source. The presence of a commercial funder does not alter the authors\' adherence to PLOS ONE policies on sharing data and materials. [^2]: Conceived and designed the experiments: MM GPC RWK MC MF FL. Performed the experiments: MM FDV GG CC. Analyzed the data: MM FL. Contributed reagents/materials/analysis tools: GG CC. Wrote the paper: MM AT TM FL.

1. Introduction {#sec1} =============== Vanadium was widely used as a therapeutic agent in the late eighteenth century, treating a variety of ailments including anemia, tuberculosis, rheumatism, and diabetes \[[@B1], [@B2]\]. Vanadium compounds exhibit various biological and physiological effects in the human body. Vanadium compounds have been extensively studied for their diverse biological activities such as antitumor, antibacterial, and insulin-enhancing effects and potential capabilities as DNA structural probes \[[@B3], [@B4]\]. The coordination chemistry of oxovanadium is highly ligand dependent and more important in biological systems \[[@B5]\] as well as catalytic systems \[[@B6], [@B7]\]. Due to the d1 configuration, vanadium(IV) ionic species are easily identified by EPR spectroscopy. Due to less toxicity \[[@B8], [@B9]\], the Schiff base complexes of the vanadyl ion are topic of many research reports \[[@B10], [@B11]\]. In Europe, vanadium is often used as a natural treatment for diabetes. Vanadium has been found in human studies to imitate the effects of insulin in our bodies. This ability may be useful for some of those with diabetes, a natural method to help lower blood sugar, take less insulin, or in some instances stop taking insulin altogether \[[@B12], [@B13]\]. It is noticeable that complexation of vanadium with organic ligands minimizes unfavorable effects of its inorganic salts such as vanadyl sulfate while even maintains its potential benefits \[[@B14]\]. Furthermore, mimicking the biological activities in natural systems can be achieved by vanadium complexes which contain oxygen and nitrogen donor ligands; so identification of the structure of these complexes is regarded important \[[@B15]--[@B17]\]. Bioinorganic chemistry is a fast developing field of modern chemistry that uses Schiff bases and their transition metal complexes for a variety of applications, e.g., in biological, medical, and environmental sciences. This work is interested in preparation of a series of perimidine derivatives by various substituents. New vanadyl complexes will be prepared and well characterized by using different techniques. CT-DNA binding will be tested along the organic series. Theoretical implementation will be accomplished over all prepared compounds by different standard programs. Antitumor activity will be scanned over all new prepared compounds for comparison. 2. Experimental Work {#sec2} ==================== 2.1. Chemicals Used {#sec2.1} ------------------- Chemicals essential for preparation of perimidine derivatives such as 1,8-diaminonaphthalene, ethylbenzoyl acetate, 4-methoxyaniline, aniline, 4-chloroaniline, 3-chloroaniline, 4-nitroaniline, NaNO~2~, NaOH, HCl, and dioxane were purchased from Fluka and used without previous treatments. Also, VOSO~4~·xH~2~O salt used for the complexation process was commercially available from Sigma-Aldrich. All handled solvents were from Merck and used without previous purification. 2.2. Synthesis {#sec2.2} -------------- ### 2.2.1. Synthesis of Compounds **3a--e** (Perimidine Derivatives) {#sec2.2.1} Ligands **3a--e** were synthesized as reported in the literature \[[@B18]\] from coupling reaction of compound 1 (2.5 mmol) in ethanol (20 mL) with the appropriate arenediazonium chloride 2 in the presence of sodium hydroxide (2.5 mmol) in the ice bath at 0--5°C. The whole mixture was then left in a refrigerator overnight. The precipitated solid was filtered off, washed with water, and finally crystallized from dioxane/EtOH to give the respective hydrazones **3a--e** ([Scheme 1](#sch1){ref-type="fig"}). ^1^H·NMR and mass spectra are displayed in Figures [1](#fig1){ref-type="fig"}, [2](#fig2){ref-type="fig"}, [S1](#supplementary-material-1){ref-type="supplementary-material"}, and [S2](#supplementary-material-1){ref-type="supplementary-material"}. The analysis is matching completely with that reported in the literature \[[@B16]\]. The structural forms of new perimidine compounds are displayed in Figures [3(a)](#fig3){ref-type="fig"} and [3(b)](#fig3){ref-type="fig"}. 1. *2-\[N-phenyl-2-oxo-2-phenylethanehydrazonoyl\]-1H-perimidine (* ***3a*** *) (L* ^*1*^). IR *υ*: 3402, 3194 (2NH), and 1616 (CO) cm^−1^. ^1^H·NMR (DMSO-d~6~) d: 6.72--7.80 (m, 16H, Ar--H) and 13.62 (s, 2H, 2NH). MS *m*/*z* (%): 390 (M, 22), 285 (2), 166 (5), 140 (7), 127 (1), 105 (100), 93 (8), and 77 (35). Anal. calcd. for C~25~H~18~N~4~O (390.42). 2. *2-\[N-(4-methoxyphenyl)-2-oxo-2-phenylethanehydrazonoyl\]-1H-perimidine (* ***3b*** *) (L* ^*2*^). IR *υ*: 3333, 3167 (2NH), and 1680 (CO) cm^−1^. ^1^H·NMR (DMSO-d~6~) *δ*: 3.58 (s, 3H, OCH~3~), 7.59--7.97 (m, 15H, ArH), and 12.20 (br s, 2H, 2NH). MS *m*/*z* (%): 420 (M, 5), 419 (9), 193 (12), 166 (12), 126 (14), 107(17), 105 (100), 92 (31), and 77 (75). Anal. calcd. for C~26~H~20~N~4~O~2~ (420.47). 3. *2-\[N-(4-chlorophenyl)-2-oxo-2-phenylethanehydrazonoyl\]-1H-perimidine (* ***3c*** *) (L* ^*3*^). IR *υ*: 3422, 3206 (2NH), and 1612 (CO) cm^−1^. ^1^H·NMR (DMSO-d~6~) d: 6.73--7.77 (m, 15H, Ar--H) and 13.27 (s, 2H, 2NH). MS *m*/*z* (%): 426 (M 2, 5), 425 (M 1, 6), 424 (M, 14), 140 (4), 127 (5), 111 (2), 105 (100), and 77 (37). Anal. calcd. for C~25~H~17~ClN~4~O (435.44). 4. *2-\[N-(4-*nitrophenyl*)-2-oxo-2-phenylethanehydrazonoyl\]-1H-perimidine (* ***3d*** *) (L* ^*4*^). IR *υ*: 3356, 3198 (2NH), and 1670 (CO) cm^−1^. ^1^H·NMR (DMSO-d~6~) d: 6.64--8.21 (m, 15H, Ar--H) and 12.60 (s, 2H, 2NH). MS *m*/*z* (%): 435 (M, 8), 238 (34), 138 (13), 167 (11), 106 (45), 105 (58), 93 (100), 77 (44), and 66 (76). Anal. calcd. for C~25~H~17~N~5~O~3~ (424.88). 5. *2-\[N-(3-*chlorophenyl*)-2-oxo-2-phenylethanehydrazonoyl\]-1H-perimidine (* ***3e*** *) (L* ^*5*^). IR *υ*: 3229, 3167 (2NH), and 1622 (CO) cm^−1^. ^1^H·NMR (DMSO-d~6~) d: 6.70--7.91 (m, 15H, Ar--H) and 13.02 (s, 2H, 2NH). MS *m*/*z* (%): 426 (M2, 7), 425 (M1, 9), 424 (M, 16), 194 (7), 166 (7), 140 (5), 127 (4), 105 (100), 111 (4), and 77 (36). Anal. calcd. for C~25~H~17~ClN~4~O (424.88). ### 2.2.2. Synthesis of VO(II) Complexes {#sec2.2.2} New VO(II) complex series was synthesized by using variable derivatives from perimidine ligands. Equimolar (3 mmol) values were used from the perimidine ligand and dissolved fully in dioxane; after that, it was mixed with VOSO~4~·xH~2~O which dissolves in the dioxane/H~2~O mixture. The weighted molar ratio value from vanadyl salt was calculated attributing to its anhydrous weight. After ≈5 h reflux, 0.5 g sodium acetate was added after dissolving in a little amount of bi-distilled water to precipitate the complexes. Each precipitate was separated out on hot, filtered off, washed several times with ethanol and diethyl ether, and finally dried in a vacuum desiccator. 2.3. DNA Binding Study {#sec2.3} ---------------------- The binding attitudes of perimidine derivatives towards calf thymus DNA (CT-DNA) will be studied by using the spectroscopy method. CT-DNA (50 mg) was dissolved by stirring overnight in double deionized water (pH = 7.0) and must be kept at 4°C. Bi-distilled water was used to prepare the buffer (5.0 mM tris(hydroxymethyl)-aminomethane and 50 mM NaCl, pH = 7.2). Tris-HCl buffer was prepared in deionized water. DNA buffering solution gave absorbance ratio at 260/280 nm by 1.8--1.9, and this indicates the absence of protein from DNA \[[@B19], [@B20]\]. Applying the UV-Vis technique, the DNA concentration was determined (5.10 × 10^−4^ M) using its known molar absorptivity coefficient value (6600 M^−1^·cm^−1^ at 260 nm). At room temperature, 200--900 nm is the wavelength range used, and in 1 cm quartz cuvette, a fixed concentration (2.0 × 10^−5^ M in dioxane) from each ligand was utilized. A scanning process was done after adding CT-DNA by a gradual way from 0.00 to ≈2.18 × 0^−4^ mol·L^−1^. The same DNA amount added to the ligand solution was added also to the reference cell to delete the absorbance of free DNA. A significant binding constant (*K* ~b~) for interaction between ligands towards CT-DNA was determined by using the following equation: \[DNA\]/(*є* ~a~ − *є* ~f~) = \[DNA\]/(*є* ~b ~− *є* ~f~) + 1/*K* ~b~ (*є* ~a~ − *є* ~f~) \[[@B21]\], where \[DNA\] is the concentration of CT-DNA in base pairs, *є* ~a~ is the extinction coefficient observed for A/\[compound\] at the used DNA concentration, and *є* ~f~ is the extinction coefficient for each free compound (HL^1−5^) in the solution. Moreover, *є* ~b~ is the extinction coefficient of the compound when fully bond to DNA. In plots of \[DNA\]/(*є* ~a~ − *є* ~f~) vs. \[DNA\], *K* ~b~ is given by the following ratio: slope/intercept. 2.4. Antitumor Influence {#sec2.4} ------------------------ The evaluation of cytotoxicity of candidate anticancer drugs will be performed using the most effective, available SRB method. All molecules and their derivatives will be tested for their toxicity on different cancer cell lines. In an attempt to evaluate the impact, the samples were prepared with different concentrations: 0.01, 0.1, 1, 10, and 100 *µ*g/ml, respectively. The cells were cultured in the mixture of samples and media (RPMI-FBS + samples) for 72 h; after that, cytotoxicity impact was evaluated compared to the response of doxorubicin as a positive control. The cytotoxic effect of the composites and ligands will be tested against different cancer cell lines (HepG2, MCF-7, and HCT116) as donor cancer cell lines by means of the SRB cytotoxicity test. To avoid the contamination, the RPMI media of the cells were supplemented with 100 *µ*g/ml streptomycin and 100 units/ml penicillin with 10% FBS and incubated at a 5% CO~2~ incubator. Growing cells were collected using the trypsin enzyme and then counted using the cell counter in order to distribute equally the number of cells to each well of 69-well plates. The cells will incubate under sterile conditions with different concentrations of both ligands and composites for 72 hours, and subsequently, treated cells and untreated cells and the positive control were fixed with 10% TCA (trichloroacetic acid) and kept at 4°C for 1 h. After washing few times, fixed and washed cells were stained with 0.4% SRB stain solution for ten minutes, and subsequently, the cells were washed with 1% glacial acetic acid. To dissolve SRB-stained cells, Tris-HCl was used. To detect the density of remaining colors, a plate reader will be used at 540 nm wavelength. In order to determine the IC~50~ value, statistical analysis was accomplished through SigmaPlot version 14.0. The advantage of prepared compounds as potential drugs against different cancer cells was investigated. 2.5. Physical Techniques {#sec2.5} ------------------------ ### 2.5.1. Elemental Analysis {#sec2.5.1} The element contents (carbon, hydrogen, and nitrogen) were determined at the Micro-Analytical Unit of Cairo University. Vanadium, sulfate, and chloride contents were evaluated by known standard methods \[[@B22]\] through complexometric and precipitation methods. ### 2.5.2. Conductivity Measurements {#sec2.5.2} Applying the Jenway 4010 conductivity meter, the molar conductivity of freshly prepared 1.0 × 10^−3^ mol/cm^3^ in DMSO solutions was estimated. ### 2.5.3. X-Ray Diffraction and SEM {#sec2.5.3} X-ray diffraction manners were recorded on the Rigaku diffractometer using Cu/K*α* radiation. Scanning electron microscopy (SEM) images were obtained by using Joel JSM-6390 equipment. ### 2.5.4. IR, ^1^H·NMR, and ^13^CNMR Spectra {#sec2.5.4} IR spectra were obtained using the JASCO FT/IR-4100 spectrophotometer from 400 to 4000 cm^−1^ in the KBr disc, while ^1^H·NMR spectra were recorded in deuterated dimethyl sulfoxide using the Varian Gemini 300 NMR spectrometer. ### 2.5.5. Mass spectra {#sec2.5.5} Mass spectra were recorded on GCMS-QP1000 EX (Shimadzu) and GCMS 5988-A. ### 2.5.6. ESR Analysis {#sec2.5.6} ESR spectra of VO(II)-powdered complexes were obtained on the Bruker EMX spectrometer working in the X-band (9.60 GHz) with 100 kHz modulation frequency. The microwave power was set at 1 mW, and modulation amplitude was set at 4 Gauss. The low field signal was obtained after 4 scans with a 10-fold increase in the receiver gain. A powder spectrum was obtained in a 2 mm quartz capillary at ordinary temperature. ### 2.5.7. UV-Vis Spectra and Magnetic Measurements {#sec2.5.7} Electronic spectra for all compounds were recorded using the UV~2~ Unicam UV/Vis spectrophotometer in the DMSO solvent. Magnetic susceptibility values for VO(II) complexes were conducted by the Johnson Matthey magnetic susceptibility balance at room temperature. ### 2.5.8. Thermal Analysis {#sec2.5.8} The Shimadzu thermogravimetric analyzer (20--900°C) at 10°C·min^−1^ heating rate under nitrogen was used for thermal analysis. Theoretical treatments (modeling and docking) were accomplished by known standard programs. ### 2.5.9. Antitumor Activity {#sec2.5.9} Antitumor activity was conducted at the Regional Center for Mycology and Biotechnology. 2.6. Computational {#sec2.6} ------------------ ### 2.6.1. DFT/Hartree--Fock Study {#sec2.6.1} Implementing the Gaussian 09 software \[[@B23]\], the structural optimization process was accomplished over pyrimidine ligands and their VO(II) complexes in the gas phase. Two known methods were found as the most suitable one for the optimization process. The output files were visualized by the GaussView program \[[@B24]\]. According to the numbering scheme, DFT parameters were extracted using frontier energy gaps (*E* ~HOMO~ and *E* ~LUMO~) for all investigated compounds. Moreover, other significant computations were taken from log files as oscillator strength, excitation energy, charges assigned for coordinating atoms, and some bond lengths. ### 2.6.2. QSAR Computation {#sec2.6.2} New perimidine compounds were treated for the optimization process to give the best structural forms. HyperChem (v8.1) software is the tool used for such a purpose. The preoptimization process was executed by molecular mechanics force field (MM^+^) accompanied by semiempirical AM1 for the soft adjustment procedure. This process was accomplished without fixing any parameter till the equilibrium state for geometric structures. A system for minimizing energy was employed the Polak--Ribiere conjugated gradient algorithm. The QSAR process leads to computing essential parameters including the partition coefficient (log *P*). Log *P* value is considered the essential indicator used to predict the biological activity for optimized compounds \[[@B25]\]. ### 2.6.3. Docking Computation {#sec2.6.3} Applying AutoDockTools 4.2 by using Gasteiger partial charges which added over the elements of pyrimidine ligands, the simulation procedure was executed to give a view on the biological behavior of compounds. Rotatable bonds were cleared, and nonpolar hydrogen atoms were conjoined. Interaction occurred between inhibitors (ligands) and protein receptors (4c3p, 3bch, and 4zdr) for breast, colon, and liver cancer proteins. The docking process was accomplished after addition of fundamental hydrogen atoms, Kollman united atom-type charges, and salvation parameters \[[@B26]\]. Affinity (grid) maps of ×× Å grid points and 0.375 Å spacing were generated applying the AutoGrid program \[[@B27]\]. Van der Waals forces and electrostatic terms were obtained. This is done by applying autodock parameter set-dependent and distance-dependent dielectric functions, respectively. The docking process was executed using the Solis and Wets local search method and Lamarckian genetic algorithm (LGA) \[[@B28]\]. Initial position, orientation, and torsions of the inhibitor molecule were set indiscriminately. All rotatable torsions were expelled during the docking process. Each experiment is the mean value of 10 different runs that are set close after the maximum of 250000 energy assessments. 150 is the used population size. During the process, the translational step of 0.2 Å, quaternion, and torsion steps of 5 were applied. 3. Results and Discussion {#sec3} ========================= 3.1. Physical Properties {#sec3.1} ------------------------ Essential analytical and physical data for ligands and their VO(II) complexes are summarized in [Table 1](#tab1){ref-type="table"}. All investigated complexes are nonhygroscopic in nature, having high melting point (\>300°C). The elemental analysis proposes 1 : 2 (HL : M) molar ratio as the general formula for all complexes. All complexes are completely soluble in DMSO or DMF solvents. The conductivity measured is 5.66--14.22 Ω^−1^·cm^2^·mol^−1^. Such conductivity values are attributed to the nonconducting character of tested complexes \[[@B29]\]. This coincides with sulfate anion which favors covalent attachment with metal ions inside the coordination sphere. 3.2. Comparative IR Study {#sec3.2} ------------------------- The assignments of all characteristic bands for five perimidine ligands and their VO(II) complexes are summarized in [Table 2](#tab2){ref-type="table"}. *ν*(NH), *δ*(NH), *ν*(C=N), and *ν*(C=O) are the significant functional bands for coordinating groups which appear in narrow regions observed in all derivatives. This may refer to the far effect of the aromatic substituent on bond movement inside such groups. A comparative study of ligands and their VO(II) complexes reveals the following observations: (1) lower-shifted appearance of former bands is considered a strong evidence for contribution of C=O, NH, and C=N groups in coordination towards two central atoms. (2) New bands appeared at 1368--1434 and 1140--1179 cm^−1^ assigned for *ν* ~as~(SO~4~) and *ν* ~s~(SO~4~), respectively, through bidentate attachment \[[@B30]\]. (3) Other bands appearing at ≈760 and ≈690 cm^−1^ are attributed to *δ* ~r~(H~2~O) and *δ* ~w~(H~2~O), respectively, for crystal water molecules. (4) *ν*(M-L) bands appeared at the low wavenumber region belonging to M-O and M-N bonds. These spectral observations suggest a tetradentate mod of coordination towards two vanadyl atoms. Also, the band observed at 966--1074 cm^−1^ range assigns for *ν*(V = O), significantly pointing to the square-pyramidal configuration \[[@B31]\]. 3.3. Electronic Spectra and Magnetic Measurements {#sec3.3} ------------------------------------------------- Electronic transition bands and magnetic moment values are aggregated in [Table 3](#tab3){ref-type="table"}. UV-Vis spectra were recorded qualitatively in the DMSO solvent to gain smoothly absorption curves. Intraligand transition bands appearing at 31,250--38,168, 25,974--30,769, and 17,544--19,157 cm^−1^ are attributed to *n* ⟶ σ^*∗*^, *π* ⟶ *π* ^*∗*^, and *n* ⟶ *π* ^*∗*^ transitions, respectively, inside variable groups \[[@B32]\]. A structural condensed conjugation of chromophores leads to appearance of deep colors for all perimidine ligands. This is accompanied with the appearance of the *n* ⟶ *π* ^*∗*^ band in the middle of the visible region. VO(II) complex spectra display intraligand transitions suffer shift due to coordination. The appearance of charge transfer bands attributes to O ⟶ V and N ⟶ V transitions. Also, new significant d-d transition bands were observed at ≈15,300 and 12,800 cm^−1^ assigned for ^2^B~2~g ⟶ ^2^B~1~g (*E* ~2~, *υ* ~2~) and ^2^B~1~g ⟶ ^2^Eg (*E* ~1~, *υ* ~1~), respectively. These bands are attributed to transition inside the square-pyramidal configuration ([Figure 4](#fig4){ref-type="fig"}). Reduced magnetic moment values (*μ* ~eff~ = 1.65--1.68 BM) recorded for all complexes support the proposal of binuclear complexes \[[@B33]\]. 3.4. ESR Spectra {#sec3.4} ---------------- ESR spectra ([Figure 5](#fig5){ref-type="fig"}, for example) of VO(II) solid complexes were obtained and investigated to verify the structural forms of them. All spectra demonstrated an eight-line pattern, which attributes to the analogous and vertical ingredients g-tensors and hyperfine (hf) A-tensors. Spin Hamiltonian parameters and molecular orbital values were calculated and are represented in [Table 4](#tab4){ref-type="table"}. The analogous and vertical ingredients are well resolved. Nitrogen super-hyperfine splitting is not observed, which points to the presence of single electron in the *d* ~xy~ orbital. The pattern suggests that *g* and *A* are axially symmetric in nature. The factors *A* and *g* appear to be in covenant with the values commonly known for vanadyl complex in the square-pyramidal geometry. *G* factor, which is expressed by *G* = (*g* ~//~ ~ ~− 2.0023)/(*g* ~⊥ ~− 2.0023) = 4, measures the exchange interaction between metal centers. In agreement with Hathaway \[[@B34], [@B35]\], *G* \> 4 shows negligible exchange interaction, while *G* \< 4 is the vice versa. An observable reduction of the values calculated (1.71--2.79) proposes strong interaction inside binuclear complexes \[[@B36], [@B37]\]. This interaction affects the magnetic moment value of complexes which suffers observable reduction. The tendency of *A* ~11~ to decrease with increasing *g* ~11~ is an index for tetrahedral distortion (*f*=*g* ~//~/*A* ~//~) \[[@B38]--[@B40]\]. The molecular orbital coefficients *α* ^2^ and *β* ^2^ are calculated by$$\begin{matrix} {\beta^{2} = \frac{7}{6}\left( {- \frac{A_{11}}{P} + \frac{A_{\bot}}{P} + g_{11} - \frac{5}{14}g_{\bot} - \frac{9}{14}g_{\text{e}}} \right),} \\ {\alpha^{2} = \frac{2.0023 - \Delta g}{8\beta^{2}\lambda},\quad\text{where  }\Delta g = \left( {g_{\bot} - g_{||}} \right) \times 10^{- 3}.} \\ \end{matrix}$$ The hyperfine conjunction disciplinarians were calculated by taking A~//~ and A~⊥~ as negative, which gave positive values of *β* ^2^ and *α* ^2^. The calculated *α* ^2^ and *β* ^2^ values introduce the highly ionic character of in-plane *σ*- and *π*-bonding. The electronic transition spectra display two significant bands at ≈15,300 and 12,800 cm^−1^ assigned for ^2^B~2~g ⟶ ^2^B~1~g (*E* ~2~, *υ* ~2~) and ^2^B~1~g ⟶ ^2^Eg (*E* ~1~, *υ* ~1~), respectively. Assume pure d-orbitals by using first- and second-order perturbation theories. The parameters attributing to transition energy are called the spin Hamiltonian parameters and calculated by the following expression: *g* ~⊥~ = *g* ~e~ − (2*λ*/*E* ~2~), where *g* ~e~ is the free-electron *g* value (2.0023). Using *E* ~2~ value, the spin-orbital coupling constant (*λ*) was evaluated (138.18). A value for *λ* of 250 cm^−1^ is reported \[[@B41]\] for free V^+4^ ion. The high reduction in the magnitude of *λ* for the double-bonded oxovanadium complex (V=O)^+2^ is attributed to substantial *π*-bonding. However, the value falls inside the logical borders announced. The orbital reduction factors, namely, *K* ~//~ and *K* ~⊥~, are also calculated using ^2^ *K* ~//~ = (*g* ~//~ − 2.00277) *E*/8 *λ* and ^2^ *K* ~⊥~ = (*g* ~⊥~ − 2.00277) *E*/2 *λ*. For pure *σ*-bonding, *K* ~//~ ≈ *K* ~⊥~ ≈ −0.77, while ^2^ *K* ~// ~\> ^2^ *K* ~⊥~ signifies in-plane *σ*-bonding, with ^2^ *K* ~⊥ ~\< ^2^ *K* ~//~ accounting for out-of-plane *π*-bonding \[[@B42]\]. ### 3.4.1. Calculation of Dipole Term (*p*) {#sec3.4.1} Dipolar term values can be determined by$$\begin{matrix} {p = \frac{7\left( {A_{11} - A_{\bot}} \right)}{6 + \left( {3/2} \right)\left( {\lambda/E_{1}} \right)}.} \\ \end{matrix}$$ If *A* ~11~ is taken to be negative and *A* ~⊥~ positive, the value of *p* will be more than 270 *G*, which is far from the expected value. Thus, the signs of both *A* ~11~ and *A* ~⊥~ are used as negative and are indicated in the form of the isotropic hf constant (*A* ~o~). McGarvey theoretically accomplished the *p* value as +136 *G* for vanadyl complexes which does not deviate much from the expected value. ### 3.4.2. Calculation of MO Coefficients and Bonding Parameters {#sec3.4.2} The *g* values observed are different from the electronic value (2.0023). This assigns to spin-orbit interaction of the *d* ~xy~ ground state level. The isotropic and anisotropic *g* and *A* parameters were calculated using the following equations: *A* ~o~ = (*A* ~//~ + 2*A* ~⊥~)/3 and *g* ~o~ = (*g* ~//~ + 2*g* ~⊥~)/3. Taking *A* ~11~ and *A* ~⊥~ to be negative values, the *K* expression is *K* = −(*A* ~o~/*p*) − (*g* ~e~ ~ ~− *g* ~o~). Thus, *K* (Fermi contact term) can be determined. The Fermi contact term, *k*, is a sense of polarization exerted by the uneven apportionment of d-electron density on the inner core s-electron. 3.5. X-Ray Diffraction {#sec3.5} ---------------------- X-ray diffraction patterns were executed over 10° \< 2*θ* \< 90° range (Figures [6](#fig6){ref-type="fig"} and [S3](#supplementary-material-1){ref-type="supplementary-material"}) for ligands under study. This technique gives a considerable view about dynamics of the crystal lattice in solid compounds. Using standard methods, a comparative study of patterns with reactants reflects the purity of isolated compounds \[[@B43]\]. Also, significant parameters related to crystalline compounds can be calculated using the high-intense peak (full width at half maximum (FWHM)). The crystallinity appearing with the LH ligand reflects the isolation of a strictly known irregular crystallite, while the amorphous appearance of others reflects the indiscriminate orientation of atoms inside the 3D space. 2*θ* (21.18), *d* spacing (4.1910), FWHM (0.2454), relative intensity (%) (857), and particle size (6.003 *Ε*) were calculated for LH compounds. The crystallite size was computed by utilizing the Debye--Scherrer equation: *β* = 0.94 *λ*/(S cos *θ*), where *S* is the crystallite size, *θ* is the diffraction angle, *β* is FWHM, and Cu/K*α* (*λ*) = 1.5406 Å. The d-spacing between inner crystal planes was extracted from the Bragg equation: *nλ* = 2dsin(*θ*) at *n* = 1. The size calculated falls in the nanometer range (nm) which expects a widespread application especially for the biological field. Also, crystal strain (*ε*, 5.027) was calculated by *β* = (*λ*/*S*  cos  *θ*) − *ε*  tan  *θ*, while dislocation density (*δ*, 0.0277) was computed by *δ* = 1/*S* ^2^ \[[@B44]\]. The dislocation density and strain are the aspects for network dislocation in compounds. The lower values of them indicate high quality of compounds. The SEM tool is used to give a clear view about the habit and surface morphology of all studied compounds ([Figure S4](#supplementary-material-1){ref-type="supplementary-material"}). The images of paramagnetic compounds are not strictly resolved because an insufficient electron beam can meet the surface to provide well resolution. Subsequently, the determination of particle size in an accurate way is strongly absent. It was known about this study that the crystals were grown up from just a single one to several accumulated distributables with particle sizes starting with few nanometers to many hundreds. The formation of extended crystals over a rocky shape may happen by two nucleation processes: by distribution and by piling up of layers which are grown. It was pointed to that if the rate of growth along the *C*-axis is fast and a great number of grown nuclei are active across the axis in comparison with vertical to the *C*-axis, the crystals will be extended over patches \[[@B45]\]. The attitude displayed on different crystals may be due to the growth along the strongest bond through anisotropy included in crystal structures. When the amount exceeds to a certain limit, the result is evolution of plates and rock shapes. It is credible to assume that the environmental conditions change the nature and shape of the morphology. Moreover, the rock and plates shaping compounds may have excellent activity towards different applications due to their broad surface area \[[@B46]\]. The homogeneous morphology observed indicates the obtained strict-defined crystals are free from metal ions on the external surface. 3.6. Thermal Study {#sec3.6} ------------------ The degradation behaviors of all perimidine compounds and their VO(II) complexes were tested. The proposed degradation insights corresponding to all decomposition stages are tabulated ([Table 5](#tab5){ref-type="table"}). The treated perimidine compounds start their successive decomposition at low temperature (≈60°C) in three stages. A sequenced complete degradation of the organic compound was recorded with or without carbon atoms residue. VO(II)-perimidine complexes display an observable thermal stability for the organic compounds coordinated. The degradation stages varied in between three and four stages. The first degradation process starts at 40--80°C temperature range which starts with the removal of water molecules and is followed by decomposition of the coordinating ligand. Variable residue was proposed with the complexes degradation process but all agree with the presence of biatomic metals. An acceptable conformity between calculated and found weight losses percentage may reflect the exact determination of stage borders. 3.7. DNA Binding {#sec3.7} ---------------- Appling the spectrophotometric titration method, the binding mod of perimidine derivatives towards CT-DNA was investigated. Electronic absorption of freshly prepared solutions was obtained at 25°C over 200--800 nm range, with a reference solution for each concentration. Scanned solutions include fixed ligand concentration (2 × 10^−5^ M) with a regular increase of DNA added. The effective binding constant for the interaction of the organic derivatives with DNA was obtained based on observable changes in absorption at 418, 420, 420, 385, and 410 nm for LH, LOMe, LNO~2~, L^4^, and L^5^, respectively. A regular increase of DNA amount added to the ligand solution leads to the bathochromic effect for the significant ligand band assigned for transition inside interacting groups. This band is minimized gradually as appeared clearly with the aggregated spectra for each derivative. This minimization is followed by appearance of the slightly shifted peak (1-2 nm) from the free ligand peak, which assigns for the binding complex and suffers a gradual increase in absorbance. This is considered as a sufficient indicator of coupled DNA helix stabilization, after the interaction process. Such an investigation suggests the coupling for binding sites through electrostatic attraction or occluded in major and minor grooves inside DNA. Also, the bathochromic effect can be investigated and explained based on two bases: broad surface area of perimidine molecules and the presence of planar aromatic chromophore, which facilitate well binding towards CT-DNA. This groove binding leads to structural reorganization of CT-DNA. This requires a partial disassembling or deterioration of double helix at the exterior phosphate, which leads to formation of cavity suitable for entering compounds \[[@B47]\]. The bathochromic feature observed is directly proportional to electron withdrawing character for substituents and their position. The binding constants (*K* ~b~) for the five derivatives were calculated by known spectral relationships \[[@B19]\] for L^1^, L^2^, L^3^, L^4^, and L^5^ as 6.10 × 10^4^, 6.07 × 10^4^, 6.75 × 10^4^, 7.99 × 10^4^, and 8.80 × 10^4^ M^−1^. According to Hammett\'s constants (*σ* ~R~), essential correlation against intrinsic constants will be conducted ([Figure 7](#fig7){ref-type="fig"}), and the relation verifies the direct relation in between \[[@B48]\]. 3.8. Computational {#sec3.8} ------------------ ### 3.8.1. DFT/Hartree--Fock Study {#sec3.8.1} Applying the Gaussian 09 software, the optimization process was executed over all new compounds till reaching the best configuration. A known standard method was used for this purpose. Essential parameters will be extracted from the energy levels of frontiers (HOMO and LUMO). The energy gap between *E* ~HOMO~ and *E* ~LUMO~ will give an excellent view about the character of the tested compound. The biological behavior and the ligational mode are most significant features concluded. The frontier images of perimidine ligands and their VO(II) complexes are shown in Figures [8(a)](#fig8){ref-type="fig"} and [8(b)](#fig8){ref-type="fig"}, respectively. HOMO-level images display the concentration over the perimidine ring which includes two donor centers, while the LUMO-level images display the concentration over the other side in molecules including the other two coordination sites. This view introduces a good electron relocation between donor atoms which smoothens the donation of coordinating centers. On the other side, the two levels in VO(II) complexes represent the concentration around the two central atoms mainly. This may offer the good role of VO atoms in the application feature interested in this research. This may happen through the smooth charge transfer process that includes the complexes. Electronegativity (*χ*), chemical potential (*μ*), global hardness (*η*), global softness (*S*), global electrophilicity index (*ω*), and absolute softness (ϭ) were calculated by using known standard equations \[[@B49], [@B50]\]. Toxicity and reactivity of compounds can be clarified by using the electrophilicity index (*ω*) value. This index gives a clear insight about the expected biological attitude of tested perimidine compounds in comparison with their VO(II) complexes and, also, measures the firmness of the compound which takes an extra negative charge from the environment. Also, the firmness and reactivity of compounds can be tested from two opposite indexes (*η* and ϭ) \[[@B42], [@B51]\]. *(1) Some Quantum Parameters*. Some important quantum parameters are calculated for all treated compounds attributing to frontier energy gaps and are displayed in [Table 6](#tab6){ref-type="table"}. The computed results of ligands introduce the following notices: (i) the degree of converged softness recorded for perimidines offers their compatible flexibility towards the coordination. (ii) Electrophilicity index (*χ*) and electronic chemical potential index (*μ*) have two different signs. This is evidence for the ability of compounds to acquire electrons from the surrounding by the following order: L^4^ \> L^3^ \> L^5^ \> L^1^ \> L^2^ ligands. This arrangement agrees by an excellent way with the priority of electron withdrawing substituents (Cl and NO~2~) in para position which facilitates the compound electron affinity. Whenever, the extracted data assigning for VO(II) complexes introduce the following observations: (i) frontier energy gaps are completely minimized from original perimidines leading to red shift inside electronic transitions. Such a behavior may clarify the effect of metal atoms (vanadyl) in stabilizing the compounds. This reduction is preferable in biological attitude of compounds \[[@B52]\]. (ii) The absolute softness values in complexes were enhanced than the ligand values which predicated their high biological activity. From calculated energy gaps, Hammett\'s relation displays a significant effect of the p-substituent on *δE* values of ligands or their complexes by two reverse features ([Figure 9](#fig9){ref-type="fig"}). *(2) Some Log File Parameters*. Essential log file data are summarized and presented in [Table 7](#tab7){ref-type="table"}. The allowed data are varied in between the free ligands and their complexes, due to the difference in methods used for the treatment. A suitable method used for the organic ligand appeared unsuitable for its VO(II) complex. A comparative investigation introduced the following notices: (i) a general reduction in the charges computed for coordinating atoms (O^19^, N^11^, N^15^, and N^16^). This is due to their participation in coordination with VO(II) atoms. (ii) The computed bond lengths appearing with four perimidine derivatives are comparable with each other except for the L^3^ ligand. This displays the inductive effect of the p-substituent (nitro group) on the elongation of bond lengths attributing to the affected function groups. (iii) Oscillator strength values (range 0--1) of complexes are commonly minimized than those of their corresponding ligands. This may indicate the effect of the metal atom (vanadyl) in facilitating the absorption or reemission of electromagnetic radiations inside complex molecules \[[@B53]\]. The values are close to zero and not 1; this may suggest low excitation energy values needed for electronic transitions. (iv) Also, an increase in dipole moment values of complexes over ligands indicates high polarity of covalent bonds surrounding two central atoms except for the L^2^ complex. This may refer to the significant difference between all substituents from the methoxy group which has electron-donating feature in opposite with the others \[[@B54]\]. ### 3.8.2. QSAR Calculations {#sec3.8.2} Using the HyperChem (v8.1) program, essential QSAR parameters are calculated and tabulated ([Table 8](#tab8){ref-type="table"}). This computation gives a clear view about some statistics belonging to coordinating agents. Log *P* value is an indication for the biological feature of the tested compound by a reverse relation \[[@B55]\]. The values are arranged by the following order: L^1^ (2.53) \> L^4^ = L^5^ (2.31) \> L^2^ (1.53) \> L^3^ (−1.64). Partition coefficient (log *P*) values introduce a distinguish biological activity may appear with the L^3^ ligand. ### 3.8.3. Docking Computation {#sec3.8.3} Simulation technique is a new revolution process served in different applications. Drug design is a complicated process that needs significant facilities to establish a view about the expected efficiency of proposed drugs. In last decades, the docking computation process between the proposed drug (inhibitor) and the infected cell proteins is the concern in drug industrial research. AutoDockTools 4.2 software was used for this approach. 4c3p, 3bch, and 4zdr are the BDP files for breast, colon, and liver cancer cell proteins which are used for the docking process with five perimidine derivatives. The extracted energies over PDB files (a format using the Gaussian 09 software) are presented in [Table 9](#tab9){ref-type="table"}. Scanning for the energy values introduces the following observations: (i) there is no interaction observed with the five inhibitors towards 3bch colon cancer protein. (ii) The degree of interaction towards breast colon protein (4c3p) is arranged as L^5^ \> L^1^ \> L^2^ \> L^4^, while the arrangement towards 4zdr (liver cancer protein) is as L^5^ \> L^1^ \> L^4^ \> L^3^ \> L^2^. This result displays the priority of L^5^ and L^1^ ligands in the inhibition process towards breast and liver carcinoma cell lines through a strong interaction (Figures [10](#fig10){ref-type="fig"} and [S5](#supplementary-material-1){ref-type="supplementary-material"}) \[[@B55]\]. Dissociation constant (p*K* ~a~) calculated is considered the biopharmaceutical measure of drug-likeness compounds. This constant helps in understanding the ionic form of the drug along the pH range. High p*K* ~a~ values (\>10) reflect their ionization which facilitates their diffusion across the cell membrane to give a well inhibition process. Also, highly reduced energies were recorded for 4c3p and 4zdr receptors. Positive sign of electrostatic energy recorded clarifies high stability of interacting complexes. HP plots (Figures [11](#fig11){ref-type="fig"} and [S6](#supplementary-material-1){ref-type="supplementary-material"}) as well as 2D plots ([Figure S7](#supplementary-material-1){ref-type="supplementary-material"}) display prolonged H-bonding appearing with L^5^ and LH ligands. This verifies the degree of interaction proposed on extracted energies. Also, high surface area recorded with breast or liver cancer protein complexes introduces a good degree of H-interaction. And hp, 2D, and surface area data verify the absence of interaction recorded with colon cancer protein. 3.9. Antitumor Efficiency {#sec3.9} ------------------------- The results obtained by screening all prepared compounds for comparison confirmed that the complexes exhibit more cytotoxicity against HepG2, MCF-7, and HCT116 *in vitro*. The IC~50~ values are displayed in [Table 10](#tab10){ref-type="table"} and in Figures [12](#fig12){ref-type="fig"} and [S8](#supplementary-material-1){ref-type="supplementary-material"}. Cells were treated with various concentrations of compounds and incubated for 48 h \[[@B56]\]. Cytotoxicity is considered as a good anticancer parameter if the influence induced apoptotic pathways inside the cell. Apoptotic may be detected by many parameters like the activation of caspase family, DNA fragmentation, or morphology of the cell. The sample L^4^ + VO(II) was the best impacted complex on liver, breast, and colon cancer cell lines with IC~50~ values of 1.66, 3.42, and 1.27, respectively, while its relative ligand (L^4^) impact was moderate at all cancer types. Hence, these results showed that the present study\'s effort to improve and enhance the effect of new complexes has achieved a clear, acceptable, and respectable success because the effect was enhanced for 15, 7, and 22 times, respectively, compared to the ligand with clear signs that it is going to be very close to the positive control. On the contrary, unfortunately, the same results were not detected in other complexes; they went in the contrary way: instead, they increased the impact they decreased it dramatically. What our results tell us clearly is that neither VO(II) nor ligands alone can act as an anticancer candidate drug, while only one complex can present that effect. So, the mechanism of action is not related to the ligand or to VO(II) itself, as far as related to the complex itself. 4. Conclusion {#sec4} ============= This paper presents new VO(II) complexes derived from a series of perimidine ligands. This study focuses on the effect of substituents on the chemistry and applicability of complexes. All the new compounds were well characterized by all possible tools. The complexes were found in a nanoscale comfortably. The different theoretical implementations gave a view about the biological feature of the investigated compounds in a comparative way. The docking process displays the high interaction of organic derivatives against breast cancer, while the experimental investigation displays the priority of the L^4^-VO(II) complex against all carcinomas tested. The binding efficiency of ligands towards CT-DNA was tested. Binding constant (*K* ~b~) values are in agreement with the electron-drawing character of the p-substituent which displayed high *K* ~b~ values. The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through General Research project under grant number (G.R.P-124-38). Data Availability ================= The data used to support the findings of this study are available from the corresponding author upon request. Conflicts of Interest ===================== The authors declare that they have no conflicts of interest regarding the publication of this paper. Supplementary Materials {#supplementary-material-1} ======================= ###### Figure S1: ^1^H·NMR spectra of L^4^ and L^5^ perimidine ligands. Figure S2: mass spectra of L^3^ and L^4^ ligands. Figure S3: X-ray patterns of four perimidine ligands. Figure S4: SEM images of perimidine ligands and their VO(II) complexes. Figure S5: interacting protein-inhibitor complexes for L^1^, L^2^, L^3^, L^4^, and L^5^ with 4c3p (1) and 3bch (2) receptors (A--E, respectively). Figure S6: interacting hp plot: LH, LOMe, LNO~2~, LClP, and LClM with 4c3p(1) and 3bch(2) receptors (A--E, respectively). Figure S7: 2D plot forms: L^1^, L^2^, L^3^, L^4^, and L^5^ with 4c3p (1), 3bch (2), and 4zdr (3) receptors (A--E, respectively). Figure S8: dose response curves of perimidine-VO(II) complexes against MCF-7, HCT116, and HepG2 cancer cells. ###### Click here for additional data file. {#sch1} {#fig1} {#fig2} {#fig3} {#fig4} {#fig5} {#fig6} {#fig7} {#fig8} {#fig9} {#fig10} {#fig11} {#fig12} ###### Significant analytical and physical data of perimidine compounds and their VO(II) complexes. Compounds (formula weight) (calcd./found) Color Elemental analysis (%) calcd. (found) ------------------------------------------------------- ------------ --------------------------------------- ------------- --------------- --------------------------- --------------- (**1**) (C~25~H~18~N~4~O) (L^1^) (390.44/390.42) Dark brown 76.91 (76.90) 4.65 (4.66) 14.35 (14.35) --- --- (**2**) \[(VO)~2~ (SO~4~)~2~ (L^1^)\]H~2~O (734.46) Dark brown 40.88 (40.88) 2.74 (2.73) 7.63 (7.65) 26.16 (26.16) 13.87 (13.88) (**3**) (C~26~H~20~N~4~O~2~) (L^2^) (420.47/420.44) Dark brown 74.27 (74.27) 4.79 (4.79) 13.32 (13.31) --- --- (**4**) \[(VO)~2~ (SO~4~)~2~ (L^2^)\]2H~2~O (782.51) Dark green 39.91 (39.90) 3.09 (3.10) 7.16 (7.17) 24.55 (24.54) 13.02 (13.03) (**5**) (C~25~H~17~N~5~O~3~) (L^3^) (435.44/435.42) Dark brown 68.96 (68.95) 3.93 (3.93) 16.08 (16.09) --- --- (**6**) \[(VO)~2~ (SO~4~)~2~ (L^3^)\]H~2~O (779.46) Dark green 38.52 (38.52) 2.46 (2.46) 8.99 (8.97) 24.65 (24.66) 13.07 (13.05) (**7**) (C~25~H~17~N~4~OCl) (L^4^) (424.88/424.86) Dark brown 70.67 (70.66) 4.03 (4.02) 13.19 (13.18) 8.34 (8.35) --- (**8**) \[(VO)~2~ (SO~4~)~2~ (L^4^)\]H~2~O (768.90) Dark green 39.05 (39.05) 2.49 (2.48) 7.29 (7.28) 24.99 (25.02)/4.61 (4.63) 13.25 (13.26) (**9**) (C~25~H~17~N~4~OCl) (L^5^) (424.88/424.86) Dark brown 70.67 (70.68) 4.03 (4.05) 13.19 (13.18) 8.34 (8.35) --- (**10**) \[(VO)~2~ (SO~4~)~2~ (L^5^)\]3H~2~O (804.93) Dark brown 37.30 (37.31) 2.88 (2.88) 6.96 (6.95) 23.87 (23.88)/4.40 (4.41) 12.66 (12.67) ###### Significant IR spectral bands (cm^−1^) of perimidine compounds and their VO(II) complexes. Compounds *ν* ~NH,~ *ν* ~OH~ *δ* ~NH~ *υ* ~C=O~ *ν* ~C=N~ *ν* ~as(SO4)~ *ν* ~s(SO4)~ *δ*r(H~2~O), *δ*w(H~2~O) *ν* ~V=O~ *ν* ~M-O~ *ν* ~M-N~ ---------------------------------------------- -------------------- ---------- ----------- ----------- --------------- -------------- -------------------------- ----------- ----------- ----------- (**1**) (C~25~H~18~N~4~O) (L^1^) 3155 1473 1618 1518 --- --- --- --- --- --- (**2**) \[(VO)~2~ (SO~4~)~2~ (L^1^)\]H~2~O 3110, 3350 1470 1596 1514 1420 1142 765, 670 966 588 476 (**3**) (C~26~H~20~N~4~O~2~) (L^2^) 3177 1470 1597 1508 ---- --- --- --- --- --- (**4**) \[(VO)~2~ (SO~4~)~2~ (L^2^)\]2H~2~O 3100, 3372 1447 1592 1502 1411 1146 765, 697 1074 572 515 (**5**) (C~25~H~17~N~5~O~3~) (L^3^) 3150 1473 1620 1538 --- --- --- --- --- --- (**6**) \[(VO)~2~ (SO~4~)~2~ (L^3^)\]H~2~O 3105, 3382 1447 1616 1517 1411 1179 743, 697 966 600 508 (**7**) (C~25~H~17~N~4~OCl) (L^4^) 3160 1474 1614 1518 --- --- --- --- --- --- (**8**) \[(VO)~2~ (SO~4~)~2~ (L^4^)\]H~2~O 3100, 3420 1471 1624 1510 1434 1150 755, 637 985 610 550 (**9**) (C~25~H~17~N~4~OCl) (L^5^) 3150 1473 1620 1518 --- --- --- --- --- --- (**10**) \[(VO)~2~ (SO~4~)~2~ (L^5^)\]3H~2~O 3054, 3384 1446 1616 1512 1368 1140 754, 689 1074 589 508 ###### Electronic transitions of perimidine compounds and their VO(II) complexes. Compounds *μ* ~eff~ (BM) d-d transition bands (cm^−1^) Intraligand and charge transfer (cm^−1^) ---------------------------------------------- ---------------- ------------------------------- ------------------------------------------ (**1**) (C~25~H~18~N~4~O) (L^1^) --- --- 31,746; 26,316; 23,923; 18,868 (**2**) \[(VO)~2~ (SO~4~)~2~ (L^1^)\]H~2~O 1.66 15,290; 12800 35,714; 29,412; 25,641; 24,272; 17,857 (**3**) (C~26~H~20~N~4~O~2~) (L^2^) --- --- 38,168; 28,249; 23,810; 19,048 (**4**) \[(VO)~2~ (SO~4~)~2~ (L^2^)\]2H~2~O 1.68 15,393; 12750 37,037; 30,769; 26,316; 23,256; 17,544 (**5**) (C~25~H~17~N~5~O~3~) (L^3^) --- --- 36,364; 30,303; 26,316; 23,810; 19,157 (**6**) \[(VO)~2~ (SO~4~)~2~ (L^3^)\]H~2~O 1.66 15,873; 12830 37,037; 28,571; 26,667; 18,182 (**7**) (C~25~H~17~N~4~OCl) (L^4^) --- --- 31,746; 25,974; 18,587 (**8**) \[(VO)~2~ (SO~4~)~2~ (L^4^)\]H~2~O 1.67 15,385; 12,800 35,714; 30,303; 25,974; 24,390; 17,857 (**9**) (C~25~H~17~N~4~OCl) (L^5^) --- --- 31,250; 26,316; 24,390; 18,182 (**10**) \[(VO)~2~ (SO~4~)~2~ (L^5^)\]3H~2~O 1.65 15,873; 12780 35,714; 30,769; 25,000; 23,256; 18,868 ###### Spin Hamiltonian parameters of all VO(II) complexes (A and *p* x10^−4^). Complex *g* ~//~ *g* ~⊥~ *g* ~o~ *A* ~11~ *F* *A* ~⊥~ *A* ~o~ *G* *p* *k* ^2^ *K* ~//~ ^2^ *K* ~⊥~ *α* ^2^ *β* ^2^ --------- ---------- --------- --------- ---------- -------- --------- --------- ------ -------- ------- -------------- ------------- --------- --------- (**1**) 1.93 1.96 1.95 167 115.57 66 99.67 1.71 117.52 0.796 −0.843 −1.981 1.959 0.9357 (**2**) 1.94 1.97 1.96 170 114.12 71 104.00 1.93 115.19 0.861 −0.724 −1.512 1.490 0.9435 (**3**) 1.92 1.96 1.95 168 114.28 69 105.67 1.95 115.19 0.865 −0.961 −1.985 1.964 0.9243 (**4**) 1.94 1.98 1.97 171 113.45 73 105.67 2.79 114.00 0.895 −0.727 −1.055 1.033 0.9395 (**5**) 1.93 1.97 1.96 171 112.86 72 105.00 2.24 115.19 0.869 −0.841 −1.515 1.494 0.9318 ###### Estimated TG data of perimidine compounds and all VO(II) complexes. Compound Steps Temp. range (°C) Decomposed Weight loss; calcd. (found %) ------------------------------------- ---------------- --------------------------------- --------------------------------- ------------------------------- L^1^ 1^st^ 45.1--120.5 -\[C~6~H~6 ~+ N~2~\] 27.18 (27.16) 2^nd^ 122.2--410.1 -\[C~6~H~5 ~+ CO\] 26.92 (26.95) 3^rd^ 410.3--670.2 -\[C~8~H~7~N~2~\] 33.59 (33.54) Residue   4C 12.31 (12.35) \[(VO)~2~ (SO~4~)~2~ (L^1^)\]H~2~O 1^st^ 80.3--120.3 -\[H~2~O + SO~4~\] 15.53 (15.55) 2^nd^ 120.6--391.7 -\[SO~4 ~+ C~6~H~6 ~+ N~2~\] 27.53 (27.55) 3^rd^ 391.9--798.8 -\[C~19~H~12~N~2~\] 36.53 (36.50) Residue   V~2~O~3~ 20.41 (20.40) L^2^ 1^st^ 65.6--156.1 -\[C~6~H~5~OCH~3~\] 25.72 (25.71) 2^nd^ 156.6--299.9 -\[C~6~H~5 ~+ CO + N~2~\] 31.66 (31.69) 3^rd^ 301.0--663.2 -\[C~9~H~7~N~2~\] 34.05 (33.89) Residue   3C 8.57 (8.71) \[(VO)~2~ (SO~4~)~2~ (L^2^)\]2H~2~O 1^st^ 42.1--135.1 -\[2H~2~O + 2SO~4~\] 29.16 (29.16) 2^nd^ 136.1--270.1 -\[C~6~H~5~OCH~3 ~+ N~2~\] 17.40 (17.29) 3^rd^ 271.0--485.4 -\[C~6~H~5~\] 9.85 (9.94) 4^th^ 485.6--797.9 -\[C~13~H~7~N~2~\] 24.43 (24.45) Residue   V~2~O~3~ 19.15 (19.16) L^3^ 1^st^ 63.66--230.51 -\[C~6~H~6~ + NO~2~\] 28.50 (27.90) 2^nd^ 231.21--410.11 -\[C~7~H~5 ~+ CO + N~2~\] 33.33 (33.31) 3^rd^ 410.52--650.64 -\[C~11~H~6~N~2~\] 38.16 (38.79) \[(VO)~2~ (SO~4~)~2~ (L^3^)\]H~2~O 1^st^ 79.2--140.6 -\[H~2~O + SO~4~\] 14.63 (14.62) 2^nd^ 141.9--278.9 -\[SO~4~ + C~6~H~5 ~+ CO\] 25.81 (25.78) 3^rd^ 279.1--479.5 -\[NO~2 ~+ C~6~H~6 ~+ N~2~\] 19.52 (19.53) 4^th^ 480.11--798.8 -\[C~12~H~6~N~2~\] 22.86 (22.90) Residue   V~2~O~2~ 17.18 (17.17) L^4^ 1^st^ 64.65--145.46 -\[C~6~H~5~Cl + CO + N~2~\] 39.68 (39.68) 2^nd^ 145.68--326.78 -\[C~6~H~5 ~+ N~2~\] 24.74 (24.75) 3^rd^ 330.12--680.23 -\[C~12~H~7~\] 35.58 (35.57) \[(VO)~2~ (SO~4~)~2~ (L^4^)\]H~2~O 1^st^ 69.1--256.1 -\[H~2~O + C~6~H~5~Cl + SO~4~\] 29.48 (29.65) 2^nd^ 256.9--484.1 -\[SO~4~ + CON~2 ~+ C~6~H~5~\] 29.80 (29.81) 3^rd^ 484.6--789.4 -\[C~10~H~7~N~2~\] 20.18 (19.98) Residue   V~2~O~2 ~+ 2C 20.54 (20.56) L^5^ 1^st^ 62.3--169.6 -\[C~6~H~5 ~+ N~2~\] 24.74 (24.71) 2^nd^ 160.1--371.9 -\[C~6~H~5~Cl + CO + N~2~\] 39.68 (39.59) 3^rd^ 372.6--666.8 -\[C~9~H~7~\] 27.10 (27.19) Residue   3C 8.48 (8.51) \[(VO)~2~ (SO~4~)~2~ (L^5^)\]3H~2~O 1^st^ 42.1--266.3 -\[3H~2~O + C~6~H~5~Cl\] 20.70 (20.71) 2^nd^ 266.38--482.5 -\[2SO~4~ + CON~2 ~+ C~6~H~5~\] 40.41 (40.61) 3^rd^ 482.9--793.5 -\[C~8~H~7~N~2~\] 16.29 (16.36) Residue   V~2~O~2 ~+ 4C 22.60 (22.32) ###### Energy parameters (eV) using the DFT/B3LYP method of optimized structures. Compound *E* ~H~ *E* ~L~ (*E* ~H~ − *E* ~L~) *E* ~L~ − *E* ~H~ *x* *µ* *η* *S* (eV^−1^) *ω* ϭ --------------- ---------- ---------- --------------------- ------------------- ---------- ---------- ---------- -------------- ---------- ------------- L^1^ −0.17417 −0.07574 −0.0984 0.09843 0.124955 −0.12496 0.049215 0.024608 0.158628 20.31900843 L^1 ^+ VO(II) −0.20433 −0.19545 −0.0089 0.00888 0.19989 −0.19989 0.00444 0.00222 4.499551 225.2252252 L^2^ −0.17142 −0.07426 −0.0972 0.09716 0.12284 −0.12284 0.04858 0.02429 0.155307 20.58460272 L^2^ + VO(II) −0.20163 −0.19237 −0.0093 0.00926 0.197 −0.197 0.00463 0.002315 4.191037 215.9827214 L^3^ −0.21252 −0.05654 −0.156 0.15598 0.13453 −0.13453 0.07799 0.038995 0.11603 12.82215669 L^3^ + VO(II) −0.21881 −0.21008 −0.0087 0.00873 0.214445 −0.21445 0.004365 0.002183 5.267658 229.0950745 L^4^ −0.25291 −0.05654 −0.1964 0.19637 0.154725 −0.15473 0.098185 0.049093 0.121912 10.18485512 L^4^ + VO(II) −0.20433 −0.19545 −0.0089 0.00888 0.19989 −0.19989 0.00444 0.00222 4.499551 225.2252252 L^5^ −0.17808 −0.0842 −0.0939 0.09388 0.13114 −0.13114 0.04694 0.02347 0.183188 21.30379207 L^5^ + VO(II) −0.19719 −0.16607 −0.0311 0.03112 0.18163 −0.18163 0.01556 0.00778 1.060073 64.26735219 ###### Considerable bond lengths, charges, dipole moment (*D*), oscillator strength (*ʄ*), and excitation energies (*E*). Compound O^19^ N^11^ N^15^ N^16^ C^18^--O^19^ C^12^--N^11^ C^14^--N^15^ N^15^--N^16^ V^1^ V^2^ *D* (Debye) *E* (nm) *ʄ* --------------- ----------- ----------- ----------- ----------- -------------- -------------- -------------- -------------- ---------- ---------- ------------- ---------- -------- L^1^ −0.415473 −0.555221 −0.349927 −0.275635 1.224561 1.384229 1.287795 1.366391 --- --- 5.1769 567.81 0.0316 L^1^ + VO(II) −0.410181 −0.282766 −0.316313 −0.248429 --- --- --- --- 0.933201 0.929331 11.5667 16513.7 0.003 L^2^ −0.410237 −0.555363 −0.348134 −0.275746 --- --- --- --- --- --- 6.4963 576.26 0.0403 L^2^ + VO(II) −0.420038 −0.397120 −0.322648 −0.250261 --- --- --- --- 0.927552 0.915069 3.5504 31387.7 0.0008 L^3^ −0.416751 −0.044378 −0.050930 0.113237 1.317259 2.076019 1.772715 1.281712 --- --- 5.3595 7613.39 0.002 L^3^ + VO(II) −0.414856 −0.387699 −0.320321 −0.254947         0.955124 0.952544 16.6899 17266.3 0.0032 L^4^ −0.313357 −0.358945 −0.031267 −0.294059 1.223609 1.404653 1.286454 1.367672 --- --- 4.4684 315.92 0.4055 L^4^ + VO(II) −0.410181 −0.282766 −0.316313 −0.248429 --- --- --- --- 0.933201 0.929331 11.5667 16513.7 0.003 L^5^ −0.354631 −0.480428 −0.209492 −0.312115 1.224058 1.384450 1.286411 1.367122 --- --- 3.9661 600.17 0.043 L^5^ + VO(II) −0.405024 −0.408894 −0.304735 −0.210573 --- --- --- --- 0.905245 0.730601 8.9706 20988.6 0.002 ###### QSAR computation for optimized structures of perimidine compounds. Function L^1^ L^2^ L^3^ L^4^ L^5^ ------------------------------- --------- --------- --------- --------- --------- Surface area (approx.) (Å^2^) 425.73 488.36 496.79 464.04 465.53 Surface area (grid) (Å^2^) 623.02 661.15 661.91 644.29 642.82 Volume (Å^3^) 1060.57 1138.87 1134.04 1105.99 1105.72 Hydration energy (kcal/mol) −8.29 −9.97 −17.83 −8.00 −8.02 Log P 2.53 1.53 −1.64 2.31 2.31 Reactivity (Å^3^) 132.87 139.25 138.92 137.59 137.59 Polarizability (Å^3^) 45.32 47.80 47.62 47.25 47.25 Mass (amu) 390.44 420.47 436.45 424.89 424.89 ###### Docking energy values (kcal/mol) of perimidine compounds (HL) and protein receptors complexes. Ligands p*K*a Receptor Est. free energy of binding Est. inhibition constant (*K* ~i~) (*µ*M) vdW + bond + desolving energy Electrostatic energy Total intercooled energy Frequency Interacting surface --------- --------- ---------- ----------------------------- ------------------------------------------- ------------------------------- ---------------------- -------------------------- ----------- --------------------- L^1^ 10.96 4c3p −7.92 1.57 −9.29 −0.06 −9.34 30% 859.778 3bch +355.37 --- +349.42 +0.06 +349.48 10% 665.36 4zdr −4.72 345.45 −5.97 −0.05 −6.02 20% 723.695 L^2^ 10.96 4c3p −7.75 2.09 −9.13 −0.13 −9.26 10% 983.377 3bch +490.76 --- +473.39 +0.00 +473.39 10% 662.71 4zdr −4.32 686.85 −5.97 −0.02 −5.99 20% 758.018 L^3^ 10.95 4c3p +647.56 --- +644.74 +0.01 +644.75 10% 718.318 3bch +709.10 --- +699.61 −0.05 +699.56 10% 661.43 4zdr −4.66 385.21 −6.46 +0.07 −6.39 10% 621.389 L^4^ 10.96 4c3p −7.28 4.62 −8.57 −0.03 −8.60 20% 929.747 3bch +552.25 --- +549.59 −0.03 +549.56 30% 710.605 4zdr −4.67 376.04 −6.13 −0.19 −6.32 20% 595.541 L^5^ 10.95 4c3p −8.41 683.74 −9.81 −0.00 −9.81 20% 925.161 3bch +663.87 --- +661.94 +0.01 +661.95 10% 703.598 4zdr −4.84 284.49 −6.39 +0.04 −6.35 10% 690.838 ###### IC~50~ of some tested compounds against liver (HepG2), breast (MCF-7), and colon (HCT116) cancer cell lines. Cell type IC~50~ (*µ*g/ml) ----------- ------------------ ------- ------- ------- ------- ------- ------- ------ ------- ------- ------ MCF-7 19.68 23.06 25.92 93.92 15.50 \>100 24.96 3.42 11.44 \>100 0.60 HepG2 19.79 19.94 27.23 55.67 11.01 \>100 28.25 1.27 9.91 \>100 0.34 HCT116 19.15 22.93 13.27 95.17 15.53 \>100 26.24 1.66 23.30 \>100 0.39 [^1]: Academic Editor: Spyros P. Perlepes

Background {#Sec1} ========== During the past few decades, fuzzy technique has been widely used in nonlinear system modelling, especially for systems with incomplete plant information. Thus, a number of significant results have been reported to solve the different problems of fuzzy systems, such as stability analysis (Chadli et al. [@CR2]; Su et al. [@CR19], [@CR21]), filter design (Shi et al. [@CR17]; Su et al. [@CR20], [@CR22]), robust control (Mourad et al. [@CR13]; Tian et al. [@CR26]), etc. Among these conclusions, it is worth mentioning that, the problem of Hankel-norm output feedback controller design for T--S fuzzy stochastic systems have been investigated in Su et al. ([@CR21]). For a class of T--S fuzzy switched systems with stochastic perturbation, the dissipativity-based filtering problem was considered in Shi et al. ([@CR17]). In addition, the fault detection filtering problem have been solved for nonlinear switched stochastic system in the T--S fuzzy framework in Su et al. ([@CR22]). Recently, a wider class of fuzzy systems that are described by the singular form have been studied, where the model is the extended of T--S fuzzy systems (Taniguchi et al. [@CR23]). It is known that a singular model can describes a practical system better than a standard dynamic model. So fuzzy singular model provides a new way to the analysis and synthesis of the nonlinear singular system and can be found in many applications, because it can combine the flexibility of fuzzy logic theory and fruitful linear singular system theory into a unified framework to approximate complex nonlinear singular systems, for details see Fridman ([@CR3]) and Lin et al. ([@CR9]). Meanwhile, time delays always exist in many dynamical systems and delays are the sources of poor stability and deteriorated performance of a system. Therefore, lots of stability analysis and control synthesis results (Wang et al. [@CR27]; Mourad et al. [@CR13]; Huang [@CR6]; Han et al. [@CR4]; Zhang et al. [@CR33]) have been reported for T--S fuzzy singular systems with time-delay. It should be pointed out that almost all of the existing results on fuzzy systems with time delays, the maximum allowable delay bound has been used as an important performance index for measuring the conservatism of the obtained conditions. On the other hand, in order to reduce the conservativeness of the delay-dependent criteria for fuzzy systems, input--ouput approach (Su et al. [@CR19]; Zhao et al. [@CR35]), delay partitioning method (Yang et al. [@CR31]; Xia et al. [@CR28]), convex combination technique (Su et al. [@CR20]; An and Wen [@CR1]; Peng and Fei [@CR15]; Park et al. [@CR14]), and free weighting matrices approach (Souza et al. [@CR18]; Liu et al. [@CR10]; Tian et al. [@CR26]) have been well used. The most noteworthy is the delay partitioning approach: the delay interval is divided into multiple uniform or non-uniform segments. It has been proved that less conservative results may be expected with the increasing of delay-partitioning segments. Recently, by non-uniformly dividing the time delay into multiple segments, An and Wen ([@CR1]) has established less conservative delay-dependent stability criteria than those in Li et al. ([@CR7]) using a convex way for uncertain T--S fuzzy systems with interval time-varying delay. Based on the input--output technique and delay partitioning approach, some new stability conditions of discrete-time T--S fuzzy systems with time delays have been proposed by applying scaled small-gain theorem in Su et al. ([@CR19]) , while an induced $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ell _{2}$$\end{document}$ performance is guaranteed. On the basis of delay-partitioning approach and new integral inequality established by reciprocally convex approach in Park et al. ([@CR14]) and Peng and Fei ([@CR15]) has developed less conservative stability criteria than those in Peng et al. ([@CR16]), Lien et al. ([@CR8]) and Tian and Chen ([@CR24]) for uncertain T--S fuzzy delay system. However, an important characteristic of fuzzy singular systems is the possible impulse behavior, which is harmful to the physical system and is undesired in system control. It's means that the aforementioned delay partitioning approach and the obtained results can not be directly applied to fuzzy singular system with additional algebraic constraints, because it requires considering not only stability, but also regularity and impulse-free conditions. Therefore, the motivation of this study is mainly focus on how to improve the delay partitioning approach and reduce the conservativeness of existing results for fuzzy singular systems because of its theoretical and practical significance. More recently, some research works on stability analysis (Mourad et al. [@CR13]; Zhang et al. [@CR33]; Chadli et al. [@CR2]; Wang et al. [@CR27]) and controller design (Zhu et al. [@CR36]; Ma et al. [@CR12]; Zhao et al. [@CR34]; Han et al. [@CR4]) have been extended for T--S fuzzy singular systems with time-varying delay. In Zhang et al. ([@CR33]), the problems of delay-dependent stability and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$H_{\infty }$$\end{document}$ control were discussed utilizing model transformation techniques, but model transformation may lead to considerable conservativeness. In Han et al. ([@CR4]), the problems of sliding mode control for fuzzy descriptor systems were presented using delay partitioning approach, but the time-delay is constant. Using free-weight matrix method, Mourad et al. ([@CR13]) discussed the problems of delay-dependent stability and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$L_{2}-L_{\infty }$$\end{document}$ control, however, the free-weighting matrices may be redundant and increase the computational burden in case of stability analysis for deterministic delay systems. In Chadli et al. ([@CR2]), by using quadratic method, sufficient conditions on stability and stabilization were proposed in terms of LMI for uncertain T--S fuzzy singular systems. Based on delay partitioning approach, some less conservative stability and stabilization criteria for fuzzy singular systems with time-varying delay have been investigated in Wang et al. ([@CR27]). In Ma et al. ([@CR12]), a delay-central-point method was presented to develop less conservative delay-dependent conditions for memory dissipative control for fuzzy singular time-varying delay systems under actuator saturation. It is well-known that the challenges of deriving a less conservative result are to construct an appropriate LKF that includes more useful state information and to reduce the enlargement in bounding the derivative of LKF as much as possible. Inspired by the methods mentioned above, when revisiting the stability problem for T--S fuzzy singular systems with interval time-varying delay, we find that the existing works still leave plenty of room for improvement on the reduction of conservatism for the following reasons. (1) All the given stability conditions in Han et al. ([@CR4]), Mourad et al. ([@CR13]), and Wang et al. ([@CR27]) are not all in strict LMIs form due to equality constraints, which cannot be solved directly using standard LMI procedures; (2) In Wang et al. ([@CR27]), the integral item $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$-\int _{t-\tau _{2}}^{t-\tau _{1}}{\dot{x}}^{T}(s)E^{T}RE{\dot{x}}(s)ds$$\end{document}$ is directly magnified as $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$-(\tau _{2}-\tau (t))\int _{t-\tau _{2}}^{t-\tau (t)}{\dot{x}}^{T}(s)E^{T}RE{\dot{x}}(s)ds-(\tau (t)-\tau _{1})\int _{t-\tau (t)}^{t-\tau _{2}}{\dot{x}}^{T}(s)E^{T}RE{\dot{x}}(s)ds$$\end{document}$, some useful time-varying delay-dependent integral items are ignored in the derivation of results; (3) More free-weighting matrices are employed to deduce the stabilization results in Yang et al. ([@CR31]) and Mourad et al. ([@CR13]), which have not considered the gain variations might be caused by the inaccuracies of controller implementation. The objective of this paper is to revisit the delay-dependent stability analysis and give new stabilization criteria by improved delay partitioning approach. The main contributions of this paper lie in that, firstly, by seeking an appropriate $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$, a maximum admissible upper bound of the time delay can be obtained for T--S fuzzy singular systems with interval time-varying delay. The tunable parameter $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$ which divide $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[\tau _{1},\tau _{2}]$$\end{document}$ into two variable subintervals plays a crucial role in reducing the conservativeness of stability conditions. Secondly, new LKF is established by partitioning time delay $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[0,\tau _{1}]$$\end{document}$ into *N* segments, and the time-varying delay $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x(t-\frac{n}{N}\tau _{1})$$\end{document}$ is included in the LKF, which takes fully account of the relationship between the state vectors $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x(t-\frac{n}{N}\tau _{1})$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x(t-\tau _{\rho })$$\end{document}$. Thirdly, some new results on tighter bounding inequalities have been employed to reduce the enlargement in bounding the derivative of LKF when designing the controller with linear fractional uncertainties. Then, the newly developed conditions of stability and stabilization are expected to be less conservative than the previous ones. The rest of this paper is organized as follows. The system description and some useful lemmas are presented in "[Problem formulation](#Sec2){ref-type="sec"}" section. In "[Main results](#Sec3){ref-type="sec"}" section, we show the results on stability conditions and fuzzy controller design scheme. In "[Numerical examples](#Sec6){ref-type="sec"}" section, several numerical examples are given to demonstrate the effectiveness and merits of the proposed methods. Finally, a brief conclusion is drawn in "[Conclusion](#Sec5){ref-type="sec"}". Notations: Throughout the paper, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathbb{R}}^{n}$$\end{document}$ denotes the n-dimensional real Euclidean space; *I* denotes the identity matrix; the superscripts *T* and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$-1$$\end{document}$ stand for the matrix transpose and inverse, respectively; notation $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$X>0(X\ge 0)$$\end{document}$ means that matrix *X* is real symmetric positive definite (positive semi-definite); $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Vert \cdot \Vert$$\end{document}$ is the spectral norm. If not explicitly stated, all matrices are assumed to have compatible dimensions for algebraic operations. The symbol "$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$*$$\end{document}$" stands for matrix block induced by symmetry. Problem formulation {#Sec2} =================== Consider a class of nonlinear singular system with interval time-varying delay, which can be represented by the following extended T--S fuzzy singular model:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \left\{ \begin{aligned} &E{\dot{x}}(t)=\sum _{i=1}^{r}\mu _{i}(\xi (t))\{ (A_{i}+\Delta A_{i}(t))x(t)+(A_{\tau i}+\Delta A_{\tau i}(t))x(t-\tau (t))+B_{i}u(t)\} \\ &x(t)=\sum _{i=1}^{r}\mu _{i}(\xi (t))\phi _{i}(t), \quad \forall t\in [-\tau _{2},0]. \end{aligned}\right. \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x(t)\in {\mathbb{R}}^{n}$$\end{document}$ is the state vector, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u(t)\in {\mathbb{R}}^{m}$$\end{document}$ is the control input vector. The fuzzy basis functions are given by $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu _{i}(\xi (t))=\beta _{i}(\xi (t))/\sum _{j=1}^{r}\beta _{j}(\xi (t))$$\end{document}$ with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta _{i}(\xi (t))= \prod _{i=1}^{p}M_{ij}(\xi (t))$$\end{document}$, where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$M_{ij}$$\end{document}$ is fuzzy sets, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$M_{ij}(\xi _{j}(t))$$\end{document}$ represents the grade of membership of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\xi _{j}(t)$$\end{document}$ in $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$M_{ij}$$\end{document}$. Here, it is easy to find that $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta _{i}(\xi (t))\ge 0, (i=1,2,\ldots ,r)$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sum _{j=1}^{r}\beta _{j}(\xi (t))>0$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu _{i}(\xi (t))\ge 0, (i=1,2,\ldots ,r)$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sum _{j=1}^{r}\mu _{j}(\xi (t))=1$$\end{document}$ for all $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t>0$$\end{document}$, *r* is the number of IF--THEN rules. $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\xi _{1}(t),\ldots ,\xi _{p}(t)$$\end{document}$ are the premise variables, which do not depend on the input variable *u*(*t*). $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi _{i}(t)$$\end{document}$ is a vector-valued initial continuous function defined on the interval $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[-\tau _{2},0]$$\end{document}$. $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$E\in {\mathbb{R}}^{n\times n}$$\end{document}$ is a constant matrix, which may be singular, that is, rank$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(E)=g\le n$$\end{document}$. $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$A_{i}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$A_{\tau i}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$B_{i}$$\end{document}$ are the constant real matrices of appropriate dimensions. $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta A_{i}(t)$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta A_{\tau i}(t)$$\end{document}$ denote the norm-bounded parameter uncertainties in the system and are defined as:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}{}[\Delta A_{i}(t) \ \Delta A_{\tau i}(t)]=M_{i}F(t)[N_{1i} \ N_{2i}] \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$M_{i}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N_{1i}$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N_{2i}$$\end{document}$ are known matrices, *F*(*t*) is unknown time-varying matrix, which satisfies $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F^{T}(t)F(t)\le I$$\end{document}$. The delay $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau (t)$$\end{document}$ in above systems is assumed to be interval time varying and satisfies$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \tau _{1} \le \tau (t)\le \tau _{2}, \quad \dot{\tau }(t)\le d \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{2}$$\end{document}$ and *d* are constants. Before proceeding further, we will introduce some definitions and lemmas to be needed in the development of main results throughout this paper. Consider an unforced singular time-delay system described by$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \left\{ \begin{aligned} &E{\dot{x}}(t)= Ax(t)+A_{\tau }x(t-\tau (t)) \\ &x(t)= \phi (t), t\in [-\tau _{2},0] \\ \end{aligned} \right. \end{aligned}$$\end{document}$$ **Definition 1** {#FPar1} ---------------- (Xu et al. [@CR30])The pair (*E*, *A*) is said to be regular if $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\det (sE-A)$$\end{document}$ is not identically zero.The pair (*E*, *A*) is said to be impulse free if $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$deg (\det (sE-A))= rank(E)$$\end{document}$.The pair (*E*, *A*) is said to be asymptotically stable, if all roots of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\det (sE-A)=0$$\end{document}$ lie inside the unit disk with center at the origin.The delayed singular system ([4](#Equ4){ref-type=""}) is said to be admissible if the pair (*E*, *A*) is regular, impulse free and asymptotically stable. **Definition 2** {#FPar2} ---------------- (Xu et al. [@CR30])The singular system ([4](#Equ4){ref-type=""}) is said to be regular and impulse free if the pair (*E*, *A*) is regular and impulse free.The singular system ([4](#Equ4){ref-type=""}) is said to be asymptotically stable, if for any $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\varepsilon >0$$\end{document}$, there exists a scalar $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta (\varepsilon )>0$$\end{document}$ such that for any compatible initial conditions, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi (t)$$\end{document}$ with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$sup_{-\tau (t)\le t\le 0}\Vert \phi (t)\Vert <\delta (\varepsilon )$$\end{document}$, the solution *x*(*t*) of ([4](#Equ4){ref-type=""}) satisfies $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Vert x(t)\Vert <\varepsilon$$\end{document}$ for $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t\ge 0$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\lim _{t\rightarrow \infty }x(t)=0$$\end{document}$. **Lemma 3** {#FPar3} ----------- (Liu [@CR11]) *For any positive semi-definite matrices*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$X=(X_{ij})_{3 \times 3}\ge 0$$\end{document}$, *the following integral inequality holds:*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} -\int _{t-\tau (t)}^{t}&{\dot{x}}^{T}(s)X_{33}{\dot{x}}(s)ds \le \int _{t-\tau (t)}^{t} \beta (t,s) \begin{bmatrix} X_{11}&\quad X_{12}&\quad X_{13} \\ X_{12}^{T}&\quad X_{22}&\quad X_{23} \\ X_{13}^{T}&\quad X_{23}^{T}&\quad 0 \end{bmatrix}\beta ^{T}(t,s)ds \end{aligned}$$\end{document}$$*where*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta (t,s)=[x^{T}(t)\ x^{T}(t-\tau (t)) \ {\dot{x}}^{T}(s) ]$$\end{document}$. **Lemma 4** {#FPar4} ----------- (Han [@CR5]) *For any constant matrix*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$X\in {\mathbb{R}}^{n\times n}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$X=X^{T}>0$$\end{document}$, *scalar*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$r>0$$\end{document}$, *and vector function*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\dot{x}}:[-r,0]\rightarrow {\mathbb{R}}^{n}$$\end{document}$*such that the following integration is well defined, then*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} -r\int _{-r}^{0}{\dot{x}}^{T}(t+s)&X{\dot{x}}(t+s)ds \le [x^{T}(t)\ x^{T}(t-r)] \begin{bmatrix} -X&\quad X \\ X&\quad -X \end{bmatrix} \begin{bmatrix} x(t) \\ x(t-r) \end{bmatrix} \end{aligned}$$\end{document}$$ **Lemma 5** {#FPar5} ----------- (Xie [@CR29]) *Given a symmetric matrix*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Omega$$\end{document}$*and matrices*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Gamma$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Xi$$\end{document}$*with appropriate dimensions,*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Omega +\Gamma \Delta \Xi +\Xi ^{T}\Delta ^{T}\Gamma ^{T}<0$$\end{document}$*for all*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta$$\end{document}$*satisfying*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta ^{T}\Delta \le I$$\end{document}$, *if and only if there exists a scalar*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\varepsilon >0$$\end{document}$*such that*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Omega +\varepsilon \Gamma \Gamma ^{T}+\varepsilon ^{-1}\Xi ^{T}\Xi <0$$\end{document}$ **Lemma 6** {#FPar6} ----------- (Fridman [@CR3]) *If a functional*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V:C_{n}[-\tau ,0]\rightarrow {\mathbb{R}}$$\end{document}$*is continuous and*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x(t,\phi )$$\end{document}$*is a solution to* ([4](#Equ4){ref-type=""}), *we define*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}(\phi )=\lim \nolimits _{h\rightarrow 0^{+}}sup \frac{1}{h}(V(x(t+h,\phi ))-V(\phi ))$$\end{document}$. *Denote the system parameters of* ([4](#Equ4){ref-type=""}) *as*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} (E,\ A, \ A_{\tau })=\Bigg (\begin{bmatrix} I_{g}&\quad 0 \\ 0&\quad 0 \end{bmatrix}, \begin{bmatrix} A_{11}&\quad A_{12} \\ A_{21}&\quad A_{22} \end{bmatrix}, \begin{bmatrix} A_{\tau 11}&\quad A_{\tau 12} \\ A_{\tau 21}&\quad A_{\tau 22} \end{bmatrix}\Bigg ) \end{aligned}$$\end{document}$$*Assume that the singular system* ([4](#Equ4){ref-type=""}) *is regular and impulse free,*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$A_{22}$$\end{document}$*is invertible,*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho (A_{22}^{-1}A_{\tau 22})<1$$\end{document}$. *Then, the system* ([4](#Equ4){ref-type=""}) *is stable if there exists positive numbers*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha , \mu , \nu$$\end{document}$*and a continuous function,*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V:C_{n}[-\tau ,0]\rightarrow {\mathbb{R}}$$\end{document}$, *such that*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \mu \Vert \phi _{1}(0)\Vert ^{2}\le V(\phi )\le \nu \Vert \phi \Vert ^{2}, \dot{V}(x_{t})\le -\alpha \Vert x_{t}\Vert ^{2} \end{aligned}$$\end{document}$$*where*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{t}=x(t+\theta )$$\end{document}$*with*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\theta \in [-\tau , 0]$$\end{document}$*and*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi =[\phi _{1}^{T} \ \phi _{2}^{T}]$$\end{document}$*with*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi _{1}\in {\mathbb{R}}^{q}$$\end{document}$. Main results {#Sec3} ============ Delay-dependent admissibility {#Sec4} ----------------------------- In this section, we suggest to develop a delay-dependent stability condition for the nominal unforced fuzzy singular system of ([1](#Equ1){ref-type=""}), which can be written as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \left\{ \begin{aligned} &E{\dot{x}}(t)= A(t)x(t)+A_{\tau }(t)x(t-\tau (t)) \\ &x(t)= \phi (t), t\in [-\tau _{2},0] \end{aligned}\right. \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$A(t)=\sum _{i=1}^{r}\mu _{i}(\xi (t))A_{i}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$A_{\tau }(t)=\sum _{i=1}^{r}\mu _{i}(\xi (t))A_{\tau i}$$\end{document}$. In order to derive a maximum admissible upper bound of system ([5](#Equ5){ref-type=""}), the delay interval $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[\tau _{1},\tau _{2}]$$\end{document}$ is divided into two subintervals with unequal width as Case I: $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[\tau _{1},\tau _{\rho }]$$\end{document}$ and Case II: $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[\tau _{\rho },\tau _{2}]$$\end{document}$, where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{\rho }=\tau _{1}+\rho \delta$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta =\tau _{2}-\tau _{1}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0<\rho <1$$\end{document}$. Based on the Lyapunov--Krasovskii stability theorem, the following result is obtained. ### **Theorem 7** {#FPar7} *For the given scalars*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{2}$$\end{document}$, *dand tuning parameter*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$, *system* ([5](#Equ5){ref-type=""}) *is admissible for any time-varying delay*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau (t)$$\end{document}$*satisfying* ([3](#Equ3){ref-type=""}), *if there exist matrices*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Q_{n}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W_{n}>0\; (n=1,2,\ldots ,N)$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Lambda ^{T}(Y_{ij})_{3\times 3}\Lambda =\hat{Y}\ge 0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Lambda ^{T}(Z_{ij})_{3\times 3}\Lambda =\hat{Z}\ge 0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Lambda ={\mathrm{diag}}\{E,E,E\}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{2}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{3}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R_{2}>0$$\end{document}$, *some appropriate dimension matricesS*, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{2}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{3}$$\end{document}$*and the constant matrixRsatisfying*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$E^{T}R=0$$\end{document}$*such that the following set of LMIs hold:*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Theta ^{i}=\begin{bmatrix} \Theta _{11}^{i}&\quad \Theta _{12}^{i}\\ *&\quad \Theta _{22}^{i} \end{bmatrix}<0 \end{aligned}$$\end{document}$$*and*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} R_{1}-Y_{33}\ge 0, \quad R_{2}-Z_{33}\ge 0 \end{aligned}$$\end{document}$$*where*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Theta _{11}^{i}&=\begin{bmatrix} \Theta _{1,1}^{i}&\quad E^{T}W_{1}E&\quad \cdots&\quad 0 \\ *&\quad \Theta _{2,2}&\quad \cdots&\quad 0 \\ \vdots&\quad \vdots&\quad \ddots&\quad \vdots \\ *&\quad *&\quad \cdots&\quad \Theta _{n,n} \end{bmatrix} \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Theta _{12}^{i}&=\begin{bmatrix} 0&\quad 0&\quad \Theta _{(1,N3)}^{i}&\quad 0&\quad \Theta _{(1,N5)}^{i} \\ 0&\quad 0&\quad 0&\quad 0&\quad 0 \\ \vdots&\quad \vdots&\quad \vdots&\quad \vdots&\quad \vdots \\ E^{T}W_{N}E&\quad 0&\quad 0&\quad 0&\quad 0 \\ \end{bmatrix} \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Theta _{22}^{i}&=\begin{bmatrix} \Theta _{(N1,1)}&\quad \Theta _{(N1,2)}&\quad \Theta _{(N1,3)}&\quad 0&\quad 0 \\ *&\quad \Theta _{(N2,2)}&\quad \Theta _{(N2,3)}&\quad \Theta _{(N2,4)}&\quad 0 \\ *&\quad *&\quad \Theta _{(N3,3)}&\quad \Theta _{(N3,4)}&\quad \Theta _{(N3,5)}^{i} \\ *&\quad *&\quad *&\quad \Theta _{(N4,4)}&\quad 0 \\ *&\quad *&\quad *&\quad *&\quad \Theta _{(N5,5)} \\ \end{bmatrix} \end{aligned}$$\end{document}$$*with*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Theta _{1,1}^{i}&=P_{2}^{T}A_{i}+A_{i}^{T}P_{2}+Q_{1}+S_{1}-E^{T}W_{1}E \nonumber \\ \Theta _{n,n}&=-Q_{n-1}-E^{T}W_{n-1}E+Q_{n}-E^{T}W_{n}E \nonumber \\ \Theta _{(1,N3)}^{i}&=P_{2}^{T}A_{\tau i}, \Theta _{(1,N5)}^{i}=E^{T}P_{1}+SR^{T}-P_{2}^{T}+A_{i}^{T}P_{3} \nonumber \\ \Theta _{(N1,1)}&=-Q_{N}-E^{T}W_{N}E+S_{2}+\rho \delta \hat{Y}_{11}+\hat{Y}_{13}+\hat{Y}_{13}^{T} \nonumber \\ \Theta _{(N2,2)}&=S_{3}-S_{2}+ \rho \delta \hat{Y}_{22}-\hat{Y}_{23}-\hat{Y}_{23}^{T}+(1-\rho )\delta \hat{Z}_{11}+\hat{Z}_{13}+\hat{Z}_{13}^{T} \nonumber \\ \Theta _{(N3,5)}&=A_{\tau i}^{T}P_{3}, \Theta _{(N4,4)}=-S_{3}+ (1-\rho ) \delta \hat{Z}_{22}-\hat{Z}_{23}-\hat{Z}_{23}^{T} \nonumber \\ \Theta _{(N5,5)}&=\sum _{n=1}^{N}h^{2}W_{n}+\rho \delta R_{1}+(1-\rho )\delta R_{2}-P_{3}-P^{T}_{3} \end{aligned}$$\end{document}$$***Case I****when*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1} \le \tau (t) \le \tau _{\rho }$$\end{document}$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Theta _{(N1,3)}&=\Theta ^{T}_{(N2,3)}=\rho \delta \hat{Y}_{12}-\hat{Y}_{13}+\hat{Y}_{23}^{T}, \Theta _{(N3,4)}=0 \nonumber \\ \Theta _{(N2,4)}&=(1-\rho )\delta \hat{Z}_{12}-\hat{Z}_{13}+\hat{Z}_{23}^{T}, \Theta _{(N1,2)}=0 \nonumber \\ \Theta _{(N3,3)}&=-(1-d)S_{1}+\rho \delta \hat{Y}_{11}+\hat{Y}_{13}+\hat{Y}_{13}^{T}+\rho \delta \hat{Y}_{22}-\hat{Y}_{23}-\hat{Y}_{23}^{T} \end{aligned}$$\end{document}$$***Case II****when*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{\rho } \le \tau (t) \le \tau _{2}$$\end{document}$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Theta _{(N1,2)}&=\rho \delta \hat{Y}_{12}-\hat{Y}_{13}+\hat{Y}_{23}^{T}, \Theta _{(N2,4)}=\Theta _{(N1,3)}=0\nonumber \\ \Theta _{(N2,3)}&=\Theta _{(N3,4)}=(1-\rho )\delta \hat{Z}_{12}-\hat{Z}_{13}+\hat{Z}_{23}^{T}\nonumber \\ \Theta _{(N3,3)}&=-(1-d)S_{1}+(1-\rho )\delta (\hat{Z}_{11}+\hat{Z}_{22})+\hat{Z}_{13}+\hat{Z}_{13}^{T} -\hat{Z}_{23}-\hat{Z}_{23}^{T} \end{aligned}$$\end{document}$$ ### *Proof* {#FPar8} The proof of this theorem is divided into two parts. The first one is concerned with the regularity and the impulse free characterizations, and the second one treats the stability property of system ([5](#Equ5){ref-type=""}). Since rank$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(E)=g\le n$$\end{document}$, there must exist two invertible matrices $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$G \in {\mathbb{R}}^{n\times n}$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$H \in {\mathbb{R}}^{n\times n}$$\end{document}$ such that$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \tilde{E}=GEH=\begin{bmatrix} I_{g}&\quad 0 \\ 0&\quad 0 \end{bmatrix} \end{aligned}$$\end{document}$$Similar to ([14](#Equ14){ref-type=""}), we define$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \tilde{A}_{i}=GA_{i}H=\begin{bmatrix} \tilde{A}_{i11}&\quad \tilde{A}_{i12} \\ \tilde{A}_{i21}&\quad \tilde{A}_{i22} \end{bmatrix}, \quad {\tilde{P}}=G^{-T}P_{2}H=\begin{bmatrix} \tilde{P}_{11}&\quad \tilde{P}_{12} \\ \tilde{P}_{21}&\quad \tilde{P}_{22} \end{bmatrix} \end{aligned}$$\end{document}$$Since $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Theta ^{i}<0$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Q_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{1}>0$$\end{document}$, we can formulate the following inequality easily:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Upsilon _{i} =A_{i}^{T}P_{2}+P_{2}^{T}A_{i}-E^{T}W_{1}E<0 \end{aligned}$$\end{document}$$Then, pre- and post-multiplying $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Upsilon _{i}<0$$\end{document}$ by $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$H^{T}$$\end{document}$ and *H*, respectively, ([16](#Equ16){ref-type=""}) yields$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \tilde{\Upsilon }_{i}=\tilde{A}_{i}^{T}\tilde{P}+\tilde{P}^{T}\tilde{A}_{i}-H^{T}E^{T}W_{1}EH =\begin{bmatrix} \tilde{\Upsilon }_{11}&\quad \tilde{\Upsilon }_{12} \\ *&\quad \tilde{A}_{i22}^{T}\tilde{P}_{22}+\tilde{P}^{T}_{22}\tilde{A}_{i22} \end{bmatrix}<0 \end{aligned}$$\end{document}$$Since $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tilde{\Upsilon }_{11}$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tilde{\Upsilon }_{12}$$\end{document}$ are irrelevant to the results of the following discussion, the real expression of these two variables are omitted here. From Eq. ([17](#Equ17){ref-type=""}), it is easy to see that$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \tilde{A}_{i22}^{T}\tilde{P}_{22}+\tilde{P}^{T}_{22}\tilde{A}_{i22}<0 \end{aligned}$$\end{document}$$Since $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu _{i}(\xi (t))\ge 0$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sum _{i=1}^{r}\mu _{i}(\xi (t))=1$$\end{document}$, we have$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \sum _{i=1}^{r}\mu _{i}(\xi (t))(\tilde{A}_{i22}^{T} \tilde{P}_{22}+\tilde{P}^{T}_{22}\tilde{A}_{i22})<0 \end{aligned}$$\end{document}$$This implies that $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sum _{i=1}^{r}\mu _{i}(\xi (t))\tilde{A}_{i22}$$\end{document}$ is nonsingular. Therefore, the unforced fuzzy singular system ([5](#Equ5){ref-type=""}) is regular and impulse free. Next, we will show the stability of the system ([5](#Equ5){ref-type=""}). Similar to ([14](#Equ14){ref-type=""})--([15](#Equ15){ref-type=""}), we define$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} GA_{\tau i}H&= \begin{bmatrix} \tilde{A}_{\tau i,11}&\quad \tilde{A}_{\tau i,12} \\ \tilde{A}_{\tau i,21}&\quad \tilde{A}_{\tau i,22} \end{bmatrix},\quad G^{-T}W_{1}G^{-1}=\begin{bmatrix} \tilde{W}_{1,11}&\quad \tilde{W}_{1,12} \\ \tilde{W}_{1,21}&\quad \tilde{W}_{1,22} \end{bmatrix} \\ H^{T}Q_{1}H&=\begin{bmatrix} \tilde{Q}_{1,11}&\quad \tilde{Q}_{1,12} \\ \tilde{Q}_{1,21}&\quad \tilde{Q}_{1,22} \end{bmatrix},\quad G^{-T}Y_{ij}G^{-1}=\begin{bmatrix} \tilde{Y}_{ij,11}&\quad \tilde{Y}_{ij,12} \\ \tilde{Y}_{ij,21}&\quad \tilde{Y}_{ij,22} \end{bmatrix} \\ H^{T}S_{1}H&= \begin{bmatrix} \tilde{S}_{1,11}&\quad \tilde{S}_{1,12} \\ \tilde{S}_{1,21}&\quad \tilde{S}_{1,22} \end{bmatrix},\quad G^{-T}Z_{ij}G^{-1}=\begin{bmatrix} \tilde{Z}_{ij,11}&\quad \tilde{Z}_{ij,12} \\ \tilde{Z}_{ij,21}&\quad \tilde{Z}_{ij,22} \end{bmatrix} \end{aligned}$$\end{document}$$If condition ([6](#Equ6){ref-type=""}) holds, we have$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \sum _{i=1}^{r}\mu _{i}\begin{bmatrix} P_{2}^{T}A_{i}+A_{i}^{T}P_{2}+Q_{1}+S_{1}-E^{T}W_{1}E&\quad P_{2}^{T}A_{\tau i} \\ *&\quad \Theta _{(N3,3)} \end{bmatrix}<0 \end{aligned}$$\end{document}$$Pre-multiplying and post-multiplying the preceding inequality by $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{bmatrix} H^{T} \ H \end{bmatrix}$$\end{document}$ and its transpose, respectively, since $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Q_{1}>0$$\end{document}$ and with definitions ([18](#Equ18){ref-type=""}), we can obtain$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \begin{bmatrix} \star&\quad \star&\quad \star&\quad \star \\ \star&\quad \sum _{i=1}^{r}\mu _{i}\Big (\tilde{P}_{22}^{T}\tilde{A}_{i22}+\tilde{A}_{i22}^{T}\tilde{P}_{22} \Big )+\tilde{Q}_{1,22}+\tilde{S}_{1,22}&\quad \star&\quad \tilde{P}_{22}^{T}\sum _{i=1}^{r}\mu _{i}\tilde{A}_{\tau i,22} \\ \star&\quad \star&\quad \star&\quad \star \\ \star&\quad \sum _{i=1}^{r}\mu _{i}\tilde{A}_{\tau i,22}^{T}\tilde{P}_{22}&\quad \star&\quad -(1-d)\tilde{S}_{1,22} \end{bmatrix}<0 \end{aligned}$$\end{document}$$which implies that$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \begin{bmatrix} \sum \mu _{i}\Big (\tilde{P}_{22}^{T}\tilde{A}_{i22}+\tilde{A}_{i22}^{T}\tilde{P}_{22} \Big )+\tilde{S}_{1,22}&\quad \tilde{P}_{22}^{T}\sum \mu _{i}\tilde{A}_{\tau i,22} \\ \sum _{i=1}^{r}\mu _{i}\tilde{A}_{\tau i,22}^{T}\tilde{P}_{22}&\quad -(1-d)\tilde{S}_{1,22} \end{bmatrix}<0 \end{aligned}$$\end{document}$$Then, pre-multiplying and post-multiplying ([20](#Equ20){ref-type=""}) by $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[-\vartheta ^{T}\ I]$$\end{document}$ and its transpose, respectively, ([20](#Equ20){ref-type=""}) yields $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta ^{T}\tilde{S}_{1,22}\vartheta -(1-d)\tilde{S}_{1,22}<0$$\end{document}$, which shows that $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho (\vartheta )<1$$\end{document}$ holds for all allowable $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu _{i}$$\end{document}$ with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta =\big (\sum _{i=1}^{r}\mu _{i}\tilde{A}_{i22}\big )^{-1} \big (\sum _{i=1}^{r}\mu _{i}\tilde{A}_{\tau i22}\big )$$\end{document}$. Then, we define the following Lyapunov--Krasovskii functional for the unforced fuzzy singular system ([5](#Equ5){ref-type=""}),$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} V(x_{t},t)=V_{1}(t)+V_{2}(t)+V_{3}(t) \end{aligned}$$\end{document}$$where$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} V_{1}(t)&= x^{T}(t)E^{T}P_{1}Ex(t) \\ V_{2}(t)&= \sum _{n=1}^{N}\int _{t-nh}^{t-(n-1)h}x^{T}(s)Q_{n}x(s)ds+\int _{t-\tau (t)}^{t}x^{T}(s)S_{1}x(s)ds \\&\quad+\int _{t-\tau _{\rho }}^{t-\tau _{1}} x^{T}(s)S_{2}x(s)ds +\int _{t-\tau _{2}}^{t-\tau _{\rho }} x^{T}(s)S_{3}x(s)ds \\ V_{3}(t)&=\sum _{n=1}^{N}\int _{-nh}^{-(n-1)h}\int _{t+\theta }^{t} {\dot{x}}^{T}(s)hE^{T}W_{n}E{\dot{x}}(s)dsd\theta \\&\quad+\int _{-\tau _{\rho }}^{-\tau _{1}}\int _{t+\theta }^{t}{\dot{x}}^{T}(s) E^{T}R_{1}E{\dot{x}}(s)dsd\theta +\int _{-\tau _{2}}^{-\tau _{\rho }}\int _{t+\theta }^{t}{\dot{x}}^{T}(s) E^{T}R_{2}E{\dot{x}}(s)dsd\theta \end{aligned}$$\end{document}$$where the unknown matrices $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{2}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{3}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R_{2}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Q_{n}>0$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W_{n}>0(n=1,2,\ldots ,N)$$\end{document}$ are to be determined. Here, in order to reduce the conservativeness and give a set of strict LMIs stability conditions for system ([5](#Equ5){ref-type=""}), we rewrite ([5](#Equ5){ref-type=""}) as follows:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \left\{ \begin{aligned} &{{\bar{E}}}{\dot{{\bar{x}}}}(t)= \bar{A}(t){{\bar{x}}}(t)+\bar{A}_{\tau }(t){{\bar{x}}}(t-\tau (t)) \\ &{{\bar{x}}}(t)= \bar{\phi }(t), t\in [-\tau _{2},0] \end{aligned}\right. \end{aligned}$$\end{document}$$where $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\bar{E}}=\begin{bmatrix}E&\quad 0 \\ 0&\quad 0 \end{bmatrix}, \bar{A}(t)=\begin{bmatrix}0&\quad I \\ A(t)&\quad -I \end{bmatrix}, \bar{A}_{\tau }(t)=\begin{bmatrix}0&\quad 0 \\ A_{\tau }(t)&\quad 0 \end{bmatrix},{\bar{x}}(t)=\begin{bmatrix} x(t) \\ E{\dot{x}}(t) \end{bmatrix}$$\end{document}$$. Then, we have$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} V_{1}(t)=x^{T}(t)(E^{T}P_{1}E+E^{T}RS^{T})x(t)= {{\bar{x}}}^{T}(t){\bar{E}}^{T}\bar{P}{{\bar{x}}}(t) \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{P}=\begin{bmatrix}P_{1}E+RS^{T}&0 \\ P_{2}&P_{3} \end{bmatrix}$$\end{document}$ with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\bar{E}}^{T}\bar{P}=\bar{P}^{T}{\bar{E}}$$\end{document}$. Therefore, the time derivatives of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{1}(t)$$\end{document}$ along the trajectories of the systems ([22](#Equ22){ref-type=""}) satisfies$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \dot{V}_{1}(t)&= {{\bar{x}}}^{T}(t)[\bar{P}^{T}\bar{A}(t)+\bar{A}^{T}(t)\bar{P}] {{\bar{x}}}(t)+ 2{\bar{x}}^{T}(t)\bar{P}^{T}\bar{A}_{\tau }(t){\bar{x}}(t-\tau (t))\\&= \begin{bmatrix} x^{T}(t)&\quad x^{T}(t-\tau (t))&\quad (E{\dot{x}}(t))^{T} \end{bmatrix} \Phi (t) \begin{bmatrix} x(t) \\ x(t-\tau (t)) \\ E{\dot{x}}(t) \end{bmatrix} \\ \end{aligned}$$\end{document}$$where$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Phi (t)=\begin{bmatrix} A(t)P_{2}+P_{2}A(t)&\quad P_{2}A_{\tau }(t)&\quad E^{T}P_{1}+SR^{T}-P^{T}_{2}+A^{T}(t)P_{3} \\ \star&\quad 0&\quad A^{T}_{\tau }(t)P_{3} \\ \star&\quad \star&\quad -P^{T}_{3}-P_{3} \end{bmatrix} \end{aligned}$$\end{document}$$ Similarly, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{2}(t)$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{3}(t)$$\end{document}$ along the trajectories of the systems ([5](#Equ5){ref-type=""}) satisfy$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \dot{V}_{2}(t)&=\sum _{n=1}^{N}x^{T}(t-(n-1)h)Q_{n}x(t-(n-1)h) \\&\quad -\sum _{n=1}^{N}x^{T}(t-nh)Q_{n}x(t-nh)+x^{T}(t)S_{1}x(t) \\&\quad-(1-\dot{\tau }(t))x^{T}(t-\tau (t))S_{1}x(t-\tau (t))+x^{T}(t-\tau _{1})S_{2}x(t-\tau _{1}) \\&\quad - x^{T}(t-\tau _{\rho })S_{2}x(t-\tau _{\rho }) +x^{T}(t-\tau _{\rho })S_{3}x(t-\tau _{\rho })-x^{T}(t-\tau _{2})S_{3}x(t-\tau _{2}) \\ \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}\dot{V}_{3}(t)&= {\dot{x}}^{T}(t)E^{T} \left(\sum _{n=1}^{N}h^{2}W_{n}+\rho \delta R_{1}+(1-\rho )\delta R_{2}\right)E {\dot{x}}(t) \\&\quad- \sum _{n=1}^{N}\int _{t-nh}^{t-(n-1)h}{\dot{x}}^{T}(s)hE^{T}W_{n}E{\dot{x}}(s)ds \\&\quad-\int _{t-\tau _{\rho }}^{t-\tau _{1}}{\dot{x}}^{T}(s)E^{T}(R_{1}-Y_{33})E{\dot{x}}(s)ds-\int _{t-\tau _{\rho }}^{t-\tau _{1}}{\dot{x}}^{T}(s)E^{T}Y_{33}E{\dot{x}}(s)ds \\&\quad-\int _{t-\tau _{2}}^{t-\tau _{\rho }}{\dot{x}}^{T}(s)E^{T}(R_{2}-Z_{33})E{\dot{x}}(s)ds -\int _{t-\tau _{2}}^{t-\tau _{\rho }}{\dot{x}}^{T}(s)E^{T}Z_{33}E{\dot{x}}(s)ds \end{aligned}$$\end{document}$$ For convenience of notations, in the sequel, we denote $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat{Y}_{ij}=E^{T}Y_{ij}E$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat{Z}_{ij}=E^{T}Z_{ij}E$$\end{document}$. Then, for the Case I, when $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1} \le \tau (t) \le \tau _{\rho }$$\end{document}$, the following equations are true:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}&- \int _{t-\tau _{\rho }}^{t-\tau _{1}}{\dot{x}}^{T}(s)\hat{Y}_{33}{\dot{x}}(s)ds -\int _{t-\tau _{2}}^{t-\tau _{\rho }}{\dot{x}}^{T}(s)\hat{Z}_{33}{\dot{x}}(s)ds \nonumber \\&\quad = -\int _{t-\tau _{\rho }}^{t-\tau (t)}{\dot{x}}^{T}(s)\hat{Y}_{33}{\dot{x}}(s)ds-\int _{t-\tau (t)}^{t-\tau _{1}}{\dot{x}}^{T}(s)\hat{Y}_{33}{\dot{x}}(s)ds\nonumber \\&\qquad -\int _{t-\tau _{2}}^{t-\tau _{\rho }}{\dot{x}}^{T}(s)\hat{Z}_{33}{\dot{x}}(s)ds \end{aligned}$$\end{document}$$By utilizing Lemma [3](#FPar3){ref-type="sec"} and the Leibniz--Newton formula, we have$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} -\int _{t-\tau _{\rho }}^{t-\tau (t)}{\dot{x}}^{T}(s)\hat{Y}_{33}{\dot{x}}(s)ds&\le x^{T}(t-\tau (t))\left[\rho \delta \hat{Y}_{11}+\hat{Y}_{13}+\hat{Y}_{13}^{T}\right] x(t-\tau (t)) \\&\quad + 2x^{T}(t-\tau (t))\left[\rho \delta \hat{Y}_{12}-\hat{Y}_{13}+\hat{Y}_{23}^{T}\right]x(t-\tau _{\rho }) \\&\quad +x^{T}(t-\tau _{\rho })\left[\rho \delta \hat{Y}_{22}-\hat{Y}_{23}-\hat{Y}_{23}^{T}\right]x(t-\tau _{\rho }) \end{aligned}$$\end{document}$$Similarly, we obtain$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} - \int _{t-\tau (t)}^{t-\tau _{1}}{\dot{x}}^{T}(s)\hat{Y}_{33}{\dot{x}}(s)ds&\le x^{T}(t-\tau _{1})\left[\rho \delta \hat{Y}_{11}+\hat{Y}_{13}+\hat{Y}_{13}^{T}\right]x(t-\tau _{1}) \\&\quad +2x^{T}(t-\tau _{1})\left[\rho \delta \hat{Y}_{12}-\hat{Y}_{13}+\hat{Y}_{23}^{T}\right] x(t-\tau (t)) \\&\quad +x^{T}(t-\tau (t))\left[\rho \delta \hat{Y}_{22}-\hat{Y}_{23}-\hat{Y}_{23}^{T}\right] x(t-\tau (t)) \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} - \int _{t-\tau _{2}}^{t-\tau _{\rho }}{\dot{x}}^{T}(s)\hat{Z}_{33}{\dot{x}}(s)ds & \le x^{T}(t-\tau _{\rho }) \left[(1-\rho )\delta \hat{Z}_{11}+\hat{Z}_{13}+\hat{Z}_{13}^{T}\right] x(t-\tau _{\rho }) \\&+2x^{T}(t-\tau _{\rho })\left[(1-\rho )\delta \hat{Z}_{12}-\hat{Z}_{13}+\hat{Z}_{23}^{T}\right] x(t-\tau _{2}) \\&+x^{T}(t-\tau _{2}) \left[(1-\rho )\delta \hat{Z}_{22}-\hat{Z}_{23}-\hat{Z}_{23}^{T}\right] x(t-\tau _{2}) \end{aligned}$$\end{document}$$Substituting ([25](#Equ25){ref-type=""})--([28](#Equ28){ref-type=""}) into ([24](#Equ24){ref-type=""}), by Lemma [4](#FPar4){ref-type="sec"}, a straightforward computation gives$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \dot{V}(t)& \le \zeta ^{T}(t)\Theta (t)\zeta (t) -\int _{t-\tau _{\rho }}^{t-\tau _{1}}{\dot{x}}^{T}(s)E^{T}(R_{1}-Y_{33})E{\dot{x}}(s)ds \\&- \int _{t-\tau _{2}}^{t-\tau _{\rho }}{\dot{x}}^{T}(s)E^{T}(R_{2}-Z_{33})E{\dot{x}}(s)ds \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\zeta ^{T}(t)=[x^{T}(t) \ x^{T}(t-h) \ \cdots \ x^{T}(t-\tau _{1}) \ x^{T}(t-\tau _{\rho }) \ x^{T}(t-\tau (t)) \ x^{T}(t-\tau _{2}) \ (E{\dot{x}}(t))^{T}]$$\end{document}$. When $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R_{1}-Y_{33}\ge 0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R_{2}-Z_{33} \ge 0$$\end{document}$, and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}\le \tau (t) \le \tau _{\rho }$$\end{document}$, the last two terms in ([29](#Equ29){ref-type=""}) are all less than 0. Therefore, if the conditions ([6](#Equ6){ref-type=""})--([7](#Equ7){ref-type=""}) hold, there exist $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha >0$$\end{document}$ such that $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{V}(x_{t})<\alpha \Vert x_{t}\Vert$$\end{document}$. By Lemma [6](#FPar6){ref-type="sec"}, we conclude that the unforced fuzzy singular system ([5](#Equ5){ref-type=""}) is stable. For the Case II, when $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{\rho } \le \tau (t) \le \tau _{2}$$\end{document}$, the following equations are true:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}&-\int _{t-\tau _{\rho }}^{t-\tau _{1}}{\dot{x}}^{T}(s)\hat{Y}_{33}{\dot{x}}(s)ds -\int _{t-\tau _{2}}^{t-\tau _{\rho }}{\dot{x}}^{T}(s)\hat{Z}_{33}{\dot{x}}(s)ds\\&\quad =-\int _{t-\tau _{\rho }}^{t-\tau _{1}}{\dot{x}}^{T}(s)\hat{Y}_{33}{\dot{x}}(s)ds -\int _{t-\tau _{2}}^{t-\tau (t)}{\dot{x}}^{T}(s)\hat{Z}_{33}{\dot{x}}(s)ds-\int _{t-\tau (t)}^{t-\tau _{\rho }}{\dot{x}}^{T}(s)\hat{Z}_{33}{\dot{x}}(s)ds \end{aligned}$$\end{document}$$Then, the proof can be completed in a similar formulation to Case I and is omitted here for simplification. Therefore, if LMIs ([6](#Equ6){ref-type=""})--([7](#Equ7){ref-type=""}) hold, the fuzzy singular system ([5](#Equ5){ref-type=""}) is admissible for the Cases I and II, respectively. This completes the proof. $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\square$$\end{document}$ For uncertain T--S fuzzy system of ([5](#Equ5){ref-type=""}), the following result can be easily derived by applying Lemma [5](#FPar5){ref-type="sec"} and Schur complement. ### **Corollary 8** {#FPar9} *For the given scalars*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{2}$$\end{document}$, *dand*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$, *the uncertain fuzzy system of* ([5](#Equ5){ref-type=""}) *is robustly admissible for any time-varying delay*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau (t)$$\end{document}$*satisfying* ([3](#Equ3){ref-type=""}), *if there exist matrices*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Q_{n}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W_{n}>0\;(n=1,2,\ldots ,N)$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Lambda ^{T}(Y_{ij})_{3\times 3}\Lambda =\hat{Y}\ge 0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Lambda ^{T}(Z_{ij})_{3\times 3}\Lambda =\hat{Z}\ge 0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Lambda ={\mathrm{diag}}\{E,E,E\}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{2}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{3}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R_{2}>0$$\end{document}$, *and positive scalars*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\varepsilon _{1i}$$\end{document}$, *some appropriate dimension matricesS*, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{2}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{3}$$\end{document}$*and the constant matrixRsatisfying*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$E^{T}R=0$$\end{document}$*such that the following set of LMIs hold:*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}&\bar{\Theta }^{i}=\begin{bmatrix} \Theta ^{i}&\quad \Gamma _{1i}^{T}&\quad \Omega _{1i}^{T} \\ *&\quad -\varepsilon _{i}I&\quad 0 \\ *&\quad *&\quad -\varepsilon _{i}I \end{bmatrix} < 0 \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}&R_{1}-Y_{33}\ge 0, \quad R_{2}-Z_{33}\ge 0 \end{aligned}$$\end{document}$$*with*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Omega _{1i}&=[N_{1i} \quad 0 \quad \cdots \quad 0 \quad 0 \quad 0 \quad N_{2i} \quad 0 \quad 0 ] \\ \Gamma _{1i}&=[M_{i}^{T}P_{2} \quad 0 \quad \cdots \quad 0 \quad 0 \quad 0 \quad 0 \quad 0 \quad M_{i}^{T}P_{3} ] \\ \end{aligned}$$\end{document}$$*and*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Theta ^{i}$$\end{document}$*are defined in Theorem* [7](#FPar7){ref-type="sec"}. ### *Remark 9* {#FPar10} Different from the results in Yang et al. ([@CR31]), Zeng et al. ([@CR32]), Souza et al. ([@CR18]), Peng et al. ([@CR16]), Peng and Fei ([@CR15]), Liu et al. ([@CR10]) and An and Wen ([@CR1]), by dividing the constant part of time-varying delay $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[0, \tau _{1}]$$\end{document}$ into *N* segments, that is, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[0,\frac{1}{N}\tau _{1}]$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[\frac{1}{N}\tau _{1}, \frac{2}{N}\tau _{1}]$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\cdots$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[\frac{N-1}{N}\tau _{1},\tau _{1}]$$\end{document}$, we define different energy functional $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Q_{n}(n=1,2,\ldots ,N)$$\end{document}$ in each different delay subinterval segment. Because the piecewise Lyapunov function candidates are much richer than the globally quadratic functions, so the obtained stability criteria based on this method can further reduce the conservativeness of analysis and synthesis. ### *Remark 10* {#FPar11} Since the interval $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[\tau _{1}, \tau _{2}]$$\end{document}$ is divided into two unequal variable subintervals $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[\tau _{1},\tau _{\rho }]$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[\tau _{\rho },\tau _{2}]$$\end{document}$ in which $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$ is a tunable parameter, it is clear that the LKF defined in Theorem [7](#FPar7){ref-type="sec"} is more general and simple than Zhang et al. ([@CR33]) and Mourad et al. ([@CR13]) by seeking a appropriate $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$ satisfying $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$0<\rho <1$$\end{document}$. For different $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$, the LKF matrices may be different and the LMIs also may be different in stability conditions, and thus compared with the methods using the same LKF matrices (Wang et al. [@CR27]) or the uniformly dividing delay subintervals (Yang et al. [@CR31]), the variable and different LKF matrices may lead to less conservativeness. ### *Remark 11* {#FPar12} The decomposition method in Theorem [7](#FPar7){ref-type="sec"} may increase the maximum allowable upper bounds on $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{2}$$\end{document}$ for the fixed lower bound $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}$$\end{document}$, if one can set a suitable dividing point with relation to $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$. For seeking an appropriate $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$, a algorithm is given as follows:Step 1:For the given *d*, choose upper bound on $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta$$\end{document}$ satisfying ([6](#Equ6){ref-type=""})--([7](#Equ7){ref-type=""}), select this upper bound as initial value $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta _{0}$$\end{document}$ of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta$$\end{document}$.Step 2:Set step lengths, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta _{step}$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho _{step}$$\end{document}$ for $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$, respectively. Set *k* as a counter and choose $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k=1$$\end{document}$. Meanwhile, let $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta =\delta _{0}+\delta _{step}$$\end{document}$ and the initial value $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho _{0}$$\end{document}$ of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$ equals $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho _{step}$$\end{document}$.Step 3:Let $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho =k\rho _{step}$$\end{document}$, if ([6](#Equ6){ref-type=""})--([7](#Equ7){ref-type=""}) are feasible, go to Step 4; otherwise, go to Step 5.Step 4:Let $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta _{0}=\delta$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho _{0}=\rho$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k=1$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta =\delta _{0}+\delta _{step}$$\end{document}$, go to Step 3.Step 5:Let $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k=k+1$$\end{document}$, if $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k\rho _{step}<1$$\end{document}$, then go to Step 3. otherwise, stop. ### *Remark 12* {#FPar13} In order to further reduce the enlargement of the derivative of LKF, inspired by Liu ([@CR11]), a new integral inequality is employed to estimate the integral term, which will be helpful to increase the maximum admissible upper bound of time delay. Moreover, when the information of the time-derivative of delay is unknown or the time delay is not differentiable, just let $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{1}=0$$\end{document}$ and proceed in a similar way as the previous proof, some new stability criteria can be obtained from Theorem [7](#FPar7){ref-type="sec"}. Due to limited space, no more tautology here. Fuzzy controller design {#Sec5} ----------------------- In this section, based on Theorem [7](#FPar7){ref-type="sec"}, we will proposed a design method of fuzzy controller. Consider the controller gain variations might be caused by the inaccuracies of controller implementation, we employ the following controller form with PDC scheme: **Controller rule***i*: **IF**$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\xi _{1}(t)$$\end{document}$ is $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$M_{i1}$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ldots$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\xi _{p}(t)$$\end{document}$ is $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$M_{ip}$$\end{document}$**THEN**$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} u(t)=(K_{i}+\Delta K_{i}(t))x(t),\quad i=1,2,\ldots ,r. \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$K_{i}$$\end{document}$ are the local gain matrices to be determined, and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta K_{i}(t)$$\end{document}$ is the controller gain perturbations and satisfies$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Delta K_{i}(t)=M_{ai}F_{a}(t)N_{ai} \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$M_{ai}$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N_{ai}$$\end{document}$ are known matrices, and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F_{a}(t)$$\end{document}$ is an unknown time-varying matrix satisfying $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F_{a}^{T}(t)F_{a}(t)\le I$$\end{document}$. Then, the resulting closed-loop system from ([1](#Equ1){ref-type=""}) and ([32](#Equ32){ref-type=""}) can be written as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} E{\dot{x}}(t)=&\sum _{i=1}^{r}\sum _{j=1}^{r}\mu _{i}(\xi (t))\mu _{j}(\xi (t))\{ ((A_{i}+\Delta A_{i}(t))\\&+B_{i}(K_{j}+\Delta K_{j}(t)))x(t) +(A_{\tau i}+\Delta A_{\tau i}(t))x(t-\tau (t)) \} \end{aligned}$$\end{document}$$The aim of this section is to design a state feedback controller in the form of ([32](#Equ32){ref-type=""}) with the gain perturbations satisfying ([33](#Equ33){ref-type=""}), such that the closed-loop system ([34](#Equ34){ref-type=""}) is regular, impulse-free, and asymptotically stable. ### **Theorem 13** {#FPar14} *For the given scalars*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{2}$$\end{document}$, *dand tuning parameter*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$, *the closed-loop fuzzy singular system* ([34](#Equ34){ref-type=""}) *under fuzzy control* ([32](#Equ32){ref-type=""}) *is robustly admissible for any time-varying delay*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau (t)$$\end{document}$*satisfying* ([3](#Equ3){ref-type=""}), *if there exist matrices*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$X>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{j}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{P}_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{Q}_{n}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{W}_{n}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tilde{W}_{n}>0$$\end{document}$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(n=1,2,\ldots ,N)$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{S}_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{S}_{2}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{S}_{3}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{R}_{1}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{R}_{2}>0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{Y}=(\bar{Y}_{ij})_{3\times 3}\ge 0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{Z}=(\bar{Z}_{ij})_{3\times 3}\ge 0$$\end{document}$, *some positive scalars*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\varepsilon _{1i}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\varepsilon _{2ij}$$\end{document}$*and any matrices*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{S}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{P}_{2}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar{R}$$\end{document}$*with appropriate dimension such that the following set of LMIs hold for*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i,j=1,2,\ldots ,r$$\end{document}$:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \begin{bmatrix} \bar{\Xi }_{1}^{ij}&\quad \bar{\Xi }_{2}^{ij}\\ *&\quad \bar{\Xi }_{3}^{ij} \end{bmatrix} < 0 \end{aligned}$$\end{document}$$*and*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \bar{R}_{1}-\bar{Y}_{33}\ge 0, \quad \bar{R}_{2}-\bar{Z}_{33}\ge 0 \end{aligned}$$\end{document}$$*where*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \bar{\Xi }_{1}^{ij}&=\begin{bmatrix} \bar{\Xi }_{11}^{ij}&\quad \bar{\Xi }_{12}^{ij} \ \\ *&\quad \bar{\Xi }_{22}^{i} \ \end{bmatrix} \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \bar{\Xi }_{11}^{ij}&=\begin{bmatrix} \bar{\Xi }_{1,1}^{ij}&\quad \bar{W}_{1}&\quad \cdots&\quad 0 \\ *&\quad \bar{\Xi }_{2,2}&\quad \cdots&\quad 0 \\ \vdots&\quad \vdots&\quad \ddots&\quad \vdots \\ *&\quad *&\quad \cdots&\quad \bar{\Xi }_{n,n} \\ \end{bmatrix} \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \bar{\Xi }_{12}^{ij}&=\begin{bmatrix} 0&\quad 0&\quad \bar{\Xi }_{(1,N3)}^{i}&\quad 0&\quad \bar{\Xi }_{(1,N5)}^{ij} \\ 0&\quad 0&\quad 0&\quad 0&\quad 0 \\ \vdots&\quad \vdots&\quad \vdots&\quad \vdots&\quad \vdots \\ \bar{W}_{N}&\quad 0&\quad 0&\quad 0&\quad 0 \\ \end{bmatrix} \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \bar{\Xi }_{22}^{i}&=\begin{bmatrix} \bar{\Xi }_{(N1,1)}&\quad \bar{\Xi }_{(N1,2)}&\quad \bar{\Xi }_{(N1,3)}&\quad 0&\quad 0 \\ *&\quad \bar{\Xi }_{(N2,2)}&\quad \bar{\Xi }_{(N2,3)}&\quad \bar{\Xi }_{(N2,4)}&\quad 0 \\ *&\quad *&\quad \bar{\Xi }_{(N3,3)}&\quad \bar{\Xi }_{(N3,4)}&\quad \bar{\Xi }_{(N3,5)}^{i} \\ *&\quad *&\quad *&\quad \bar{\Xi }_{(N4,4)}&\quad 0 \\ *&\quad *&\quad *&\quad *&\quad \bar{\Xi }_{(N5,5)} \end{bmatrix} \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \bar{\Xi }_{2}^{ij}&=[\varepsilon _{1i}\bar{\Gamma }_{1i} \quad \bar{\Omega }_{1i}^{T}\quad \varepsilon _{2ij}\bar{\Gamma }_{2ij}\quad \bar{\Omega }_{2j}^{T}] \nonumber \\ \bar{\Xi }_{3}^{ij}&={\mathrm{diag}}\{-\varepsilon _{1i}I \ \ -\varepsilon _{1i}I \ \ -\varepsilon _{2ij}I \ \ -\varepsilon _{2ij}I \} \end{aligned}$$\end{document}$$*with*$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}&\bar{\Xi }_{1,1}^{ij}=A_{i}X+B_{i}V_{j}+(A_{i}X+B_{i}V_{j})^{T}+\bar{Q}_{1}+\bar{S}_{1}-\bar{W}_{1} \\&\bar{\Xi }_{n,n}=-\bar{Q}_{n-1}-\bar{W}_{n-1}+\bar{Q}_{n}-\bar{W}_{n}, \ n=2,3,\ldots ,N \\&\Xi _{(1,N3)}^{i}=A_{\tau i}X, \ \Xi _{(1,N5)}^{ij}=\bar{P}_{1}+\bar{S}\bar{R}^{T}-X+\lambda (A_{i}X+B_{i}V_{j})^{T} \\&\bar{\Xi }_{(N1,1)}=-\bar{Q}_{N}-\bar{W}_{N}+\bar{S}_{2}+\rho \delta \bar{Y}_{11}+\bar{Y}_{13}+\bar{Y}_{13}^{T} \\&\bar{\Xi }_{(N2,2)}=\bar{S}_{3}-\bar{S}_{2}+\rho \delta \bar{Y}_{22}-\bar{Y}_{23}-\bar{Y}_{23}^{T} +(1-\rho )\delta \bar{Z}_{11}+\bar{Z}_{13}+\bar{Z}_{13}^{T} \\&\bar{\Xi }_{(N3,5)}^{i}=\lambda X^{T}A_{\tau i}^{T}, \ \bar{\Xi }_{(N4,4)}=-\bar{S}_{2}+(1-\rho ) \delta \bar{Z}_{22}-\bar{Z}_{23}-\bar{Z}_{23}^{T} \\&\bar{\Xi }_{(N5,5)}=h^2\tilde{W}_{n}+\rho \delta R_{1}+(1-\rho \delta )R_{2}-\lambda \left(X+X^{T}\right) \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}&\bar{\Gamma }_{1i}=[M_{i}^{T} \quad 0 \quad \cdots \quad 0 \quad 0 \quad 0 \quad 0 \quad 0 \quad \lambda M_{i}^{T} ]^{T} \\&\bar{\Gamma }_{2ij}=[M_{aj}^{T}B_{i}^{T} \quad 0 \quad \cdots \quad 0 \quad 0 \quad 0 \quad 0 \quad 0 \quad \lambda M_{aj}^{T}B_{i}^{T} ]^{T} \\&\bar{\Omega }_{1i}=[N_{1i}X \quad 0 \quad \cdots \quad 0 \quad 0 \quad 0 \quad N_{2i}X \quad 0 \quad 0 ] \\&\bar{\Omega }_{2j}=[N_{aj}X \quad 0 \quad \cdots \quad 0 \quad 0 \quad 0 \quad 0 \quad 0 \quad 0 ] \end{aligned}$$\end{document}$$***Case I:****when*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1} \le \tau (t) \le \tau _{\rho }$$\end{document}$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \bar{\Xi }_{(N1,3)}&=\bar{\Xi }^{T}_{(N2,3)}=\rho \delta \bar{Y}_{12}-\bar{Y}_{13}+\bar{Y}_{23}^{T}, \bar{\Xi }_{(N3,4)}=0 \nonumber \\ \bar{\Xi }_{(N2,4)}&=(1-\rho )\delta \bar{Z}_{12}-\bar{Z}_{13}+\bar{Z}_{23}^{T}, \bar{\Xi }_{(N1,2)}=0 \nonumber \\ \bar{\Xi }_{(N3,3)}&=-(d-1)\bar{S}_{1}+\rho \delta (\bar{Y}_{11}+ \bar{Y}_{22})+\bar{Y}_{13}+\bar{Y}_{13}^{T}-\bar{Y}_{23}-\bar{Y}_{23}^{T} \end{aligned}$$\end{document}$$***Case II:****when*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{\rho } \le \tau (t) \le \tau _{2}$$\end{document}$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \bar{\Xi }_{(N1,2)}&=\rho \delta \bar{Y}_{12}-\bar{Y}_{13}+\bar{Y}_{23}^{T}, \bar{\Xi }_{(N2,4)}= \bar{\Xi }_{(N1,3)}=0\nonumber \\ \bar{\Xi }_{(N2,3)}&=\bar{\Xi }_{(N3,4)}=(1-\rho )\delta \bar{Z}_{12}-\bar{Z}_{13}+\bar{Z}_{23}^{T}\nonumber \\ \bar{\Xi }_{(N3,3)}&=-(1-d)\bar{S}_{1}+(1-\rho )\delta \bar{Z}_{11} +\bar{Z}_{13} +\bar{Z}_{13}^{T}+(1-\rho )\delta \bar{Z}_{22}-\bar{Z}_{23}-\bar{Z}_{23}^{T} \end{aligned}$$\end{document}$$*Moreover, if the aforementioned condition is feasible, the gain matrices of controller in the form of* ([32](#Equ32){ref-type=""}) *can be designed by*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$K_{j}=V_{j}X^{-1}$$\end{document}$. ### *Proof* {#FPar15} For the uncertain closed-loop T--S fuzzy singular system ([34](#Equ34){ref-type=""}), replacing $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$A_{i}$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$A_{\tau i}$$\end{document}$ with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$((A_{i}+\Delta A_{i}(t))+B_{i}(K_{j}+\Delta K_{j}(t)))$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$A_{\tau i }+\Delta A_{\tau i}(t)$$\end{document}$ in system ([5](#Equ5){ref-type=""}), respectively. Then, according to ([2](#Equ2){ref-type=""}) and ([33](#Equ33){ref-type=""}), the condition ([6](#Equ6){ref-type=""}) can be rewritten as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Xi ^{ij}_{1}+ \Gamma _{1i}F(t)&\Omega _{1i}+\Omega _{1i}^{T}F^{T}(t)\Gamma _{1i}^{T} +\Gamma _{2ij}F_{a}(t)\Omega _{2j}+\Omega _{2j}^{T}F^{T}_{a}(t)\Gamma _{2ij}^{T}<0 \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Xi ^{ij}_{1}=\begin{bmatrix} \ \Xi _{11}^{ij}&\quad \Xi _{12}^{ij} \ \\ \ *&\quad \Xi _{22}^{i} \ \end{bmatrix}$$\end{document}$ and$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Xi _{11}^{ij}&=\begin{bmatrix} \Xi _{1,1}^{ij}&\quad E^{T}W_{1}E&\quad \cdots&\quad 0 \\ *&\quad \Theta _{2,2}&\quad \cdots&\quad 0 \\ \vdots&\quad \vdots&\quad \ddots&\quad \vdots \\ *&\quad *&\quad \cdots&\quad \Theta _{n,n} \end{bmatrix} \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Xi _{12}^{ij}&=\begin{bmatrix} 0&\quad 0&\quad P_{2}^{T}A_{\tau i}&\quad 0&\quad \Xi _{(1,N5)}^{ij} \\ 0&\quad 0&\quad 0&\quad 0&\quad 0 \\ \vdots&\quad \vdots&\quad \vdots&\quad \vdots&\quad \vdots \\ E^{T}W_{N}E&\quad 0&\quad 0&\quad 0&\quad 0 \\ \end{bmatrix} \end{aligned}$$\end{document}$$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Xi _{22}^{i}&=\begin{bmatrix} \Theta _{(N1,1)}&\quad \Theta _{(N1,2)}&\quad \Theta _{(N1,3)}&\quad 0&\quad 0 \\ *&\quad \Theta _{(N2,2)}&\quad \Theta _{(N2,3)}&\quad \Theta _{(N2,4)}&\quad 0 \\ *&\quad *&\quad \Theta _{(N3,3)}&\quad \Theta _{(N3,4)}&\quad A_{\tau i}^{T}P_{3} \\ *&\quad *&\quad *&\quad \Theta _{(N4,4)}&\quad 0 \\ *&\quad *&\quad *&\quad *&\quad \Theta _{(N5,5)} \end{bmatrix} \end{aligned}$$\end{document}$$with$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Xi _{1,1}^{ij}&=P_{2}^{T}(A_{i}+B_{i}K_{j})+(A_{i}+B_{i}K_{j})^{T}P_{2} +Q_{1}+S_{1}-E^{T}W_{1}E \\ \Xi _{(1,N5)}^{ij}&=E^{T}P_{1}+SR^{T}-P^{T}_{2}+(A_{i}+B_{i}K_{j})^{T}(t)P_{3} \\ \Omega _{1i}&=\begin{bmatrix}N_{1i}&\quad 0&\quad \cdots&\quad 0&\quad 0&\quad 0&\quad N_{2i}&\quad 0&\quad 0 \end{bmatrix} \\ \Omega _{2j}&=\begin{bmatrix}N_{aj}&\quad 0&\quad \cdots&\quad 0&\quad 0&\quad 0&\quad 0&\quad 0&\quad 0 \end{bmatrix} \\ \Gamma _{1i}&=\begin{bmatrix}M_{i}^{T}P_{2}&\quad 0&\quad \cdots&\quad 0&\quad 0&\quad 0&\quad 0&\quad 0&\quad M_{i}^{T}P_{3} \end{bmatrix}^{T} \\ \Gamma _{2ij}&=\begin{bmatrix}M_{aj}^{T}B_{i}^{T}P_{2}&\quad 0&\quad \cdots&\quad 0&\quad 0&\quad 0&\quad 0&\quad 0&\quad M_{aj}^{T}B_{i}^{T}P_{3} \end{bmatrix}^{T} \end{aligned}$$\end{document}$$and other matrix elements such as $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Theta _{ij}$$\end{document}$ are defined in Theorem [7](#FPar7){ref-type="sec"}. By Lemma [5](#FPar5){ref-type="sec"}, we get from ([46](#Equ46){ref-type=""}) that$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \Xi ^{ij}_{1}+\varepsilon _{1i}\Gamma _{1i}\Gamma _{1i}^{T} +\varepsilon ^{-1}_{1i}\Omega _{1i}^{T}\Omega _{1i} +\varepsilon _{2ij}\Gamma _{2ij}\Gamma _{2ij}^{T} +\varepsilon ^{-1}_{2ij}\Omega _{2j}^{T}\Omega _{2j}<0 \end{aligned}$$\end{document}$$where scalars $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\varepsilon _{1ij}>0$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\varepsilon _{2ij}>0$$\end{document}$. Then, by Schur complement, inequality ([50](#Equ50){ref-type=""}) equals to$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \begin{bmatrix} \ \Xi _{1}^{ij}&\quad \Xi _{2}^{ij} \ \\ \ *&\quad \Xi _{3}^{ij} \ \end{bmatrix} < 0 \end{aligned}$$\end{document}$$where$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}&\Xi _{2}^{ij}=[\varepsilon _{1i}\Gamma _{1i} \quad \Omega _{1i}^{T}\quad \varepsilon _{2ij}\Gamma _{2ij}\quad \Omega _{2j}^{T}] \\&\Xi _{3}^{ij}={\mathrm{diag}}\{-\varepsilon _{1i}I \quad -\varepsilon _{1i}I \quad -\varepsilon _{2ij}I \quad -\varepsilon _{2ij}I \} \end{aligned}$$\end{document}$$ In order to obtain the control gain matrix, take $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{3}=\lambda P_{2}$$\end{document}$, where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\lambda$$\end{document}$ is the designing parameter and define the following matrices variables:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}&X=P_{2}^{-1},\ V_{j}=K_{j}X, \ X^{T}E^{T}P_{1}X=\bar{P}_{1}, \quad X^{-1}R^{T}X=\bar{R}\\&X^{T}Q_{n}X=\bar{Q}_{n}, X^{T}E^{T}W_{n}EX=\bar{W}_{n}, X^{T}W_{n}X=\tilde{W}_{n} \quad (n=1,2,3,\ldots ,N) \\&X^{T}S_{n}X=\bar{S}_{n},\ X^{T}R_{n}X=\bar{R}_{n}, \quad (n=1,2) \\&X^{T}E^{T}Y_{ij}EX=\bar{Y}_{ij}, \ X^{T}E^{T}Z_{ij}EX=\bar{Z}_{ij}, \quad (i=1,2,3; j=1,2,3) \\ \end{aligned}$$\end{document}$$Then, pre- and post-multiplying both sides of inequality ([51](#Equ51){ref-type=""}) with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{diag}}\{X^{T},\ldots ,X^{T},I,I,I,I\}$$\end{document}$ and its transpose, respectively, we can obtain the conditions ([35](#Equ35){ref-type=""}) and ([36](#Equ36){ref-type=""}), which means that the closed-loop fuzzy singular system ([34](#Equ34){ref-type=""}) is regular, impulse-free and stable under fuzzy control ([32](#Equ32){ref-type=""}). This completes the proof. $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\square$$\end{document}$ ### *Remark 14* {#FPar16} Different from the work in Su et al. ([@CR19]) concerned with dynamic output controller design for discrete-time T--S fuzzy delay systems, this study is mainly focused on the state feedback controller design for T--S fuzzy singular systems with time-varying delay while the gain variations may be caused by the inaccuracies of controller implementation. In addition, the input--output technique (Su et al. [@CR19]; Zhao et al. [@CR35]) is employed to reduce the conservativeness in stability analysis, however, the model transformation of the original system will result in approximation error. In this study, only need to select a appropriate $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$ in the new constructed LKF, less conservative stability and stabilization conditions can be directly obtained. In Examples 1--3, the comparison results with input--output approach in Su et al. ([@CR19]) and other methods to deal with time delays are presented to illustrate the advantages of the proposed approach. ### *Remark 15* {#FPar17} It should be mentioned that the main character of delay partitioning approach lies in that when the number of subintervals *N* is increased, the conservatism of the result decreases. Meanwhile, the computational complexity increases, see Yang et al. ([@CR31]), Wang et al. ([@CR27]) and Peng and Fei ([@CR15]). Therefore, the choice of the number of subintervals *N* generally depends on the tradeoff between the conservatism reduction and the computational burden. However, according to the examples presented in the next section, we can see that our results ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N=1$$\end{document}$) used less partitioning segments is much better than the one in Wang et al. ([@CR27]) ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N=2$$\end{document}$), Peng and Fei ([@CR15]) ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N=3$$\end{document}$) and Yang et al. ([@CR31]) ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N=3$$\end{document}$), It is means that the presented approach has higher computational efficiency, especially when the number of delay partitioning segments is large. Numerical examples {#Sec6} ================== In this section, four examples are given to demonstrate the effectiveness of the proposed approaches. The first three examples are presented to show the improvement of our results over the existing ones. The last example is used to demonstrate the applicability of the controller design method. *Example 16* {#FPar18} ------------ Consider the following time-delayed nonlinear system:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \left\{ \begin{aligned} &{\dot{x}}_{1}(t)=0.5(1-sin^{2}(\theta (t)))x_{2}(t)-x_{1}(t-\tau (t))-(1+sin^{2}(\theta (t)))x_{1}(t) \\ &{\dot{x}}_{2}(t)=sgn( | \theta (t)|-\frac{\pi }{2})(0.9cos^{2}(\theta (t))-1)x_{1}(t-\tau (t))-x_{2}(t-\tau (t)) \\&\quad\qquad-(0.9+0.1cos^{2}(\theta (t)))x_{2}(t) \end{aligned}\right. \end{aligned}$$\end{document}$$which can be exactly expressed as a nominal T--S delayed system with the following rules:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}&Rule\,1{:}\,if \theta (t)\,is \pm \frac{\pi }{2}, \quad then \ {\dot{x}}(t)=A_{1}x(t)+A_{\tau 1}x(t-\tau (t)) \\&Rule\,2{:}\,if \theta (t)\,is \ 0 , \quad then \ {\dot{x}}(t)=A_{2}x(t)+A_{\tau 2}x(t-\tau (t)) \end{aligned}$$\end{document}$$where the membership functions for above rule 1 and rule 2 are $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$h_{1}(\theta (t))=sin^{2}(\theta (t))$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$h_{2}(\theta (t))=cos^{2}(\theta (t))$$\end{document}$ with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\theta (t)=x_{1}(t)$$\end{document}$, and the system matrices are:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} A_{1}=\begin{bmatrix} -2&\quad 0 \\ 0&\quad -0.9 \end{bmatrix}, \quad A_{\tau 1}= \begin{bmatrix} -1&\quad 0 \\ -1&\quad -1 \end{bmatrix},\quad A_{2}=\begin{bmatrix} -1&\quad 0.5 \\ 0&\quad \ -1 \end{bmatrix}, \quad A_{\tau 2}= \begin{bmatrix} -1&\quad 0 \\ 0.1&\quad -1 \end{bmatrix} \end{aligned}$$\end{document}$$For this example, because the time-derivative of delay $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau (t)$$\end{document}$ is unknown and the considered systems is nonsingular, we set $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{1}=0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$E=I_{2\times 2}$$\end{document}$ in Theorem [7](#FPar7){ref-type="sec"} and choose the delay interval segmentation parameter $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho =0.7$$\end{document}$ in Case I, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho = 0.3$$\end{document}$ in Case II, respectively. The upper delay bounds $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{2}$$\end{document}$ derived by the input--output method (Zhao et al. [@CR35]), convex combination technique (An and Wen [@CR1]; Peng and Fei [@CR15]), free weighting matrices approach (Tian et al. [@CR25]; Souza et al. [@CR18]) and the improved delay partitioning method proposed in this paper are tabulated in Table [1](#Tab1){ref-type="table"} under different values of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}$$\end{document}$. It is seen from Table [1](#Tab1){ref-type="table"} that the results obtained from Theorem [7](#FPar7){ref-type="sec"} of this paper are significantly better than those obtained from the other methods. When the system matrices of rule 2 are given as Lien et al. ([@CR8]) with$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} A_{2}=\begin{bmatrix} -1.5&\quad 1 \\ 0&\quad -0.75 \end{bmatrix}, \quad A_{\tau 2}= \begin{bmatrix} -1&\quad 0 \\ 1&\quad -0.85 \end{bmatrix} \end{aligned}$$\end{document}$$the improvement of this paper is shown in Table [2](#Tab2){ref-type="table"}. It can be concluded that the obtained results in our method are less conservative than those of Souza et al. ([@CR18]), Peng et al. ([@CR16]), Tian et al. ([@CR25]), Tian and Chen ([@CR24]) and Lien et al. ([@CR8]). Moreover, it is shown in Tables [1](#Tab1){ref-type="table"} and [2](#Tab2){ref-type="table"} that the conservatism is gradually reduced with the increase of *N* while guaranteeing asymptotically stability of the considered system. ###### Example 1-maximum allowable delay bounds of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{2}$$\end{document}$ under different values of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}$$\end{document}$ with *d* unknown Methods$\documentclass[12pt]{minimal} 0 0.4 0.8 1.0 1.2 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\setminus } \tau _{1}$$\end{document}$ --------------------------------------------------------------------------- -------- -------- -------- -------- -------- Tian et al. ([@CR25]) Corollary 1 -- 1.2647 1.3032 1.3528 1.4214 An and Wen ([@CR1]) Theorem 1 1.2780 1.3030 1.3160 1.3610 1.4250 Souza et al. ([@CR18]) Corollary 4 -- 1.2836 1.3394 1.4009 1.4815 Peng and Fei ([@CR15]) Theorem 1($\documentclass[12pt]{minimal} 1.3400 1.3200 1.3200 -- 1.4200 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N=2$$\end{document}$) Peng and Fei ([@CR15]) Theorem 1($\documentclass[12pt]{minimal} 1.3800 1.3900 1.4300 -- 1.5700 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N=3$$\end{document}$) Zhao et al. ([@CR35]) Theorem 1 -- 1.3802 1.4627 -- 1.6066 Xia et al. ([@CR28]) Theorem 4 -- 1.5274 1.5361 1.5762 1.6340 $\documentclass[12pt]{minimal} 1.4841 1.6743 1.7794 1.7965 1.7805 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C II\,(\rho =0.7, N=1)$$\end{document}$ $\documentclass[12pt]{minimal} 1.4839 1.6761 1.8001 1.8403 1.8699 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C II\,(\rho =0.7, N=2)$$\end{document}$ $\documentclass[12pt]{minimal} 3.2721 2.5582 2.0346 1.8698 1.7495 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C I\,(\rho =0.3, N=1)$$\end{document}$ $\documentclass[12pt]{minimal} 3.2712 2.6034 2.1798 2.0577 1.9769 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C I\,(\rho =0.3, N=2)$$\end{document}$ ###### Example 1-maximum allowable delay bounds of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{2}$$\end{document}$ under different values of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}$$\end{document}$ with *d* unknown Methods$\documentclass[12pt]{minimal} 0.2 0.4 0.6 0.8 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\setminus } \tau _{1}$$\end{document}$ --------------------------------------------------------------------------- -------- -------- -------- -------- Tian and Chen ([@CR24]) 0.6870 0.8500 0.9460 1.0480 Lien et al. ([@CR8]) Corollary 1 0.7945 0.8487 0.9316 1.0325 Peng et al. ([@CR16]) Corollary 5 0.9119 0.9793 1.0639 1.1662 Tian et al. ([@CR25]) Corollary 1 1.1410 1.1500 1.1720 1.2090 Souza et al. ([@CR18]) Corollary 4 1.1639 1.1734 1.1994 1.2532 $\documentclass[12pt]{minimal} 1.3775 1.4419 1.4837 1.5002 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C II\,(N=1,\rho =0.95)$$\end{document}$ $\documentclass[12pt]{minimal} 1.3780 1.4447 1.4940 1.5279 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C II\,(N=2,\rho =0.95)$$\end{document}$ $\documentclass[12pt]{minimal} 2.3328 2.0765 1.8643 1.6990 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C I\,(N=1,\rho =0.35)$$\end{document}$ $\documentclass[12pt]{minimal} 2.3371 2.1024 1.9288 1.8101 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C I\,(N=2,\rho =0.35)$$\end{document}$ *Example 17* {#FPar19} ------------ Consider the following uncertain fuzzy system with two rules:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} {\dot{x}}(t)=\sum _{i=1}^{2}\mu _{i}(\xi (t))\{A_{i}x(t)+A_{\tau i}x(t-\tau (t)) \} \end{aligned}$$\end{document}$$where$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} A_{1}&=\begin{bmatrix} -2&\quad 1 \\ 0.5&\quad -0.1 \end{bmatrix},\quad A_{\tau 1}= \begin{bmatrix} -1&\quad 0 \\ -1&\quad -1 \end{bmatrix}, \quad E_{1}=\begin{bmatrix} 1.6&\quad 0 \\ 0&\quad 0.05 \end{bmatrix}\\ A_{2}&=\begin{bmatrix} -2&\quad 0 \\ 0&\quad -1 \end{bmatrix},\quad A_{\tau 2}= \begin{bmatrix} -1.6&\quad 0 \\ 0&\quad -1 \end{bmatrix},\quad E_{\tau 1}= \begin{bmatrix} 0.1&\quad 0 \\ 0&\quad 0.3 \end{bmatrix} \\ E_{2}&=\begin{bmatrix} 1.6&\quad 0 \\ 0&\quad -0.05 \end{bmatrix},\quad E_{\tau 2}= \begin{bmatrix} 0.1&\quad 0 \\ 0&\quad 0.3 \end{bmatrix},\quad D=\begin{bmatrix} 0.03&\quad 0 \\ 0&\quad -0.03 \end{bmatrix} \end{aligned}$$\end{document}$$and the membership functions for rules 1 and 2 are the same as Example [16](#FPar18){ref-type="sec"}. For various *d*, by utilizing Corollary [8](#FPar9){ref-type="sec"} and the conditions in Yang et al. ([@CR31]), Zeng et al. ([@CR32]), Liu et al. ([@CR10]) and Lien et al. ([@CR8]), the computed upper bounds that guarantee the robust stability of the considered system are summarized in Table [3](#Tab3){ref-type="table"}. It can be concluded that the result proposed in this paper is better than the aforementioned results. In addition, compared with the results in Yang et al. ([@CR31]), assume that $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i=2$$\end{document}$, there are ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$13n(n+1)/2)+7n^{2}$$\end{document}$ ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N=3$$\end{document}$) scalar decision variables and six LMIs in their Theorem 1. However, different from the delay interval $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[\tau _{1},\tau _{2}]$$\end{document}$ is divided into multiple segments, we divide the delay interval into two unequal subintervals by seeking a appropriate $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho$$\end{document}$. Thus, only $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(10n(n+1)/2)+4n^{2}$$\end{document}$ ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N=1$$\end{document}$) scalar decision variables and four LMIs are required to improve the results. Especially, when *N* is increased, less number of decision variables and LMIs may reduce the mathematical complexity and computational load. ###### Example 2-maximum allowable delay bounds of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{2}$$\end{document}$ for different values of *d* with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}=0$$\end{document}$ Methods$\documentclass[12pt]{minimal} 0 0.01 0.1 0.5 Unknown \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\setminus d$$\end{document}$ --------------------------------------------------------------------------- -------- -------- -------- -------- --------- Lien et al. ([@CR8]) Theorem 1 1.1680 1.1630 1.1220 0.9340 0.4990 Liu et al. ([@CR10]) Corollary 4 1.1920 1.1870 1.1550 1.1000 1.0500 Zeng et al. ([@CR32]) Theorem 1 ($\documentclass[12pt]{minimal} 1.3900 1.3820 1.3180 1.1320 1.1270 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m=2$$\end{document}$) Yang et al. ([@CR31]) Theorem 1 ($\documentclass[12pt]{minimal} 1.4737 -- 1.4182 1.2916 1.2299 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m=2$$\end{document}$) Yang et al. ([@CR31]) Theorem 1 ($\documentclass[12pt]{minimal} 1.6425 -- 1.5990 1.4923 1.4182 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m=3$$\end{document}$) $\documentclass[12pt]{minimal} 2.7084 2.6940 2.6007 2.2059 1.8245 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C I\,(\rho =0.45, N=1)$$\end{document}$ ###### Example 3-maximum allowable delay bounds of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{2}$$\end{document}$ for different values of *d* with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}=2$$\end{document}$ Methods$\documentclass[12pt]{minimal} 0.1 0.35 0.6 0.85 0.9 0.95 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\setminus } d$$\end{document}$ ---------------------------------------------------------------------------- -------- -------- -------- -------- -------- -------- Zhang et al. ([@CR33]) Theorem 1 3.3623 2.9810 2.6010 1.8330 1.0380 -- Mourad et al. ([@CR13]) Theorem 3 3.3685 3.1560 3.1510 3.0760 2.6750 2.0780 Wang et al. ([@CR27]) Theorem 1 ($\documentclass[12pt]{minimal} 3.5023 3.2915 3.2379 3.1321 3.9775 2.5863 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N=1$$\end{document}$) Wang et al. ([@CR27]) Theorem 1 ($\documentclass[12pt]{minimal} 3.6761 3.4755 3.3580 3.2425 3.0737 2.8257 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N=2$$\end{document}$) $\documentclass[12pt]{minimal} 3.7445 3.7553 3.7566 3.7573 3.7550 3.7546 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C II\,(\rho =0.95, N=1)$$\end{document}$ $\documentclass[12pt]{minimal} 3.8070 3.8066 3.8064 3.8045 3.8065 3.8072 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C II\,(\rho =0.95, N=2)$$\end{document}$ $\documentclass[12pt]{minimal} 5.2484 4.4255 4.1246 4.0834 4.0663 4.0552 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C I\,(\rho =0.45, N=1)$$\end{document}$ $\documentclass[12pt]{minimal} 5.3596 4.5894 4.3146 4.2472 4.2140 4.1870 \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C I\,(\rho =0.45, N=2)$$\end{document}$ *Example 18* {#FPar20} ------------ Consider a continuous fuzzy singular system composed of two rules and the following system matrices:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} E= & {} \begin{bmatrix} 1&\quad 0&\quad 0&\quad 0\\ 0&\quad 1&\quad 0&\quad 0\\ 0&\quad 0&\quad 1&\quad 0\\ 0&\quad 0&\quad 0&\quad 0 \end{bmatrix},\quad A_{1}=\begin{bmatrix} -3&\quad 0&\quad 0&\quad 0.2\\ 0&\quad -4&\quad 0.1&\quad 0\\ 0&\quad 0&\quad -0.1&\quad 0\\ 0.1&\quad 0.1&\quad -0.2&\quad -0.2 \end{bmatrix},\quad A_{\tau 1}=\begin{bmatrix} -0.5&\quad 0&\quad 0&\quad 0\\ 0&\quad -1&\quad 0&\quad 0\\ 0&\quad 0.1&\quad -0.2&\quad 0\\ 0&\quad 0&\quad 0&\quad 0 \end{bmatrix}\\ A_{2}= & {} \begin{bmatrix} -2&\quad 0&\quad 0&\quad -0.2\\ 0&\quad -2.5&\quad -0.1&\quad 0\\ 0&\quad -0.2&\quad -0.3&\quad 0\\ 0.1&\quad 0.1&\quad -0.2&\quad -0.2 \end{bmatrix},\quad A_{\tau 2}=\begin{bmatrix} -0.5&\quad 0&\quad 0&\quad 0\\ 0&\quad -1&\quad 0&\quad 0\\ 0&\quad 0.1&\quad -0.5&\quad 0\\ 0&\quad 0&\quad 0&\quad 0 \end{bmatrix} \end{aligned}$$\end{document}$$In order to compare with the existing results, supposing that $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau (t)$$\end{document}$ satisfies ([3](#Equ3){ref-type=""}) and with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}=2$$\end{document}$. Then, setting $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho =0.45$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho =0.95$$\end{document}$ in Cases I and II, respectively. Table [4](#Tab4){ref-type="table"} presents a comparison results with various *d*, which show that the stability condition in Theorem [7](#FPar7){ref-type="sec"} give less conservative results than those in Wang et al. ([@CR27]), Mourad et al. ([@CR13]) and Zhang et al. ([@CR33]). It is worth mention that the stability conditions in the aforementioned works are not in strict LMIs form due to equality constraints. However, by introducing the variable *R*, much better results are obtained by solving strict LMIs via the existing numerical convex optimization method. *Example 19* {#FPar21} ------------ Consider the following nonlinear time-delay systems borrowed from Lin et al. ([@CR9]):$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} (1+(a+\Delta a(t))cos(\theta ))\ddot{\theta }(t)&= -b\dot{\theta }^{3}(t)+c\theta (t) \\&\quad+(c_{\tau }+\Delta c_{\tau }(t)) \theta (t-\tau (t))+eu(t) \end{aligned}$$\end{document}$$The range of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\dot{\theta }(t)$$\end{document}$ is assumed to satisfy $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$|\dot{\theta }(t)|<\varphi$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\varphi =2$$\end{document}$. *u*(*t*) is the control input. $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau (t)=0.85+0.05 sin(10t)$$\end{document}$ is the time-varying delay (thus, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{1}=0.8$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau _{2}=0.9$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d=0.5$$\end{document}$). For the simulation purpose, the system parameter is given as $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$a=0.3$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$b=0.5$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e=0.2$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c=1$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{\tau }=0.8$$\end{document}$. As in Lin et al. ([@CR9]), we introduce new variables $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x(t)=[x_{1}(t)\ x_{2}(t) \ x_{3}(t)]^{T}$$\end{document}$ with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{1}(t)=\theta (t)$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{2}(t)=\dot{\theta }(t)$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{3}(t)=\ddot{\theta }(t)$$\end{document}$. The system is described by$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \begin{bmatrix} 1&\quad 0&\quad 0 \\ 0&\quad 1&\quad 0 \\ 0&\quad 0&\quad 0 \end{bmatrix}{\dot{x}}(t) =\begin{bmatrix} 0&\quad 1&\quad 0 \\ 0&\quad 0&\quad 1 \\ c&\quad -bx_{2}^{2}(t)&\quad -1-acosx_{1}(t) \end{bmatrix}x(t) + \begin{bmatrix} 0&\quad 0&\quad 0 \\ 0&\quad 0&\quad 0 \\ c_{\tau }&\quad 0&\quad 0 \end{bmatrix}x(t-\tau (t)) + \begin{bmatrix} 0 \\ 0 \\ e \end{bmatrix}u(t) \end{aligned}$$\end{document}$$Then this system can be expressed exactly by the following fuzzy singular form with respect to uncertainties described by ([2](#Equ2){ref-type=""}):$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \left\{ \begin{aligned} &E{\dot{x}}(t)=\sum _{i=1}^{3}\mu _{i}(\xi (t))\{ (A_{i}+\Delta A_{i}(t))x(t)+(A_{\tau i}+\Delta A_{\tau i}(t))x(t-\tau (t))+B_{i}u(t)\} \\ &x(t)=\sum _{i=1}^{3}\mu _{i}(\xi (t))\phi _{i}(t) \end{aligned}\right. \end{aligned}$$\end{document}$$where$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} E&=\begin{bmatrix} 1&\quad 0&\quad 0 \\ 0&\quad 1&\quad 0 \\ 0&\quad 0&\quad 0 \end{bmatrix},\quad A_{1}=\begin{bmatrix} 0&\quad 1&\quad 0 \\ 0&\quad 0&\quad 1 \\ c&\quad -b(\varphi ^{2}+2)&\quad a-1 \end{bmatrix},\quad A_{3}=\begin{bmatrix} 0&\quad 1&\quad 0 \\ 0&\quad 0&\quad 1 \\ c&\quad 0&\quad a-1 \end{bmatrix} \\ A_{2}&=\begin{bmatrix} 0&\quad 1&\quad 0 \\ 0&\quad 0&\quad 1 \\ c&\quad 0&\quad -a-1-a\varphi ^{2} \end{bmatrix},\quad A_{\tau i}=\begin{bmatrix} 0&\quad 0&\quad 0 \\ 0&\quad 0&\quad 0 \\ c_{\tau }&\quad 0&\quad 0 \end{bmatrix},\quad B_{i}=\begin{bmatrix} 0 \\ 0 \\ e \end{bmatrix},\quad (i=1,2,3) \end{aligned}$$\end{document}$$The membership functions can be chosen as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \mu _{1}(t)=\frac{x_{2}^{2}(t)}{\varphi ^{2}+2}, \quad \mu _{2}(t)=\frac{1+cos(x_{1}(t))}{\varphi ^{2}+2}, \quad \mu _{3}(t) =\frac{\phi ^{2}-x_{2}^{2}(t)+1-cos(x_{1}(t))}{\varphi ^{2}+2} \end{aligned}$$\end{document}$$Here, we set $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho =0.5$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\lambda =1$$\end{document}$ and assume that the parameters uncertainty matrices in $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta A_{i}(t)$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta A_{\tau i}(t)$$\end{document}$ in ([2](#Equ2){ref-type=""}) are given as follows:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} M_{i}&=\begin{bmatrix} 0.1&\quad 0&\quad 0 \\ 0&\quad 0.5&\quad 0 \\ 0&\quad 0&\quad 0.1 \end{bmatrix},\quad N_{11}=N_{13}=\begin{bmatrix} 0&\quad 0&\quad 0 \\ 0&\quad 0&\quad 0 \\ 0&\quad 0&\quad a \end{bmatrix} \\ N_{12}&=\begin{bmatrix} 0&\quad 0&\quad 0 \\ 0&\quad 0&\quad 0 \\ 0&\quad 0&\quad -a(\varphi ^{2}+1) \end{bmatrix},\quad N_{2i}=\begin{bmatrix} c_{\tau }&\quad 0&\quad 0 \\ 0&\quad 0.1&\quad 0 \\ 0&\quad 0&\quad 0.1 \end{bmatrix},\quad (i=1,2,3) \end{aligned}$$\end{document}$$In this example, considering the case of controller gain variation in the form of ([33](#Equ33){ref-type=""}), the parameters are given as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} M_{ai}=\begin{bmatrix} 0.1&\quad 0.1&\quad 0.2 \end{bmatrix}, \quad N_{ai}= \begin{bmatrix} 0.1&\quad 0.1&\quad 0.2 \\ 0.1&\quad 0.1&\quad 0.3 \\ 0.2&\quad 0.4&\quad 0.1 \end{bmatrix} (i=1,2,3) \end{aligned}$$\end{document}$$Then, according to Theorem [13](#FPar14){ref-type="sec"} and by solving LMIs ([35](#Equ35){ref-type=""})--([43](#Equ43){ref-type=""}) with ([44](#Equ44){ref-type=""}), we can obtain the feasible solution for Case I ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$N=1$$\end{document}$) as follows: (due to space consideration, we do not list all the matrices here)$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} X&=\begin{bmatrix} 0.2228&\quad -0.0943&\quad 0.0466 \\ -0.0943&\quad 0.1031&\quad -0.1530 \\ 0.0466&\quad -0.1530&\quad 1.4543 \\ \end{bmatrix},\quad \bar{P}_{1}=\begin{bmatrix} 0.2711&\quad -0.1386&\quad 0.2555 \\ -0.1386&\quad 0.1659&\quad -1.5745 \\ 0.2555&\quad -1.5745&\quad 23.7354 \\ \end{bmatrix} \\ \bar{Q}_{1}&=\begin{bmatrix} 0.0523&\quad -0.0247&\quad 0.0024 \\ -0.0247&\quad 0.0125&\quad -0.0083 \\ 0.0024&\quad -0.0083&\quad 4.8551 \\ \end{bmatrix},\quad \bar{S}_{1}=\begin{bmatrix} \ 0.0776&\quad -0.0373&\quad 0.0654 \\ \ -0.0373&\quad 0.0191&\quad 0.0032 \\ \ 0.0654&\quad 0.0032&\quad 3.3431 \\ \end{bmatrix} \\ \bar{S}_{2}&=\begin{bmatrix} 0.0715&\quad -0.0391&\quad -0.0026 \\ -0.0391&\quad 0.0361&\quad 0.0045 \\ -0.0026&\quad 0.0045&\quad 2.7343 \\ \end{bmatrix},\quad \bar{S}_{3}=\begin{bmatrix} 0.0731&\quad -0.0413&\quad -0.0013 \\ -0.0413&\quad 0.0433&\quad 0.0023 \\ -0.0013&\quad 0.0023&\quad 1.3438 \\ \end{bmatrix} \\ \bar{R}_{1}&=\begin{bmatrix} 0.6551&\quad -0.3024&\quad 0.2893 \\ -0.3024&\quad 0.2563&\quad -0.7339 \\ 0.2893&\quad -0.7339&\quad 3.5068 \\ \end{bmatrix},\quad \bar{R}_{2}=\begin{bmatrix} 0.5959&\quad -0.2666&\quad 0.2165 \\ -0.2666&\quad 0.2178&\quad -0.6041 \\ 0.2165&\quad -0.6041&\quad 2.9812 \\ \end{bmatrix} \\ V_{1}&=[-4.2929 \ \ \ \ \ 0.2507 \ -46.1158 ],\quad \varepsilon _{11}=0.3134, \varepsilon _{12}=0.3108,\varepsilon _{13}=0.3006 \\ V_{2}&=[-3.9275 \ -0.2329 \ -46.5635 ], \quad \varepsilon _{211}=1.5229, \varepsilon _{212}=0.2762,\varepsilon _{213}=1.5568 \\ V_{3}&=[-3.7714 \ -0.0943 \ -48.3501],\quad \varepsilon _{222}=2.3433, \varepsilon _{223}=1.5910,\varepsilon _{233}=1.5191 \\ \end{aligned}$$\end{document}$$Then, the feedback controller gains are designed as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}&K_{1}=\begin{bmatrix}-58.2677&\quad -112.7529&\quad -41.7051 \end{bmatrix} \\&K_{2}=\begin{bmatrix} -59.5466&\quad -120.1667&\quad -42.7519 \end{bmatrix} \\&K_{3}=\begin{bmatrix}-58.2903&\quad -119.4432&\quad -43.9446 \end{bmatrix} \\ \end{aligned}$$\end{document}$$Similarly, according to Theorem [13](#FPar14){ref-type="sec"} and by solving LMIs ([35](#Equ35){ref-type=""})--([43](#Equ43){ref-type=""}) with ([45](#Equ45){ref-type=""}), we can obtain that the feedback controller gains in Case II are designed as:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned}&\bar{K}_{1}=\begin{bmatrix}-39.0312 \quad -64.8855 \quad -20.9107\end{bmatrix} \\&\bar{K}_{2}=\begin{bmatrix}-39.6921 \quad -69.1777 \quad -21.6299\end{bmatrix} \\&\bar{K}_{3}=\begin{bmatrix}-38.6646 \quad -68.7853 \quad -21.6868\end{bmatrix} \\ \end{aligned}$$\end{document}$$Then, let the initial condition be $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{1}(t)=1$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{2}(t)=-1$$\end{document}$, and the unknown matrix function $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F(t)=F_{a}(t)=sin(t)$$\end{document}$. The simulation results are given in Figs. [1](#Fig1){ref-type="fig"}, [2](#Fig2){ref-type="fig"}, [3](#Fig3){ref-type="fig"}, [4](#Fig4){ref-type="fig"} and [5](#Fig5){ref-type="fig"}. Figures [1](#Fig1){ref-type="fig"} and [2](#Fig2){ref-type="fig"} plots the state trajectories of the closed-loop system with the obtained feedback gain matrices in Case I and Case II, respectively. The phase portraits of closed system are given in Figs. [3](#Fig3){ref-type="fig"}, [4](#Fig4){ref-type="fig"} and [5](#Fig5){ref-type="fig"}. From the simulation result, it can be seen that the designed fuzzy controller not only makes the closed-loop system states converge to zero, but also effectively attenuate the uncertainty as expected. Fig. 1States response of closed-loop system with designed fuzzy controller in Case IFig. 2States response of closed-loop system with designed fuzzy controller in Case IIFig. 3The phase portrait of closed-system states $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{1}(t)$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{2}(t)$$\end{document}$ in Case I and IIFig. 4The phase portrait of closed-system states $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{1}(t)$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{3}(t)$$\end{document}$ in Case I and IIFig. 5The phase portrait of closed-system states $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{2}(t)$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$x_{3}(t)$$\end{document}$ in Cases I and II Conclusion {#Sec7} ========== In this paper, the stability analysis and fuzzy stabilizing controller design for fuzzy singular systems with interval time-varying delay have been discussed. Based on improved delay partitioning method, new stability criteria for unforced fuzzy singular systems have been established. Then, the explicit expression of the desired fuzzy controller gains are also presented. All the obtained results reported in this paper are formulated in terms of strict LMIs, which can be readily solved using standard numerical software. Some numerical examples are provided to show the effectiveness of the proposed methods. All authors contributed equally and significantly in writing this manuscript. All authors read and approved the final manuscript. Acknowledgements {#d30e31710} ================ This work is supported in part by the National Natural Science Foundation of China (Grant Nos. 61533007, 61374146 and 61174215), Project 863 of China (Grant No. 2011AA060204) and IAPI Fundamental Research Funds (Grant No. 2013ZCX02-04). Competing interests {#d30e31715} =================== The authors declare that they have no competing interests.

Mark Vander Roest, Christopher Krapp, Joanne L. Thorvaldsen, Marisa S. Bartolomei, W. David Merryman. Physiol Rep, 7 (19), 2019, e14244, 10.14814/phy2.14244 **Funding Information** This work was supported by the National Institute of General Medical Sciences (GM051279 and HL007411); Fondation Leducq; and National Heart, Lung, and Blood Institute (HL135790). Introduction {#phy214244-sec-0001} ============ Calcific aortic valve disease (CAVD) is an increasingly prevalent source of cardiovascular morbidity in the elderly, but identifiable genetic causes only explain a small fraction of disease cases (Lindman et al. [2016](#phy214244-bib-0017){ref-type="ref"}; Stewart et al. [1997](#phy214244-bib-0024){ref-type="ref"}; Osnabrugge et al. [2013](#phy214244-bib-0020){ref-type="ref"}). *NOTCH1* loss of function mutation is one of the most widely studied genetic causes of CAVD, although such a mutation is not found in the majority of disease cases (Garg et al. [2005](#phy214244-bib-0009){ref-type="ref"}; Mohamed et al. [2006](#phy214244-bib-0019){ref-type="ref"}). Despite this discrepancy, alterations in the *NOTCH1* signaling pathway offer a proven mechanism for CAVD, and downstream mechanisms for valve calcification are similar for *Notch1*‐driven CAVD in mice and idiopathic CAVD in humans (Hutcheson et al. [2014](#phy214244-bib-0014){ref-type="ref"}; Chen et al. [2015](#phy214244-bib-0004){ref-type="ref"}; Clark et al. [2017](#phy214244-bib-0005){ref-type="ref"}). As a result, upstream signaling or alternative mechanisms to mimic *NOTCH1* loss of function have been sought as an explanation for idiopathic CAVD. Recently, long noncoding RNA *H19* has been found to be highly upregulated in stenotic and sclerotic human aortic valves (Hadji et al. [2016](#phy214244-bib-0011){ref-type="ref"}; Merryman and Clark [2016](#phy214244-bib-0018){ref-type="ref"}). Furthermore, *H19* was shown to competitively bind to the promoter region of *NOTCH1* in aortic valve interstitial cells, preventing P53 recruitment and subsequent *NOTCH1* transcription (Fig. [1](#phy214244-fig-0001){ref-type="fig"}A). This effectively suppresses *NOTCH1*, leading to calcification even in the absence of a *NOTCH1* mutation. However, this makes the means by which *H19* expression becomes so dramatically upregulated a critical question to understand disease initiation. {#phy214244-fig-0001} *H19* is highly conserved among mammals and is found in an imprinted locus near insulin‐like growth factor 2 (*Igf2*) (Bartolomei et al. [1991](#phy214244-bib-0002){ref-type="ref"}). This locus is epigenetically regulated by an imprinting control region (ICR), located between the two genes (Fig. [1](#phy214244-fig-0001){ref-type="fig"})B and C) (Bartolomei et al. [1991](#phy214244-bib-0002){ref-type="ref"}). On the maternally inherited allele, the ICR binds CCCTC‐binding factor (CTCF), which serves as an insulator between *Igf2* and enhancer elements downstream of *H19*. As a result, shared enhancers promote expression of *H19* while *Igf2* is silenced (Fig. [1](#phy214244-fig-0001){ref-type="fig"}B). On the paternally inherited allele, methylation of the ICR prevents CTCF binding, allowing the downstream enhancers to activate *Igf2* expression while *H19* is silenced (Fig. [1](#phy214244-fig-0001){ref-type="fig"}C) (Engel et al. [2008](#phy214244-bib-0006){ref-type="ref"}). This imprint is established during embryonic development and persists through the life of the organism, though disruptions in this epigenetic signature could lead to rapid changes in *H19* expression (Gabory et al. [2006](#phy214244-bib-0008){ref-type="ref"}). Hadji et al. showed that hypomethylation in the promoter region of *H19* was associated with increased expression in calcified human aortic valves, even though the imprint was maintained (Hadji et al. [2016](#phy214244-bib-0011){ref-type="ref"}). More recently, Agba et al. showed evidence for age‐associated hypomethylation in the *H19*/*Igf2* ICR of rats, suggesting a loss of imprint which correlated with increased *H19* expression (Agba et al. [2017](#phy214244-bib-0001){ref-type="ref"}). Together, these findings suggest an epigenetic mechanism by which *H19* may become upregulated with advanced age and lead to CAVD via the *NOTCH1* pathway, even in the absence of a genetic mutation. The goal of this study was to determine if these findings were replicated in mouse aortic valves, and if age‐related *H19* expression is a mechanism for CAVD in a mouse model. Because *H19* is known to be involved in other cardiovascular diseases, expression levels were also assessed in ventricular tissue and the aortic arch, as well as the liver, which is known to express higher levels of *H19* and was previously shown to exhibit age‐related loss of imprint. While many existing mouse models of CAVD consider only male mice, CAVD is also highly prevalent in women, thus motivating our investigation into the effect of sex on *H19*‐driven calcification (Owens et al. [2010](#phy214244-bib-0021){ref-type="ref"}; Simard et al. [2017](#phy214244-bib-0023){ref-type="ref"}). Because our primary interest was in the effects of age and sex (rather than genetic mutation or chronic injury through diet) on *H19* expression in the aortic valve, these experiments were conducted in healthy C57BL/6 mice maintained on a normal diet. Methods {#phy214244-sec-0002} ======= Mice {#phy214244-sec-0003} ---- Groups of five male and female C57BL/6 mice were purchased from Jackson Laboratory at 26, 52, and 78 weeks of age. Mice were assessed via echocardiography and euthanized for sample collection within two weeks of receipt. All procedures were performed in accordance with protocols approved by Institutional Animal Care and Use Committee (IACUC) at Vanderbilt University. Echocardiography {#phy214244-sec-0004} ---------------- Mice were anesthetized with isofluorane, and a Vevo 2100 imaging system was used to acquire parasternal, short axis M‐mode images of the heart and pulsed‐wave (PW) Doppler images of the aorta immediately distal to the aortic valve. An exemplary PW Doppler scan and corresponding flow profile traces are shown in Figure [2](#phy214244-fig-0002){ref-type="fig"}A and B. VevoLAB software was used to analyze M‐mode cardiac cycles (\~9 per image) and extract PW Doppler images. A custom MATLAB script was used to isolate and average PW Doppler cardiac cycles (\~10--30 per image) in order to compute the systolic transvalvular pressure gradient and peak systolic velocity (PSV) (Ferruzzi et al. [2018](#phy214244-bib-0007){ref-type="ref"}). Ejection fraction to velocity ratio (EFVR), a metric used as an indicator of valve disease, was calculated as EFVR = (ejection fraction)/(4\*(PSV)^2^) (Cattaneo et al. [2009](#phy214244-bib-0003){ref-type="ref"}). {#phy214244-fig-0002} Microdissection and sample collection {#phy214244-sec-0005} ------------------------------------- Mice were euthanized by carbon dioxide inhalation and promptly dissected. The systemic and pulmonary circulatory systems were flushed with sterile PBS, and the aortic valve leaflets -- connected to a minimal annulus of aorta -- were dissected away from the ventricles. Samples of liver, left ventricle, and ascending aorta were also harvested and cleaned of external connective tissue and fat. Samples were flash frozen in liquid nitrogen and stored at −80°C until RT‐PCR analysis. Nucleic acid purification {#phy214244-sec-0006} ------------------------- Tissue samples were thawed at room temperature and bead homogenized in 400 *μ*L of RLT‐plus buffer with Reagent DX to reduce foaming (Qiagen, Hilden, Germany) in Lysing Matrix D tubes (MP Biomedicals, Santa Ana, CA) until no visible tissue remained. RNA and gDNA were purified using the Qiagen AllPrep Micro kit following manufacturer's instructions and were stored at −80°C until further analysis. Real‐time PCR {#phy214244-sec-0007} ------------- Reverse transcription was performed using the SuperScript IV Reverse Transcriptase kit with oligo(dT) primer (ThermoFisher Scientific, Waltham, MA). RT‐PCR was performed with equal amounts of cDNA using iQ SYBR Green Supermix (Bio‐Rad, Hercules, CA) and gene specific primer sequences (Table [1](#phy214244-tbl-0001){ref-type="table"}). Gene expression was normalized to the geometric mean expression of *Gapdh*, *Tuba1b*, and *Actb*, which was found to be more stable than any housekeeping gene in isolation. For visual clarity, gene expression was normalized to the highest expressing sample of each gene. ###### RT‐PCR primer sequences Gene name Forward primer[1](#phy214244-note-0002){ref-type="fn"} Reverse primer[1](#phy214244-note-0002){ref-type="fn"} ----------- -------------------------------------------------------- -------------------------------------------------------- *Gapdh* ATGACAATGAATACGGCTACAG TCTCTTGCTCAGTGTCCTTG *Tuba1b* CCGGTGTCTGCTTCTATCTC CCATGTTCCAGGCAGTAGAG *Actb* CAAGCAGGAGTACGATGAGTC AACGCAGCTCAGTAACAGTC *H19* GGAATGTTGAAGGACTGAGGG GTAACCGGGATGAATGTCTGG *Igf2* CGCTTCAGTTTGTCTGTTCG GCAGCACTCTTCCACGATG *Notch1* ATGTCAATGTTCGAGGACCAG TCACTGTTGCCTGTCTCAAG *Bmp2* TTATCAGGACATGGTTGTGGAG GGGAAATATTAAAGTGTCAGCTGG Primer sequences are from 5′‐3′. John Wiley & Sons, Ltd Methylation analysis {#phy214244-sec-0008} -------------------- Pyrosequencing was performed as previously described with the following modifications. 40 ng of bisulfite treated DNA was used as input, and 5 *μ*L of the biotinylated PCR product was used for each sequencing assay (Susiarjo et al. [2013](#phy214244-bib-0025){ref-type="ref"}). Statistics {#phy214244-sec-0009} ---------- All values are presented as mean ± standard error. Two‐way ANOVA with post hoc Holm‐Sidak test for multiple comparisons was used to detect differences between age and sex. In the event that conditions of normality or equal variance were not met, one‐way ANOVA on ranks was used to detect differences due to age within each sex, and the Mann‐Whitney rank sum test was used to detect difference due to sex at a specific age. Potential correlations between measured variables were assessed by the Pearson product‐moment correlation r. For all statistical tests, a value of *P* \< 0.05 was considered significant. Results {#phy214244-sec-0010} ======= Aortic valve health diverges, but does not worsen with age {#phy214244-sec-0011} ---------------------------------------------------------- Analysis of PW Doppler images revealed no changes with age in PSV or peak transvalvular pressure gradient, although as whole cohorts by sex, male mice had higher PSV than females (*P* = 0.032) (Fig. [2](#phy214244-fig-0002){ref-type="fig"}C and D). While typical values for aortic PSV in healthy BL6 mice fall below 150 cm/sec, we identified one 78‐week‐old male mouse had a PSV over 200 cm/sec, indicative of aortic stenosis, and several older male and female mice had velocities between 150 and 200 cm/sec, indicative of early stenosis (Hinton et al. [2008](#phy214244-bib-0012){ref-type="ref"}). Male mice also had higher systolic gradients (*P* = 0.032). None of the mice showed signs of left ventricular hypertrophy, indicating that observed hemodynamic changes were early stage, prior to extensive cardiac remodeling. Curiously, the 52‐ and 78‐week‐old male mice had two and three times larger variance in PSV than younger males, indicating a wide divergence of overall valve health with increased age. Cardiac function is preserved with age {#phy214244-sec-0012} -------------------------------------- Ejection fraction was extremely consistent with age in male mice (Fig. [2](#phy214244-fig-0002){ref-type="fig"}E). Female mice exhibited increasing ejection fraction with age, such that 26‐week‐old females had lower ejection fraction than 26‐week‐old male mice (*P* = 0.042) and 78‐week‐old female mice had higher ejection fractions than 78‐week‐old males (*P* = 0.005). Seventy‐eight‐week‐old females also had higher ejection fractions than 26‐ and 52‐week‐old females (*P* = 0.017, *P* = 0.025). No differences were detected in EFVR, although males again showed trends toward divergent valve health with age (Fig. [2](#phy214244-fig-0002){ref-type="fig"}F). H19 imprint and expression are not altered by age or sex in the aortic valve {#phy214244-sec-0013} ---------------------------------------------------------------------------- Pyrosequencing of the *H19* ICR in mouse aortic valve gDNA revealed no change in average methylation fraction due to age or sex (Fig. [3](#phy214244-fig-0003){ref-type="fig"}A). Likewise, there was no observed trend toward increased *H19* expression in the valve with increasing age nor correlation between methylation fraction and *H19* expression (Fig. [3](#phy214244-fig-0003){ref-type="fig"}B and C). Rather, 26‐week‐old mice had higher average *H19* expression than either the 52‐ or 78‐week‐old mice (*P* = 0.025; *P* = 0.017). Expression of *Igf2*, which is regulated by the *H19/Igf2* ICR, and *Notch1*, which was previously shown to be repressed by *H19*, also showed no clear age or gender effect, though individual comparisons reached statistical significance (Fig. [3](#phy214244-fig-0003){ref-type="fig"}C). *Bmp2*, a driver of osteogenic calcification that has been found to be increased in calcified valves, was higher in male mice than in females overall (*P* = 0.002) and specifically at 26 and 78 weeks of age (*P* = 0.015; *P* = 0.017). To examine if *H19* may still be suppressing *Notch1* on an individual level that is not revealed in group‐averaged data, we also correlated expression of *H19* and *Notch1* in individual mice, but no significant effect of *H19* expression on *Notch1* expression was detected (Fig. [3](#phy214244-fig-0003){ref-type="fig"}D). {#phy214244-fig-0003} Liver and aortic tissue show increased H19 expression in oldest mice {#phy214244-sec-0014} -------------------------------------------------------------------- Based on other studies showing *H19* upregulation in aortic aneurysm and cardiac ischemia, as well as a study that found age‐related loss of imprint in rat liver, we also probed *H19* expression changes in the liver, left ventricle, and ascending aorta (Greco et al. [2016](#phy214244-bib-0010){ref-type="ref"}; Agba et al. [2017](#phy214244-bib-0001){ref-type="ref"}; Li et al. [2018](#phy214244-bib-0016){ref-type="ref"}) (Fig. [3](#phy214244-fig-0003){ref-type="fig"}E). The 78‐week‐old male mice had higher *H19* expression than other age males in both the liver and ascending aorta. *H19* expression trended upwards with age in female liver, while the ascending aorta of 78‐week‐old female mice showed higher expression than the other female age groups and the 78‐week‐old male mice. In left ventricular tissue, no clear pattern of age‐related *H19* upregulation was observed, though the 78‐week‐old female mice had significantly lower *H19* expression than other female age groups, and 52‐week‐old males had higher expression than other male age groups. Discussion {#phy214244-sec-0015} ========== This study investigated the effects of age and sex on hemodynamic function and *H19* expression in the aortic valve. To the best of our knowledge, this is the first cross‐sectional study of a potential CAVD‐initiating mechanism in both male and female mice that did not have a CAVD‐associated mutation or receive a chronic hypercholesteremic treatment. Furthermore, this study isolated nucleic acids from individual aortic valves, enabling expression profiling and methylation analysis without requiring sample pooling. This approach represents a novel approach to probe subtle signaling which may precede or initiate valve disease in mice without introducing the biases of specific models, whereas most studies (including our own (Clark et al. [2017](#phy214244-bib-0005){ref-type="ref"})) typically utilize specific mutations, diet, or cardiovascular injury to induce symptoms of CAVD, which may bias or obscure subtle signaling changes early in disease progression. We found that the *H19*/*Igf2* ICR does not undergo age‐related hypomethylation and *H19* is not upregulated in mouse aortic valves with age alone. This contrasts with expression data from other tissues such as liver and ascending aorta, as well as with prior studies in rats that showed loss of ICR methylation and *H19* upregulation in a tissues such as brain and skin (Agba et al. [2017](#phy214244-bib-0001){ref-type="ref"}). The rats used in that study and the mice used here included animals comparable to 60‐ to 70‐year‐old humans, well within a timeframe at which early signs of CAVD could be expected. These results suggest a mechanism for preserving *H19* imprinting and low expression levels that differs by tissue type or species. In addition to the lack of a robust increase in *H19* expression, we did not observe substantial correlation between expression of *Notch1* and *H19* in the aortic valve. Previous studies have shown that elevated *H19* can repress *NOTCH1* in human aortic valve interstitial cells and mouse brain tissue (Hadji et al. [2016](#phy214244-bib-0011){ref-type="ref"}). A potential explanation for this discrepancy is a threshold effect, in which *H19* expression must reach a certain critical level before measurably repressing *Notch1*. Further work with a titratable *H19* overexpression system may be a useful tool to answer this question. Despite the lack of strong *H19* upregulation, a few of the oldest mice showed signs of early aortic stenosis as indicated by elevated PSV and transvalvular pressure gradient. These functional indicators of stenosis even in the absence of *H19* upregulation underscore the heterogeneous nature of CAVD and suggest that *H19* is not the sole initiator of disease. Nearly 30 different mouse models of CAVD exist, and while many of these models are convergent, it is clear that multiple distinct mechanism can initiate and drive CAVD (Sider et al. [2011](#phy214244-bib-0022){ref-type="ref"}; Hutcheson et al. [2014](#phy214244-bib-0014){ref-type="ref"}; Lerman et al. [2015](#phy214244-bib-0015){ref-type="ref"}). Certain sex‐related differences emerged in this study which corroborate known statistics of human disease. For example, male mice had higher overall PSV values that trended upwards with age and higher *Bmp2* expression, indicative of onset of stenosis with activation of calcific pathways. This matches with clinical data showing that men are more likely to develop CAVD than women and that features of the disease (fibrosis and calcification) differ between sexes (Owens et al. [2010](#phy214244-bib-0021){ref-type="ref"}; Simard et al. [2017](#phy214244-bib-0023){ref-type="ref"}). Still, CAVD is relatively prevalent in women, and the differences in disease progression between sexes may direct more personalized treatment strategies. Despite this, many studies either do not distinguish between male and female mice or use only males. The work presented here included both male and female mice in order to better capture any differences in *H19* regulation and valve stenosis. Although age‐related upregulation of *H19* was not found in the aortic valve, it was confirmed in the liver and reported for the first time in the ascending aorta. It was also not found to increase in the left ventricle, showing a high degree of tissue specificity in overall expression regulation. Many studies have identified *H19* as a biomarker or driver of other cardiovascular diseases such as aortic aneurysm, smooth muscle cell apoptosis, endothelial cell aging, and ischemic heart failure (Greco et al. [2016](#phy214244-bib-0010){ref-type="ref"}; Li et al. [2018](#phy214244-bib-0016){ref-type="ref"}; Hofmann et al. [2019](#phy214244-bib-0013){ref-type="ref"}). Our findings of tissue‐specific differences in age‐related upregulation of *H19* may inform future work looking at disease initiating events in these other pathologies. This study is not without its limitations. One of the biggest challenges was the sample size -- both of individual aortic valves and the overall size of the cohort. To obtain testable quantities of RNA and DNA, valves were used entirely for nucleic acid extraction. Alternate methodologies such as histology, immunohistochemistry, or *in situ* hybridization may have revealed more information about the extent of valve remodeling and stenosis. Furthermore, the high cost of raising or purchasing aged mice prevented higher numbers in each cohort, which may have increased the statistical power and significance of these results. Finally, the methylation sites tested in this study were within the ICR, which is typically considered to be the site of most epigenetic regulation of *H19* expression and was shown to be hypomethylated with age in work by Agba et al. ([2017](#phy214244-bib-0001){ref-type="ref"}). However, Hadji et al. report a stronger association of *H19* expression with a particular CpG site in the promoter region of *H19* (Hadji et al. [2016](#phy214244-bib-0011){ref-type="ref"}). Despite this difference, we did not observe the increase in *H19* expression that was clearly reported in their work. This work shows a lack of age‐related epigenetic dysregulation of *H19* in mouse aortic valves, even while other tissue types demonstrated consistent upregulation of *H19* expression. Nevertheless, echocardiographic metrics and changes in gene expression in individual mice showed signs of valve remodeling and early stenosis. Together, these results show that *H19* loss of imprint and subsequent upregulation are not common in the aortic valve of old mice, and that increased *H19* levels do not appear to be a prerequisite for early stage valve disease. Conflict of Interest {#phy214244-sec-0017} ==================== No conflicts of interest, financial or otherwise, are declared by the authors. We thank the Vanderbilt University Mouse Cardiovascular Pathophysiology Core which performed echocardiography. We also thank Matthew Bersi and Alison Schroer for their editorial help in manuscript preparation.

Background {#Sec1} ========== Falls due to balance and gait impairments are one of the most important health and quality of life issues in the elderly. Parkinson Disease (PD) is responsible for more falls than any other chronic disease and imposes a heavy burden on over 3 % of people over 65 years \[[@CR1]\]. Until recently, gait and balance were largely perceived as automated, biomechanical processes that did not require cortical control. However, work over the past decade has demonstrated the important relationships between balance/gait/falls and cognition \[[@CR2], [@CR3]\]. In fact, 60 % of older people with cognitive impairment fall annually, approximately twofold more than cognitively intact peers, and the worse the cognitive deficits, particularly executive dysfunction, the more often people fall \[[@CR4]--[@CR6]\]. These studies support the notion that mobility and cognition are connected, perhaps because mobility relies upon common cortical-subcortical networks subserving cognition and balance. Executive function, defined as a set of higher order cognitive processes that control, integrate, organize and maintain other cognitive abilities, is often altered in people with PD. \[[@CR7]--[@CR9]\] Specific deficits include response inhibition, set switching and updating of working memory \[[@CR10]--[@CR12]\]. All of these executive components are required for functional mobility in everyday environments and the relationship between such function with balance and rehabilitation has not been explored in people with PD \[[@CR2], [@CR13]\]. Imaging studies also suggest a link between structural and functional changes in the frontal lobe (a region commonly associated with cognitive function) and mobility. A recent European study of 415 older people used diffusion tensor imaging (DTI) to show that parkinsonian signs of slow walking speed, and falls are related to white matter loss in the frontal cortex, but not the basal ganglia \[[@CR14]--[@CR16]\]. Likewise, a recent systematic review that included eighty-six published studies using various imaging modalities to relate neuroimaging to mobility reported that there were consistent finding across imaging modalities linking frontal lobe measures with mobility performance \[[@CR17]\]. Specifically, this targeted review supports an increased cortical control of gait in aging, reduced volume in several regions of grey and white matter that relate to impaired mobility and consistent neuroimaging findings that reveal the basal ganglia, parietal and frontal cortices and cerebellum are related to mobility outcomes \[[@CR17]\]. Recent work identifying the locomotor neural network, which includes the i) supplementary motor area, ii) subthalamic nucleus, iii) mesencephalic and iv) cerebellar locomotor regions, also provides evidence for reduced structural and altered functional connectivity in people with PD \[[@CR18], [@CR19]\]. Though mobility and cognitive function may be related, rehabilitation of such deficits typically remains separate. There is some evidence that cognitive training may improve motor function and that mobility training may improve cognitive function in the elderly but a recent meta-analysis on this topic revealed limited and low quality studies \[[@CR20]\]. Although many individual studies of exercise and rehabilitation interventions report success in improving balance and gait in people with PD, the overall effect size of many interventions are sometimes minimal, often not reaching the minimally important change and/or minimal detectable change levels \[[@CR20]--[@CR25]\]. The limited success of rehabilitation treatment for mobility problems in people with PD may be because current physical therapy treatment does not directly address deficits related to frontal cortex dysfunction, such as cognition and cognitive control of balance and gait. We recently documented that rehabilitation using the Agility Boot Camp (ABC) training resulted in multiple improvements in mobility such as turning, gait speed, sit-to-stand, and balance in people with PD. \[[@CR26]\] With our increasing interest in the interaction of cognition and mobility, we adapted the ABC program to incorporate additional cognitive, particularly executive function, challenges known to be impaired in people with PD, now called Agility Boot Camp-Cognition (ABC-C). The purpose of this study is to 1) determine if people with PD can improve mobility and/or cognition after partaking in the ABC-C program compared to a control intervention of education and 2) determine if cognition and postural, cognitive, and brain posture/locomotor circuitry deficits predict responsiveness to the cognitively challenging Agility Boot Camp (ABC-C) rehabilitation. Our underlying hypothesis is that frontal lobe connections with the basal ganglia and brainstem posture/locomotor centers play a large role in postural deficits in people with PD and that postural impairments will be related to executive cognitive impairments. We will also determine which postural, cognitive and circuitry impairments predict efficacy of cognitively-challenging mobility rehabilitation. Methods/design {#Sec2} ============== The study is a cross-over, randomized, controlled trial design to determine responsiveness to the ABC-C exercise program. The study will include 120 people with PD (Fig. [1](#Fig1){ref-type="fig"}). People will be randomized into either an exercise (case) or education (control) 6-week intervention period. They will cross over after 6 weeks to receive the other treatment. Both interventions were designed to have the same frequency and will be delivered by the same exercise trainers for all sessions. Preceding the interventions, all patients will be tested on multiple measures of mobility cognition, and imaging. This same battery of tests will be administered after 6 weeks of intervention before the participants cross over into the second intervention. A final assessment will occur at the end of the second and final treatment arm (Table [2](#Tab2){ref-type="table"}). All other interventions (medication, other interventions, exercise) will be kept as stable as possible and any changes in medication will be monitored. This trial is registered at clinical trials.gov (NCT02231073) and OHSU ethics committee has approved all aspects of the study. All research is in compliance with the Helsinki Declarations.Fig. 1Consort diagram Participants {#Sec3} ------------ Inclusion criteria for PD recruitment are a) ages 50--90 years old, without major musculoskeletal or peripheral or central nervous system disorders (other than PD) that could significantly affect their balance and gait, b) no recent changes in medication, excessive use of alcohol or recreational drugs, c) no history of structural brain disease, active epilepsy, stroke or acute illness, factors affecting gait such as severe joint disease, weakness, peripheral neuropathy with proprioceptive deficits, severe peripheral vascular occlusive disease, severe musculoskeletal disorders, uncorrected vision or vestibular problems, or dementia that precludes consent to participate or ability to follow testing procedures, d) ability to stand or walk for 2 min with or without an assistive device, e) no medical condition that precludes exercise, f) no claustrophobia, severe tremor, or any health history (implanted devices, Deep Brain Stimulation, etc.) that would interfere or put the subject at risk near the powerful magnetic field of the MRI scanner. Though the majority of participants will have the diagnosis of idiopathic PD a subset of people with frontal gait disorder may be included for pilot data analysis. Participants will also be assessed at baseline on the Montreal Cognitive Assessment as a global screening of cognition. All subjects will be tested at Oregon Health & Science University. All subjects will sign an informed consent and no minors will be included in the study. Idiopathic PD inclusion criteria {#Sec4} -------------------------------- The United Kingdom Brain Bank criteria will be used. i.e., bradykinesia and at least one of the following: rest tremor (4--6 Hz), muscular rigidity, and postural instability not cause by visual, vestibular, cerebellar or proprioceptive dysfunction. Three or more of the following must be present for diagnosis of idiopathic PD: unilateral onset, rest tremor, progressive, persistent asymmetry, excellent response to levodopa, levodopa-induced dyskinesia \[[@CR27]\]. All PD subjects will be Hoehn and Yahr Levels II-IV and responsive to levodopa. Sample Size {#Sec5} ----------- We computed power to detect differences between the intervention arms in a crossover trial via analysis of variance with level of significance set to 0.05 (SAS software v9.3). We assumed baseline levels and patterns of change similar to those reported in King 2013 \[[@CR28]\]. We examined the significance of treatment effect from 1000 simulated replications of a crossover trial. The trial is comfortably powered (85 %) with 120 patients if total Mini-BESTest score increases by 2.4 points on average in the ABC arm, and only 1.3 points on average in the control arm. We predict a roughly 20 % drop-out rate, which is conservative based on our previous experience with short duration trials of rehabilitation in PD and by others \[[@CR24], [@CR25], [@CR28], [@CR29]\]. Randomization and blinding {#Sec6} -------------------------- Subjects will be randomly assigned per centralized database; Research Electronic Data Capture (REDCAP) to either Education (ED) or Exercise (EX) first after passing the phone screening procedure. A computerized block randomization in will be centrally held in the redcap database-scheduling mode. Randomization will be implemented by an independent statistician using a block size of twelve subjects. The exercise trainer (unblinded) will notify the subjects by phone. The researchers who will be performing all pre, mid and post-tests will remain blinded to group assignment throughout the duration of the study. Intervention {#Sec7} ------------ ### Exercise {#Sec8} Subjects will participate in an 80-minute, group (6 per group) exercise session led by a certified exercise trainer knowledgeable in the ABC-C program for 3 days per week for 6 weeks. Trained research assistants will spot participates with high fall risk. Although the literature does not provide a clear dose--response for balance exercise intervention, there is consensus that a challenging program at higher doses (at least 2×/week) will show improvement \[[@CR30], [@CR31]\]. The exercise protocol is an adaptation of our ABC exercise program for PD (Table [1](#Tab1){ref-type="table"}) \[[@CR26]\] The theoretical basis for ABC is based on research from our laboratory and others that identified the primary neurophysiological and cognitive constraints that limit balance and mobility in PD \[[@CR26], [@CR28]\]. The exercises are designed as a circuit to challenge movement-skills known to be impaired in PD. Stations will include: (1) Gait training (2) PWR Moves © \[[@CR32]\], (3) Agility course, (4) Lunges, (5) Boxing and (6) Tai Chi \[[@CR33]\]. Each activity was chosen for its inherent focus on multi-directional movements, dynamic postural transitions, axial mobility, big movements and whole body motor sequencing. Each station is engaged for 10--20 minutes with rest periods in between stations. Each station is systematically progressed from beginning to intermediate to advanced levels by challenging: (1) divided attention with secondary cognitive tasks, (2) response inhibition, (3) limiting external sensory cues, (4) increasing speed and resistance.Table 1Overview of exercise and education interventionsEDUCATIONEXERCISEFrequencyOne 90 min session/week, five 30 min relaxation sessions/week at home\ TOTAL: 240 min/week of education and relaxationFrequencyThree 80 min sessions/week: TOTAL: 240 min/week of exerciseTopicGoalStationTimeGoalProgressionResourcesTopic: Finding information on PD, communicating effectively with health care providers and building a support team.Gait training10Warm up, big steps, arm swingSpeed, UE support (poles), cues for big steps, ankle weights, cognitive taskSleep, Fatigue, PainTopic: Solutions to common sleep, pain and fatigue problem in PD.PWR! Moves20Aerobic, whole body sequencing, amplitude training3 levels of difficultyNutritionTopic: Healthy eating guidelines, normal serving sizes, nutrition, and meal planning.Lunging15Multi-directional, anticipatory control, dual task, executive functionSequences, visual cues, UE support, cognitive taskDifficult emotions, stress and depressionTopic: Solutions to common emotional ups and downs that accompany chronic illness.Agility10Turns, multi-directional, cognitive taskSpeed, cognitive taskCommunicationTopic: Improving communication (verbal, voice tone and body language).Boxing10Powerful movements, dual task, anticipatory movement, executive functionResponse inhibition, hand weights, cognitive taskMedicationTopic: Purposes, effects and responsibilities of common PD related medication.Tai Chi15Weight shifting, limits of stability, postural responses, step initiation3 levels of difficulty ### Education {#Sec9} The Education program is a chronic disease education program to teach patients how to live better with their chronic condition. It was developed by our research team to be specific for people with PD. It will include content and discussion of topics such as sleep, nutrition, and medication management (Table [1](#Tab1){ref-type="table"}). Classes will consist of a group of subjects (up to 6) meeting with the trainer for 90-minute session, once a week for six weeks. In order to match dose of the education intervention with the exercise intervention, participants will be provided relaxation tapes to be used at home 5 times per week for 30 minutes for an overall education dose of 240 minutes; similar to the exercise dose \[[@CR34]\]. Compliance will be recorded at each session by the exercise trainer for both exercise and education. For the education arm, participants will record compliance for the relaxation sessions in a logbook. The trainer will code progression of exercise difficulty at the end of each week to determine the level of exercise progression for each person. Additionally, participants will state the level of perceived exertion (0--10 scale) after each exercise session. People will also wear hear rate monitor during class in order to assess the aerobic level of work being performed during exercise. ### Assessment procedures {#Sec10} All people who are eligible per phone screening will come into the clinic for the informed consent process. An investigator will verbally explain the consent form, allow the person ample time to ready through the consent form and then will acknowledge consent by signing the form. All subjects will first read and sign consent forms. All outcomes will be measured in the practical OFF levodopa state (12 hours withdrawal). At baseline, people will be assessed on imaging, mobility and cognitive measures and will repeat the mobility and cognitive measures after the 1st six-week intervention and again after the 2nd six-week intervention (Table [2](#Tab2){ref-type="table"}).Table 2Secondary outcome measures by domainsDomain TestedTestDescriptionClinicalMDS-UPDRSAll sections of the Unified Parkinson's Disease Rating Scale will be used to measure related to severity of PD.NFOGQThe New FoG of Gait Questionnaire will be used to identify 'freezers' (score \>3).Activities of balance confidence (ABC)The ABC measures how balance confidence limits participating in the community.PDQ-39This 39 item questionnaire measures multiple domains of quality of life.MobilityBalancePostural sway during 30 seconds of quiet stance with and without cognitive taskTurningSmoothness of turning measured during the 1 min turning in place (360 degree turning in place) and turns during the 2 min walk with and without a cognitive task.GaitSpatial and temporal gait metrics during walking with and without dual taskImagingHigh angular resolution diffusion imagingHigh angular resolution diffusion imaging to assess white matter microstructure. Structural connectivity of the locomotor network will be assessed using probabilistic tractography.rsfcMRIAn indirect assessment of communication between spatially disparate neural regions. Analysis is restricted to neural regions comprising the locomotor network including the supplementary motor area, subthalamic nuclei, mesencephalic locomotor regions (pedunculopontine and cuneiform nuclei), and the midline cerebellar locomotor regionCognitionGeneralScales for outcome of Parkinson's Disease-Cognition (SCOPA-COG)An instrument that was designed to assess the specific 'frontal-subcortical" cognitive deficits found in Parkinson's diseaseInhibitionStroop task, flankers, Go/nogo, Stop signal taskThe ability to deliberately inhibit dominant or prepotent responses when appropriate.ShiftingSet-Shifting, Trail making taskThe ability to flexibly alter behavior when relevant changes occur in the predefined goal or in the environment.UpdatingDot counting taskThe ability to update and monitor working memory representations.VisuospatialBenton judgment of line orientation testThe ability to identify a stimulus, its orientation, and its location. Primary outcome measure -- Mini-BESTest {#Sec11} --------------------------------------- The primary outcome measure on which the intervention study was powered is the clinical *Mini-BESTest.* \[[@CR35]\] The *Mini-BESTest* \[[@CR35]\] is a sensitive measure of dynamic balance and includes 14 items (a maximum and best score of 28) \[[@CR36], [@CR37]\]. Secondary outcome measures {#Sec12} -------------------------- Secondary measures for clinical measures, mobility (gait and balance), cognition and imaging domains are listed in Table [2](#Tab2){ref-type="table"}. Clinical Measures {#Sec13} ----------------- Our clinical tests will include assessment of quality of life, balance confidence, disease severity and freezing of gait. Specifically, the Parkinson Disease Questionnaire-39 (PDQ-39) \[[@CR38]\] will be used for quality of life, the Activities-Specific Balance Confidence Scale (ABC) \[[@CR39]\] will be used for balance confidence, the Movement Disorders Society Unified Parkinson's Disease Rating Scale ( MDS UPDRS-Motor- Parts 1-IV) will be used to measure disease severity \[[@CR13], [@CR40]\] and the new freezing of gait questionnaire will be used as a self assessment of freezing of gait \[[@CR41]\]. Mobility Testing {#Sec14} ---------------- All secondary measures of mobility come from performance of tasks while subjects are instrumented with body-worn, inertial sensors. Specifically, eight wireless, synchronized, Opal inertial sensors (APDM, Inc) will be applied with elastic Velcro bands to both feet, ankles and wrists, as well as the lumbar spine and mid sternum of the torso. Inertial sensor data collected at 128Hz will be wirelessly transferred to a laptop for automatic generation of gait and balance metrics by Mobility Lab and raw data for further analysis with Matlab \[[@CR42]\]. Participants will perform tasks of quiet stance, the 2-minute walk test and the 360-degree turn test with and without a secondary, cognitive task (Table [2](#Tab2){ref-type="table"}). All participants will wear a safety belt during the walking tasks and a trained research assistant will walk along side the participants. If a participant loses his or her balance, the research assistant will assist and prevent a fall. All safety measures will be taken to ensure a secure and comfortable environment. To prevent fatigue, participant will be repeatedly reminded that he or she may take a break whenever needed. Cognitive testing {#Sec15} ----------------- Participants will complete a battery of cognitive tests to assess several dimensions of cognition. A table of all cognitive tests and a brief description is presented in Table [2](#Tab2){ref-type="table"}. Most tests are categorized into the three domains of executive function defined by Miyake and colleagues (2000): *inhibition* (Stroop color-word test, Flankers, Go/NoGo, Stop Signal Reaction Time Test) \[[@CR43]--[@CR46]\], *set shifting* (Trail Making, Shifting Task) \[[@CR45], [@CR47]--[@CR49]\] and *updating or working memory* (Dot Counting Test) \[[@CR45]\]. We will also assess general cognition for people with PD via the Scales for Outcome of Parkinson's Disease-Cognition (SCOPA-COG) and Montreal Cognitive Assessment (MoCA) \[[@CR50], [@CR51]\]. In addition, visuospatial function (Benton Judgement of Line Orientation) will be assessed. Imaging {#Sec16} ------- The subject will be led into the MRI magnet room and positioned on the MRI system bed. A 32-channel Siemens rf-receiver coil will be placed appropriately for brain MRI, and the subject will be loaded into the magnet. During imaging, the subject's head will rest on a special neck and head pillow to minimize head movements. Extra pillows under the knees and back will be used to make subjects as comfortable as possible. They will wear headphones to dampen noise during imaging and to allow subjects to hear and talk to the investigators at all times. Our procedures do not include administration of MRI contrast agents. The following protocol will then be executed: Imaging data will be acquired using a 3.0 T Siemens Magnetom Tim Trio scanner with a 12-channel head coil. We will collect one whole brain high-resolution structural T1-weighted MPRAGE sequence (sagittal, TE = 3.58 ms, TR = 2300 ms, 256 x 256 matrix, resolution = 1 mm^3^, 1 average, total scan time = 9 min-14 s). We will also collect high angular resolution diffusion imaging using an echo-planar imaging sequence (72 different gradient directions, b-value = 3,000 mm/s^2^, TR = 7100 ms, TE = 112 ms, 2.5 mm^3^ voxels, 48 slices, FOV = 230 × 230 mm). Finally, we will acquire a resting-state functional MRI (*rs*-*f*MRI) using a gradient-echo echo-planar imaging sequence (TR = 2500 ms, TE = 30 ms, FA = 90°, 3.8 mm^3^volexs, 36 slices with interleaved acquisition, FOV = 240 × 240 mm). We will acquire two 10-minutes runs, providing a total of 20 minutes of resting state data for each subject in the study. Subjects will be instructed to lie still and keep their eyes open. Head padding will be provided to help subjects keep their heads still, earplugs will protect against scanner noise, and a leg bolster will be provided for back comfort. The technician will monitor the data and collect an extra scan if head movement \> 1 mm is apparent. ### Statistical Analysis {#Sec17} First, we will compare the amount of improvement in the Mini-BESTest with an analysis of covariance (ANCOVA) model, controlling for age. It is possible that lower functioning patients will have larger adaptation to ABC-C since they start out with less function. Alternatively, it is possible that the lower functioning patients will have poorer exercise tolerance so will improve less so we will also control for baseline Mini-BESTest*.* Because we are utilizing a crossover design, the treatment effect will represent change during the ABC-C versus control rehabilitation periods regardless of whether a patient received the ABC-C intervention during period 1 or period 2. We will assess, but do not anticipate, period by treatment group interaction effects. We will also enter an interaction term into the model to assess for differential intervention effects cognitive and/or frontal lobe structural and functional connectivity status. Second, we will relate the percent improvement in the Mini-BESTest with ABC-C intervention with baseline measures of posture/gait impairments, cognitive impairments and both structural and functional connectivity of the Posture/Locomotor circuit at baseline with linear regression models. Previous studies have shown the Minimal Detectable Change of the Mini-BESTest was 3 points \[[@CR52]\]. We will also determine how many subjects move from high- to low-fall risk; a cutoff score for identifying PD fallers from non-fallers with the Mini-BESTest is 19/28 (63 %), with a sensitivity of .98 in 80 subjects \[[@CR53]\]. Discussion {#Sec18} ========== The overarching goal of this study is to determine if cognitive function and frontal brain circuitry deficits predict responsiveness to exercise. Specifically, we are interested in understanding if certain phenotypes that best predict responsiveness to high intensity, short duration agility rehabilitation with a focus on cognition will help guide therapists to identify candidates for therapy and to develop specific therapy for specific types of mobility disabilities from parkinsonism. To date, there are very few studies on cognitive contributions to gait and balance as they relate to rehabilitation, particularly in this challenging population. Currently, physical therapists do not routinely incorporate cognitive challenges for people with PD and furthermore, it is unclear if these patients will benefit from such training. If we find that executive function deficits and reduced structural and/or functional connectivity of the locomotor circuitry predict poor responses to challenging balance rehabilitation, that supports our hypothesis that frontal (and prefrontal) lobe impairments limits rehabilitation efficacy. If particular impairments of balance and gait improve more than others with ABC-C, this information will be used to improve the ABC-C intervention and will be followed by studies focused on determining which postural domains are most amenable to improvement with rehabilitation. The results from this study will further our understanding of the relationship between cognition and mobility with a focus on brain circuitry as it relates to rehabilitation potential. ABC : Agility boot camp ABC-C : Agility boot camp-cognition ABC : Activities-specific balance confidence scale ANCOVA : Analysis of covariance DTI : Diffusion tensor imaging ED : Education EX : Exercise MoCA : Montreal cognitive assessment MDS UPDRS : Movement disorders society unified Parkinson's disease rating scale PD : Parkinson disease PDQ-39 : Parkinson disease questionnaire-39 REDCAP : Research electronic data capture *rs*-*f*MRI : Resting-state functional MRI SCOPA-COG : Scales for outcome of Parkinson's disease-cognition **Competing interests** OHSU and Dr. Horak have a significant financial interest in APDM, a company that may have a commercial interest in the results of this research and technology. This potential institutional and individual conflict has been reviewed and managed by OHSU. No other authors have a competing interest. **Authors' contributions** LK participated in design of study, development of exercise intervention and drafting of manuscript, DP participated in design of study, development of exercise intervention with cognitive adaptations, editing of manuscript, MM participated in design of study, editing of manuscript, PCK participated in design of study, editing of manuscript, BF participated in design regarding imaging aspect of study, editing of manuscript, KS participated in role of cognitive function in the design of study, editing of manuscript, JN participated in study design, medical management and provided expertise on inclusion and exclusion, edited manuscript, MD participated in medical management and provided expertise in inclusion and exclusion, edited manuscript, JC designed the educational intervention and helped with study design, KWS helped with overall exercise intervention design and edited manuscript, FH conceived of the study, participated in design of study, editing of manuscript. All authors read and approved the manuscript. Research assistants including Natassja Pal, Michael Fleming and Graham Harker, Clayton Swanson and Heather Schlueter for data collections, Nancy Nelson and Nancy Barlow for their help with the exercise and education interventions, Fuzhong Li for help in the Tai chi portion and Becky Farley and Claire McLean for their help in the PWR portion of the exercise. The National Institute of Health has funded all aspects of this study and they do not have a role in study design, data collection, analysis, interpretation, writing or where we decide to submit further manuscripts.

Introduction {#s1} ============ Chronic pain is a highly prevalent and disabling condition with major impact on individuals, their significant others, and society (Turk et al., [@B76]). Prevalence rates for chronic pain range from 10 to 30% (Reid et al., [@B65]), and Major Depressive Disorder (MDD) is the most frequent psychiatric disorder in patients with chronic pain, with a 12-month prevalence ranging from 18% in population based settings up to 85% in specialized pain clinics (Bair et al., [@B3]). Since patients who suffer from both chronic pain and depression are particularly difficult to treat (Tunks, [@B75]), more effective interventions for this population are needed. Mindfulness-based interventions have recently been shown to be effective for the treatment of chronic pain with small to moderate effect sizes on pain and depression (Veehof et al., [@B79]). Mindfulness-based therapies, and particularly mindfulness based cognitive therapy (MBCT), also have been shown to be effective for relapse prevention in recurrent depression and the treatment of active depression (Hofmann et al., [@B31]; Piet and Hougaard, [@B61]; Marchand, [@B46]; Sipe and Eisendrath, [@B71]). Results from a recent pilot randomized controlled trial (RCT) suggested that MBCT may be an effective intervention for the treatment of active depression in a population with chronic pain (de Jong et al., [@B15]). Because mindfulness-based interventions seem beneficial for chronic pain and depression, the question arises how mindfulness exerts its effects. Mindfulness entails paying attention to present moment experience, including thoughts, emotions, and bodily sensations. Training body awareness is a significant component of most mindfulness-based interventions, including the body scan, in which individuals specifically pay attention to all parts of the body; and yoga, which entails paying attention to movements of the body (Kabat-Zinn, [@B40]; Segal et al., [@B70]). Body awareness has been proposed as a potential mechanism for the therapeutic effects of mindfulness and is considered an integral part of the mindfulness construct (Mehling et al., [@B49]; Hölzel et al., [@B33]; Farb et al., [@B19]). The definition of body awareness emphasizes the fact that this is a complex multi-dimensional construct: "the sensory awareness that originates from the body\'s physiological states, processes (including pain and emotion), and actions (including movement), and functions as an interactive process that includes a person\'s appraisal and is shaped by attitudes, beliefs, and experience in their social and cultural context" (Mehling et al., [@B51]). In this article, the terms body awareness and interoceptive awareness are used interchangeably. Several studies lend support to the notion of enhanced body awareness through mindfulness training. For example, meditators have been reported to show greater coherence between objective physiological data and their subjective experience---in regard to both emotional experience (Sze et al., [@B73]) and sensitivity of body regions (Fox et al., [@B22]). With regard to the heart beat perception task, which assesses the ability of subjects to accurately determine their heartbeat rate by comparing the subjectively counted heartbeats to heartbeats measured by an electrocardiogram, a number of studies with small sample sizes did not find increased interoceptive accuracy in meditators (Nielsen and Kaszniak, [@B57]; Khalsa et al., [@B43]; Melloni et al., [@B52]; Parkin et al., [@B58]). However, a large (*N* = 160), recent longitudinal study revealed that heart beat accuracy was increased after 39 weeks of a mindfulness-based contemplative intervention (Bornemann and Singer, [@B8]). Neuroimaging studies indicate mindfulness training-related changes in brain function and structure in regions that are thought to be involved in body awareness (Lazar et al., [@B45]; Hölzel et al., [@B34]; Farb et al., [@B20], [@B21]; Gard et al., [@B23]). It has been long postulated that interoceptive awareness plays an important role in the experience of emotions (James, [@B38]) and there is empirical evidence that the extent to which one can accurately perceive bodily functions has a positive relationship with the intensity of emotions (Herbert et al., [@B30]). Former studies on this topic in clinical populations have mainly focused on anxiety disorders, which demonstrated a close association with increased interoceptive awareness (Ehlers and Breuer, [@B18]). Depression often entails anhedonia and blunted emotions. In fact, body awareness has been found to be reduced in individuals with depression (Ehlers and Breuer, [@B17]; Dunn et al., [@B16]), and higher levels of depressive symptoms are associated with decreased body awareness in healthy subjects (Pollatos et al., [@B62]). Reduction in interoceptive awareness in depression is also supported by a recent neuro-imaging study, which shows reduced effective connectivity in networks involved in interoception in patients with melancholia (Hyett et al., [@B36]). A recent study revealed that body awareness therapy resulted in decreased self-rated depressive symptoms, but no changes in body awareness were found (Danielsson et al., [@B12]). Whether improvements in body awareness lead to reduced depression has yet to be established. Variations in body awareness appear to be particularly important in patients with chronic pain. Mehling et al. ([@B48]), for example, reported differences in some dimensions of interoceptive awareness between patients with current or past low back pain and mind-body trained individuals. Neuro-scientific evidence indicates that some of the brain regions activated during pain are also activated when engaging in interoceptive awareness (Craig, [@B11]). Attention styles toward chronic pain sensations are of key importance for psychological pain management (Johnston et al., [@B39]), and fMRI studies suggest that mindfulness meditation facilitates a reduction of pain through increased sensory processing (Gard et al., [@B23]). Thus, body awareness and pain perception are closely linked on a neuro-biological level, such that the enhancement of specific styles or dimensions of body awareness may facilitate the self-regulation of pain. Although body awareness is considered an integral part of the mindfulness construct, there has been a paucity of instruments that measure body awareness (Mehling et al., [@B49]). Previous body awareness questionnaires either measured non-adaptive forms of body awareness (as indicated in disorders such as panic disorder), were uni-dimensional, lacked systematic development, or did not measure body awareness specifically, but rather a more general observation ability (Mehling et al., [@B51]). The Multidimensional Assessment of Interoceptive Awareness (MAIA; Mehling et al., [@B51]) scale is a relatively new, multifaceted body awareness questionnaire that intends to fill the apparent gap. Bornemann et al. ([@B7]), who recently translated the MAIA into German and demonstrated good scale properties, found that a 3-month contemplative training that included the bodyscan and breath meditation techniques lead to changes on several scales in a sample of individuals with good psychological and physical health. Values on all scales increased following the training, and changes were significantly greater compared to a retest control group for most of the scales. While there is evidence for an effect of a mind-body intervention on body awareness as measured with the MAIA in a healthy sample, no intervention studies have been reported on effects in chronic pain patients or in depressed individuals. In the present pilot RCT we investigated the longitudinal effects of MBCT on body awareness, as measured by the MAIA in a population of patients with chronic pain and comorbid depression. It was hypothesized that MBCT enhances aspects of body awareness in this population. Furthermore, as this is the first study using the MAIA in a sample of patients with chronic pain and comorbid active depression, we investigated the reliability of the MAIA scales in this population. Methods {#s2} ======= Participants ------------ Participants in this add-on study were part of a larger clinical trial reported elsewhere (de Jong et al., [@B15]; clinicaltrials.gov id: NCT01473615). Patients were recruited from different outpatient clinics through introduction of the study by their physicians, as well as via web-based advertisements and through several online mailing lists. After phone screening and a subsequent in-person screening visit, eligible participants were offered enrollment and were randomly assigned to treatment as usual (TAU) or TAU plus mindfulness-based cognitive therapy (MBCT). English-language literate individuals aged 18 or older were eligible if they (a) had persistent chronic pain for a minimum of 3 months; (b) met the DSM-IV criteria for MDD, Dysthymic Disorder, or Depressive disorder Not Otherwise Specified (NOS); and (c) a score ≥10 on the QIDS-C~16~ scale. After initiating the study the cutoff was reduced to a QIDS-C~16~ score ≥6 (indicative of at least mild depressive symptoms) to allow more ample recruitment. Exclusion criteria were: (a) serious suicide or homicide risk; (b) current or past bipolar disorder, current psychotic symptoms, or a current or past primary psychotic disorder; (c) diagnosis of substance abuse or dependence disorder during the last 3 months; (d) general condition that impedes attendance in group interventions, such as severe personality disorders, cognitive impairment, or tendencies toward physical aggression; (e) severe and unstable medical illness including cardiovascular, hepatic, renal, respiratory, endocrine, neurological, or hematological disease; and (f) significant present meditation practice with more than 3 h of mindfulness, insight, or vipassana meditation per week. Patients were requested to keep their psychological and pharmacological treatment as stable as possible from 8 weeks before the beginning of the study until its conclusion. Seventy-one participants were screened, of which 40 were randomized to TAU or MBCT + TAU in a 1:2 ratio (Figure [1](#F1){ref-type="fig"}). Participants received \$40 for completed study participation and provided written informed consent. The study was approved by the Partners Human Research Committee, Massachusetts General Hospital (protocol 2011-P-001699/1). {#F1} Measures -------- ### Multidimensional assessment of interoceptive awareness (MAIA) The MAIA is a 32-item instrument that assesses body awareness on 6-point Likert-type scales that range from 0 (Never) to 5 (Always). It comprises eight scales, namely Noticing, Not-Distracting, Not-Worrying, Attention Regulation, Emotional Awareness, Self-Regulation, Body Listening, and Trusting (Mehling et al., [@B51]). The eight scales have been shown to have adequate to excellent internal-consistency reliabilities, with Cronbach\'s alphas from 0.66 to 0.87, and above 0.70 for five of the eight scales (Mehling et al., [@B51]). Because the current study was an add-on to a larger study, subject burden had to be kept to a minimum. For this reason only the scales deemed most relevant for the specific population were administered, namely Noticing, Not-Distracting, Attention Regulation, Emotional Awareness, and Self-Regulation. The Noticing scale assesses the awareness of comfortable, neutral, and uncomfortable body sensations. Not-Distracting refers to not ignoring or distracting oneself from uncomfortable body sensations such as pain. Attention Regulation is the ability to maintain and regulate attention to body sensations, and Emotional Awareness is defined as consciousness of the interrelation of emotions and body sensations. Self-Regulation refers to the ability to control psychological distress by consciously attending to body sensations (Mehling et al., [@B48]). The MAIA Not Worrying scale assesses worrying or feeling emotionally distressed in response to uncomfortable body sensations including pain and was not administered, since its items are similar to the Pain Catastrophizing Scale, which was included in the larger study. ### Pain catastrophizing scale (PCS) The PCS is a 13-item scale, comprised of three subscales, that measures pain catastrophizing. Pain catastrophizing is defined as "an exaggerated negative mental set brought to bear during (actual or anticipated) painful experience" (Sullivan et al., [@B72]). The PCS total score can range from 0 to 52 and has been shown to have excellent reliability (Cronbach\'s alpha = 0.87; Sullivan et al., [@B72]). ### Quick inventory of depressive symptomatology---clinician rated (QIDS-C~16~) The QIDS-C is a widely used clinician rated instrument to assess depression symptom severity. The instrument is comprised of 16 items, has good psychometric properties, score range from 0 to 27 and is sensitive to changes in depressive severity (Rush et al., [@B68]; Trivedi et al., [@B74]). Procedure --------- Eligible participants were randomly assigned to TAU (control group) or MBCT + TAU (intervention group) in a 1:2 ratio, which allowed the main project to fill the MBCT groups with participants more quickly. An independent researcher not involved in the project generated the randomization sequence in blocks of five (using the sequence generator on [www.random.org](http://www.random.org)). In order to assure equal gender distribution in both groups we stratified for gender. The intervention group received an 8-week MBCT group skills program. Interoceptive awareness, pain catastrophizing, and depression were assessed at baseline (week 0), midpoint (week 4), and endpoint (week 8). Intervention ------------ The intervention consisted of an 8-week group skills program with one 2-h mindfulness training session each week and individual exercises for homework practice. It was modeled on the MBCT program developed by Segal et al. ([@B70]), which was developed as a program to address recurrent depressive episodes and combines elements of cognitive behavioral therapy (CBT), such as psycho-education, with experiential mindfulness practices. The program is intended to teach and foster a non-judgmental, accepting attitude toward one\'s internal, and external experience. For the current study the original program was adapted to our specific population by modifying the psychoeducation and CBT elements to a depressed CP population. This included psycho-education linking CP, negative thoughts, negative emotions, and depressive behaviors such as withdrawal; identifying automatic thoughts related to CP; and paying attention to behavioral elements such as pacing of activities. We also included meditations that specifically focused on cultivating mindfulness in relationship to CP. The MBCT program was led by two instructors, an experienced licensed independent clinical social worker (LICSW) and a fellow in psychology and was provided free of charge. Subjects that were assigned to TAU were waitlisted and offered the MBCT treatment after completion of the study. TAU included all regular visits with the pain physician, psychiatrist, psychotherapist and prescribed pain and/or antidepressant medications. Statistical analyses -------------------- Differences in patient characteristics at baseline were assessed by performing independent-samples *t*-tests for continuous variables and Chi-square tests for categorical variables. To evaluate internal consistency/reliability, Cronbach\'s alphas were assessed. Inter-scale correlations were obtained for the MAIA scales and the PCS total score based on the data of all subjects at baseline. The effects of intervention/group (MBCT + TAU vs. TAU) and time (baseline vs. endpoint) on the dependent variables body awareness and pain catastrophizing were assessed by conducting repeated measures analysis of variance (rmANOVA), with time as repeated measure, treatment group as between-subjects factor, and MAIA scales and PCS as dependent variables. Assumptions of normality and homogeneity were met. Paired samples *t*-tests were conducted to compare baseline and endpoint scores on the MAIA scales and the PCS within each group. As a measure of effect size, Cohen\'s d was calculated for each pre-post change. Analyses were conducted according to a modified intention-to-treat (ITT) principle with the last observation carried forward (LOCF). When endpoint measures were missing, midpoint measures were imputed, and if midpoint data were missing, baseline data were used. Only participants who attended at least four of the eight classes were included in the analyses. The main study, to which this study was added on, revealed a significant effect of MBCT (group by time interaction) on depression as measured with the Quick Inventory of Depressive Symptomatology-Clinician rated (QIDS-C~16~) (de Jong et al., [@B15]). To explore if and how this effects is mediated by the MAIA, a multiple mediator model was tested. The model comprised group (MBCT + TAU vs. TAU) as independent variable, depression measured at week 8 as dependent variable and MAIA scales (measured at week 8) that revealed significant group by time interactions, as mediators. Depression and respective MAIA scales measured at baseline (week 0) were included as covariates. Mediation analyses were conducted with a macro by Preacher and Hayes ([@B63]) that implements a bootstrapping procedure to create confidence intervals for partial and total indirect effects. For mediation analyses only participants who participated in at least 4 classes and who had week 0 and week 8 data available were included in the analyses and 10,000 bootstrap iterations were used. All analyses were conducted with SPSS 21 (SPSS Inc., Chicago, IL, USA). Results {#s3} ======= Participant characteristics --------------------------- For the main study, 71 patients were screened, of which 40 were randomized to TAU (*n* = 14) or MBCT + TAU (*n* = 26) in a 1:2 ratio. Of those 40, 34 completed the pain catastrophizing scale (PCS) and 31 the MAIA at baseline. Of the 14 patients in the TAU, 12 had PCS and MAIA data and were included in modified ITT analyses. Of the 26 patients in MBCT + TAU, 22 had PCS and 19 MAIA data. Five of the patients with MAIA and PCS data participated in \<4 sessions of MBCT and were excluded from further analyses, resulting in 17 patients with PCS data and 14 with MAIA data in the modified ITT analyses (Figure [1](#F1){ref-type="fig"}). For the mediation analyses 11 subjects with MAIA data were in MBCT + TAU and 7 in TAU. Characteristics of participants (*N* = 29) who attended at least four classes (MBCT+TAU) or had four clinic visits (TAU), are shown in Table [1](#T1){ref-type="table"}. Types of chronic pain included: chronic back pain, neuropathic pain, osteoarthritis, fibromyalgia, and migraines. There were no significant differences in demographics and patient characteristics (see Table [1](#T1){ref-type="table"}) or in average baseline scores on the five MAIA scales or total PCS scores between the two groups at baseline (see Table [2](#T2){ref-type="table"}). For the collapsed sample that completed the MAIA, regardless of class attendance, participants (*N* = 31) were on average 50 years old (*M* = 49.45, *SD* = 10.58), and college graduates (*M* = 16.30, *SD* = 2.55 years of education). Most of the participants were female (74.2%), Caucasian (90.3%), and non-Hispanic (83.9%). Three (9.7%) participants were African American and one (3.2%) was Hispanic. The largest proportion of participants was married (46.7%) or never married (36.7%), and five were separated or divorced (16.7%). Employed participants comprised 32.3% of the sample, and disabled ones comprised 32.3% of the sample. Most participants had (MDD; 83.9%), with the remaining 16.1% suffering from Depressive Disorder not otherwise specified (NOS). 46.4% of the participants were taking Anti-Depressant medication. ###### **Participant characteristics (*N* = 29)**. **TAU** **MBCT + TAU** ***t/χ^2^*-test** ------------------------------------------------------- --------- ---------------- ------------------- ------- ------- ---- ------- Age (years) 51.67 10.08 50.06 11.68 0.39 27 0.703 Education (years)[^a^](#TN1){ref-type="table-fn"} 16.58 2.61 15.94 2.56 0.65 26 0.519 Gender (% female) 66.7 76.5 0.34 1 0.561 Race (%) 0.88 1 0.348     African-American 16.7 5.9     Caucasian 83.3 94.1 Ethnicity (%) 3.28 2 0.194     Hispanic 0.0 5.9     Non-hispanic 100.0 76.5     Unknown/Not reported 17.6 Marital status (%)[^a^](#TN1){ref-type="table-fn"} 0.933 2 0.627     Never married 25.0 37.5     Married/Live together 50.0 50.0     Separated/Divorced 25.0 12.5 Employment status (%)[^a^](#TN1){ref-type="table-fn"} 1.67 2 0.435     Employed 16.7 37.5     Disabled 41.7 37.5     Other/Not reported 41.7 25.0 Type depression (%) 0.20 1 0.653     NOS 16.7 23.5     MDD 83.3 76.5 ADM (% taking)[^b^](#TN2){ref-type="table-fn"} 50.0 35.3 0.564 1 0.453 *ADM, Anti-Depressant Medication; MDD, Major Depressive Disorder; MBCT, Mindfulness Based Cognitive Therapy; NOS, Depressive Disorder Not Otherwise Specified; SD, Standard Deviation; TAU, Treatment As Usual*. *Based on N = 28 due to missing value*. *Based on N = 27 due to missing values*. ###### **Baseline scores on MAIA and PCS**. **TAU** **MBCT + TAU** ***t*-test** --------------------------- --------- ---------------- -------------- ------- ------ ---- ------- **MAIA**      Noticing 3.13 1.26 2.86 1.01 0.60 24 0.553      Attention regulation 2.08 1.34 2.37 1.05 0.60 24 0.550      Emotional awareness 2.65 1.63 2.71 1.29 0.11 24 0.912      Self-regulation 2.10 1.42 1.98 1.24 0.23 24 0.817      Not distracting 2.14 0.80 1.95 1.23 0.45 24 0.657 **PCS** 27.17 10.67 31.82 12.28 1.06 27 0.299 *MAIA, Multidimensional Assessment of Interoceptive Awareness; PCS, Pain Catastrophizing Scale*. Scale properties ---------------- Table [3](#T3){ref-type="table"} summarizes scale means with standard deviations, range of observed values, Cronbach\'s alphas, and range of item-scale correlations of the MAIA and the PCS for the entire sample (*N* = 31 and 34 respectively) at baseline. Cronbach\'s alphas for three of the five administered MAIA scales were excellent and ranged from 0.92 (Attention Regulation) to 0.94 (Emotional Awareness). The Not-Distracting and Noticing scales had alphas of 0.72 and 0.67 respectively. The PCS had a Cronbach\'s alpha of 0.93. ###### **Scale properties (N)**. **Scale** **Number of items** **Cronbach\'s Alpha** **Range of item-scale correlations** **Mean (SD)** **Observed** ***N*** --------------------------- --------------------- ----------------------- -------------------------------------- --------------- -------------- --------- **MAIA**      Noticing 4 0.67 0.65--0.74 3.10 (1.15) 1--5 31      Attention regulation 7 0.92 0.64--0.91 2.42 (1.22) 0.1--4.57 31      Emotional awareness 5 0.94 0.85--0.93 2.73 (1.50) 0--5 31      Self-regulation 4 0.93 0.89--0.96 1.92 (1.37) 0--4.5 31      Not distracting 3 0.72 0.49--0.84 2.04 (1.06) 0.3--5 31 **PCS** 13 0.93 0.51--0.85 30.18 (11.63) 4--52 34 *MAIA, Multidimensional Assessment of Interoceptive Awareness; PCS, Pain Catastrophizing Scale; SD, Standard Deviation*. Table [4](#T4){ref-type="table"} shows Pearson correlations between the MAIA scales. The correlations ranged from 0.76 for Self-regulation and Emotional awareness and 0.71 for Emotional awareness and Noticing, to (*r* ≤ \|0.29\|) for Not Distracting, which did not correlate significantly with any other MAIA scale. ###### **Scale-scale correlations**. **Scale** **Noticing** **Attention regulation** **Emotional awareness** **Self- regulation** **Not distracting** ---------------------- ----------------------------------------- ----------------------------------------- ----------------------------------------- ---------------------- --------------------- Noticing -- Attention regulation 0.53[^\*\*^](#TN3){ref-type="table-fn"} -- Emotional awareness 0.71[^\*\*^](#TN3){ref-type="table-fn"} 0.62[^\*\*^](#TN3){ref-type="table-fn"} -- Self-regulation 0.52[^\*\*^](#TN3){ref-type="table-fn"} 0.50[^\*\*^](#TN3){ref-type="table-fn"} 0.76[^\*\*^](#TN3){ref-type="table-fn"} -- Not distracting 0.02 0.17 −0.29 −0.09 -- *Pearson product moment correlations among MAIA scales in the total sample (N = 31)*. *Correlations are significant at p \< 0.01*. *MAIA, Multidimensional Assessment of Interoceptive Awareness*. Effects of MBCT on body awareness --------------------------------- For Noticing, no significant group-by-time interaction \[*F*~(1,\ 24)~ = 0.18, *p* = 0.676, $\eta_{p}^{2}$ = 0.007\] and no significant main effect of time \[*F*~(1,\ 24)~ = 2.59, *p* = 0.121, $\eta_{p}^{2}$ = 0.097\] was found. No significant within group changes between the pre- and the post-treatment measurements were found in either group with effect sizes of the pre-post change being medium for the treatment group but small for the control group (Figure [2A](#F2){ref-type="fig"}, Table [5](#T5){ref-type="table"}). {#F2} ###### **Changes in scores for body awareness and pain catastrophizing**. **TAU** **MBCT + TAU** --------------------------- --------------- ---------------- ------- ---- ------- -------- --------------- --------------- ------- ---- ------- -------- **MAIA**      Noticing 3.13 (1.26) 3.29 (1.20) 1.30 11 0.220 +0.134 2.86 (1.01) 3.14 (1.15) 1.21 13 0.247 +0.262      Attention regulation 2.08 (1.34) 2.58 (1.36) 2.52 11 0.029 +0.371 2.37 (1.05) 2.95 (1.01) 1.46 13 0.167 +0.564      Emotional awareness 2.65 (1.63) 2.78 (1.66) 0.73 11 0.482 +0.081 2.71 (1.29) 3.43 (1.19) 2.17 13 0.049 +0.573      Self-regulation 2.10 (1.42) 2.46 (1.62) 2.49 11 0.030 +0.216 1.98 (1.24) 3.07 (1.12) 4.34 13 0.001 +0.913      Not distracting 2.14 (0.80) 1.69 (0.81) −1.85 11 0.092 −0.553 1.95 (1.23) 2.19 (1.23) 1.20 13 0.253 +0.194 **PCS** 27.17 (10.67) 25.25 (11.52) −0.57 11 0.580 −0.172 31.82 (12.28) 24.94 (12.16) −2.23 16 0.041 −0.564 *Results of paired-samples t-tests and Cohen\'s d effect sizes for pre-post changes for treatment and control group*. *MAIA, Multidimensional Assessment of Interoceptive Awareness; PCS, Pain Catastrophizing Scale; d, Cohen\'s d; SD, Standard Deviation*. For the Attention Regulation scale, no significant group-by-time interaction effect \[*F*~(1,\ 24)~ = 0.03, *p* = 0.863, $\eta_{p}^{2}$ ≤ 0.001\] was found, but a significant main effect of time was revealed \[*F*~(1,\ 24)~ = 5.34, *p* = 0.030, $\eta_{p}^{2}$ = 0.182\]. Paired sample *t*-tests showed significant, medium size increases of Attention Regulation scores in the control group. The increase in Attention Regulation in the MBCT group did not reach statistical significance, despite its large effect size (Figure [2B](#F2){ref-type="fig"}, Table [5](#T5){ref-type="table"}). For Emotional Awareness, no significant group-by-time interaction effect \[*F*~(1,\ 24)~ = 2.17, *p* = 0.153, $\eta_{p}^{2}$ = 0.083\] was found, but a significant main effect of time was revealed \[*F*~(1,\ 24)~ = 4.63, *p* = 0.042, $\eta_{p}^{2}$ = 0.162\]. Paired samples *t*-tests revealed a large and significant increase in Emotional Awareness over time within the treatment group, while the change within the control group was small and not significant (Figure [2C](#F2){ref-type="fig"}, Table [5](#T5){ref-type="table"}). A rmANOVA revealed a significant group-by-time interaction for Self-Regulation \[*F*~(1,\ 24)~ = 5.93, *p* = 0.023, $\eta_{p}^{2}$ = 0.198\]. This interaction was driven by large and significant increases in Self-Regulation over time in the treatment group and small but significant increases in the control group. Furthermore, the analyses revealed a significant main effect of time \[*F*~(1,\ 24)~ = 22.86, *p* \< 0.001, $\eta_{p}^{2}$ = 0.488\] (Figure [2D](#F2){ref-type="fig"}, Table [5](#T5){ref-type="table"}). For the Not Distracting scale, a significant group-by-time interaction was revealed \[*F*~(1,\ 24)~ = 4.87, *p* = 0.037, $\eta_{p}^{2}$ = 0.169\]. This interaction was driven by an increase in Not Distracting scores in the treatment group and a decrease within the control group, none of which reached statistical significance. No main effect of time \[*F*~(1,\ 24)~ = 0.45, *p* = 0.511, $\eta_{p}^{2}$ = 0.018\] was found (Figure [2E](#F2){ref-type="fig"}, Table [5](#T5){ref-type="table"}). For pain catastrophizing, no significant group-by-time interaction effect \[*F*~(1,\ 27)~ = 1.15, *p* = 0.294, $\eta_{p}^{2}$ = 0.041\] was revealed. The main effect of time approached significance \[*F*~(1,\ 27)~ = 3.60, *p* = 0.069, $\eta_{p}^{2}$ = 0.118\]. Analysis showed a large and significant decrease of pain catastrophizing within the MBCT group, but only a small and not significant decrease in the control group (Figure [2F](#F2){ref-type="fig"}, Table [5](#T5){ref-type="table"}). Body awareness as mediator -------------------------- A multiple mediator model as described by Preacher and Hayes ([@B63]) was tested. The model (Figure [3](#F3){ref-type="fig"}) was comprised of group (MBCT + TAU vs. TAU) as independent variable, depression measured with QIDS-C~16~ at week 8 as dependent variable, and the MAIA Not Distracting and Self-Regulation scales, measured at week 8, as mediators. QIDS-C~16~, MAIA Not Distracting and Self-Regulation scales measured at week 0 were included as covariates. ![**Mediation model**. Mediation model for the effect of group \[Mindfulness Based Cognitive Therapy + treatment as usual (TAU), vs. TAU alone\] on depression (measured with the Quick Inventory of Depressive Symptomatology---Clinician rated) through the Not Distracting and Self-Regulation scales of body awareness as measured with the Multidimensional Assessment of Interoceptive Awareness (MAIA). Analyses are based on *N* = 18 and numbers are standardized regression coefficients. ^\*^*p* \< 0.05, ^\*\*^*p* \< 0.01.](fpsyg-07-00967-g0003){#F3} Analyses resulting in bias-corrected confidence intervals (CI) based on 10,000 bootstrap iterations, revealed a significant indirect effect of group on depression through the MAIA scale Not Distracting (a~1~ × b~1~ = −3.584, 95% CI −8.880 to −0.357), but not through the Self-Regulation scale (a~2~ × b~2~ = −2.317, 95% CI −8.733 to 0.284). There also was a significant direct effect of group on depression (c′ = 4.817, *p* = 0.0485) independent of Self-Regulation or Not Distracting. These findings indicate that the effect of MBCT on depression was partially mediated by Not Distracting but not by Self-Regulation. Discussion {#s4} ========== In the present study, we investigated the effects of MBCT on body awareness as measured with the MAIA in patients with chronic pain and comorbid active depression. To our knowledge, this is the first RCT that investigates the effects of MBCT on body awareness as measured with the MAIA in patients with chronic pain and depression. The MAIA appears a reliable instrument with scales of adequate consistency in this newly studied population. In accordance with our hypothesis, MBCT resulted in increases for several dimensions of body awareness in the studied patient population. More specifically, a significantly greater increase in Self-Regulation and Not Distracting, but no changes in Noticing in the MBCT group as compared to the TAU group were observed. In addition, participants in the MBCT group, but not in the control group, had increases in Emotional Awareness. For Pain Catastrophizing, we found significant decreases within the treatment group, but not within the control group. Furthermore, mediation analyses revealed that the effect of MBCT on depression was mediated by Not Distracting, but not by Self-Regulation. We discuss these results for each dimension of body awareness separately in more detail below. Self-regulation --------------- The finding of a significantly greater increase in Self-Regulation ratings in the treatment than in the control group is in line with findings by Mehling et al. ([@B48], [@B50]). In two cross-sectional studies they showed that patients with chronic low back pain who were practicing mind-body therapies (Mehling et al., [@B48]), or yoga and meditation (Mehling et al., [@B50]) had greater self-regulatory body awareness than patients without such practice. In the cohort study (Mehling et al., [@B50]), this difference was more pronounced for the Self-Regulation dimension than for any of the other dimensions of body awareness. Similarly, Bornemann et al. ([@B7]) also found that increases following 3 months of contemplative training including the bodyscan and breath awareness meditation were largest on the Self-Regulation scale (effect size *d* = 0.72). Our study extends the previous findings by demonstrating effects through a well-established intervention (MBCT) in a patient population (chronic pain with active depression). The present findings of a change in Self-Regulation through a mindfulness intervention are consistent with a hypothesized link between mindfulness and enhanced self-management (Baer, [@B2]), a concept closely related to general self-regulation (Vohs and Baumeister, [@B80]). Our findings also converge with empirical evidence for better general self-regulation of chronic pain through a mindfulness intervention (Kabat-Zinn et al., [@B41]), as well as with evidence from paradigms with acute pain induction, in which mindfulness practice enhanced pain tolerance (Kingston et al., [@B44]; Gard et al., [@B23]), thereby having potential clinical implications. Emotional awareness ------------------- Paired samples *t*-tests revealed a large and significant increase in Emotional Awareness over time within the treatment group, while the change within the control group was small and not significant However, no significant group by time interaction was found. The significant increase in Emotional Awareness over time in the treatment group is in line with the expectation that the MBCT intervention increases the awareness of the connection between body sensations and emotional states and corresponds to results by Mehling et al. ([@B48], [@B50]). Their group found that levels of Emotional Awareness were higher in chronic pain patients with mind-body practice than in patients without such practice (Mehling et al., [@B48]). Bornemann et al. ([@B7]) also found significant increases of scores of the Emotional Awareness scale following 3 months of bodyscan and breath awareness training, but the effect size was rather small (\<0.20). Beyond the body awareness specific construct of Emotional Awareness, our findings confirm theory (Bishop et al., [@B5]; Phillipot and Segal, [@B60]) and previous evidence that suggests an association between mindfulness and general emotional awareness (Sze et al., [@B73]; Boden et al., [@B6]). In light of evidence that demonstrates that emotional awareness is linked to better clinical outcomes and quality of life (Williams et al., [@B82]; Boden et al., [@B6]), as well as to reductions in depression symptomatology and in depression-related affective and cognitive outcomes (Goldman et al., [@B25]; Arch and Craske, [@B1]; Farb et al., [@B20]), the present findings of increased Emotional Awareness in the MBCT group may be relevant for clinical practice. In the present study, the Emotional Awareness and Self-Regulation scores were strongly correlated (*r* = 0.76). Similarly, Mehling et al. ([@B48]) found a relatively high correlation between these two scales (*r* = 0.60) in patients with chronic pain, as well as in body-mind practitioners, and Bornemann et al. ([@B7]) also found a considerable interscale correlation between these two scales (*r* = 0.46). These findings may correspond to the notion of the general self-regulation concept (Baumeister et al., [@B4]; Muraven and Baumeister, [@B56]) as being closely related to emotion regulation (Vohs and Baumeister, [@B80]; Vago and Silbersweig, [@B77]). Not distracting --------------- The significant group-by-time interaction on Not Distracting was driven by a decrease in the TAU group and an increase in the MBCT + TAU group. Distracting oneself from uncomfortable body sensations or ignoring them, is a common coping strategy in chronic pain (Reid et al., [@B66]; Peres and Lucchetti, [@B59]), as well as in depression (Matheson and Anisman, [@B47]). In the intervention group, however, subjects learned to pay mindful attention to all body sensations including pain and depression-related symptoms, independent of their valence. In the control group, subjects did not learn to use this alternative approach and may have practiced the more common coping strategy of distraction so that they became "better" at ignoring and distracting themselves from uncomfortable body sensations. Correspondingly, when a sample of patients with chronic lower-back pain was compared to healthy mind-body therapy practitioners, the mind-body sample had significantly higher Not Distracting scores (Mehling et al., [@B48]). However, contrary to our findings, Mehling et al. ([@B48]) found no significant difference between the Not Distracting capacities of those patients with past or current pain that did have mind-body therapy experience and those that did not have such practice. Bornemann et al. ([@B7]) did not find that changes on the Not Distracting scale in healthy participants, following bodyscan and breath awareness training were significantly higher than those in the retest control group. These discrepancies between the studies may be due to methodological differences, differences in the interventions, and differences in the studied population. Our findings are further aligned with prior studies showing a negative association between mindfulness and experiential avoidance (Riley, [@B67]), and general distraction (Jain et al., [@B37]). In the present study, we did not find significant correlations between the Not Distracting scale and any other MAIA scales. This result corresponds to the finding by Mehling et al. ([@B48]) that Not Distracting scores were not correlated with the other scales in their large (*N* = 301--434) chronic pain sample. Bornemann et al. ([@B7]) on the other hand found that the Not Distracting scale was significantly correlated with all other scales in their sample of healthy patients. This difference might be due to their very large sample size (*N* = 1076) or due to the fact that they investigated a sample of healthy participant. These findings may indicate that in patients with chronic pain, a coping style of not distracting from pain is independent of the other aspects of body awareness. A striking finding of the present study is that Not Distracting mediates the effect of MBCT on depressive symptom reduction. This finding supports the clinical importance of the Not Distracting aspect of body awareness. Furthermore, while it has long been hypothesized that the effects of MBCT on depression are mediated by body awareness, this is to our knowledge the first study supporting such a relationship (Michalak et al., [@B54], [@B55], [@B53]; van Der Velden et al., [@B78]). Although previous research has shown that for depression, distraction may be an effective coping style, mindfulness is even more effective in reducing negative mood (Broderick, [@B9]; Huffziger and Kuehner, [@B35]). Furthermore, mindfulness has been shown to be better in reducing acute pain than distraction in individuals with high levels in pain catastrophizing (Prins et al., [@B64]). Similarly, in patients with chronic pain that had high health anxiety, paying attention to sensations resulted in greater pain relief than distraction (Hadjistavropoulos et al., [@B27]), which is in line with the notion that "one problem in chronic pain is not only the pain itself, but the 'turning away' from, the averting of attention from the regions that give rise to painful sensations, either through deliberate distraction, or by thinking *about* the pain (conceptually) rather than experiencing the sensations directly" (Williams, [@B81]). More in general, experiential avoidance is associated with a wide variety of psychopathology (Hayes et al., [@B29]) and coping strategies that work contrary to avoidance, such as mindfulness are related to better mental health outcomes (Williams et al., [@B82]). Together these findings suggest that the Not Distracting aspect of body awareness may be an important predictor of depressive symptoms and potentially of mental health in general, and that it can be cultivated through mindfulness based interventions. Noticing and attention regulation --------------------------------- The present study did not detect significant changes in the awareness of uncomfortable, comfortable, and neutral body sensations as a result of the MBCT intervention. However, the effect sizes of the pre-post changes in the treatment group were of medium size, whereas the increases in the control group only had a small effect size. These findings contrast earlier findings where large differences in MAIA Noticing scores were found between individuals with chronic pain who had mind-body practice and those patients who did not have such practice (Mehling et al., [@B51]). Our findings are in line with those of Bornemann et al. ([@B7]), who despite a very large sample size did not find a significantly greater increase in the contemplative training group than in the rest control group. No significant group by time interaction was revealed for Attention Regulation, indicating that MBCT did not result in increases of the ability to sustain and control attention to body sensations. The absence of a significant increase of Attention Regulation in the MBCT group is surprising, given that previous studies found that mind-body therapy practicing patients with back pain had higher scores of Attention Regulation than non-practicing patients (Mehling et al., [@B51], [@B48]), and that bodyscan and breath awareness training lead to large effects on Attention Regulation in healthy participants (effect size *d* = 0.54; Bornemann et al., [@B7]). It remains an open question whether the absence of significantly greater increases in Noticing and Attention Regulation in MBCT + TAU than in TAU is specific for the studied patient population and intervention, or just a lack of power. Larger studies are warranted. Pain catastrophizing -------------------- The absence of a group by time interaction for pain catastrophizing indicates that there were no greater changes in pain catastrophizing in the MBCT group than in the control group. However, the main effect of time was approaching significance and was driven by a large and significant decrease within the MBCT group and a small, non-significant decrease in the control group. Consistent with the significant decrease in pain catastrophizing within the MBCT group, Mehling et al. ([@B48]) showed that pain patients who practiced mind-body therapies worried less about their pain or other uncomfortable body sensations than individuals without mind-body practice. Furthermore, our findings are aligned with a growing body of evidence showing that in patients with chronic pain, mindfulness and participation in mindfulness-based interventions are related to decreases in pain catastrophizing (e.g., Schutze et al., [@B69]; Cassidy et al., [@B10]; Garland et al., [@B24]; Day et al., [@B13]) but see (de Boer et al., [@B14]). Limitations ----------- The present study has several limitations. First, this pilot study had a relatively small sample size, resulting in low power to detect true effects. Yet despite this limitation, some significant effects were found. However, it must be mentioned that because of the small sample size we chose a less conservative statistical approach and did not correct for multiple comparisons. Second, because this was a pilot study, we used TAU as a control group, which does not adequately control for "placebo effects" secondary to non-specific factors like attention in patient-clinician interactions (Goyal et al., [@B26]). This means that, theoretically, the clinical improvement could be due to non-specific factors like attention and expectations about improvement and not due to the specific effects of MBCT. We controlled for attention as much as possible by providing equal numbers of office and phone visits with the clinician for both the MBCT group and TAU group. In future larger studies it would be imperative to compare MBCT to an active non-specific control group, such as group sessions including psycho-education and gentle stretching exercises without any mindfulness components, as in the study of Hoge et al. ([@B32]). Third, the MAIA is a relatively new scale that requires further refinement and validation in different populations. Fourth, because the intervention produced skills and understanding of concepts that were relatively new to participants at baseline, subjects may have reached a different understanding of the concepts enquired by the MAIA items post-treatment. While the first three limitations can be addressed by larger studies with active control groups and future versions of the MAIA, the last limitation is probably inherent to interventions that result in a change of perspective and thus more difficult to address. Using behavioral data might help to validate self-report instruments in these circumstances. Finally, because there were too many missing midpoint data, we conducted mediation analysis only on the endpoint data, which decreases the degree of causal specificity (Kazdin, [@B42]). However, Hayes argues that mediation analysis is still valuable, even when data collection of the mediator and outcome variable is at the same time point (Hayes, [@B28]). The findings on the mediation analysis in this study should be interpreted as tentative and purely hypothesis generating. Future larger studies with MBCT in this population with the assessment of the MAIA scales of potential mediators at multiple time points are warranted, to permit more conclusive statements. Conclusion {#s5} ========== In summary, our data suggest that MBCT can increase several dimensions of body awareness as measured with the MAIA in patients with chronic pain and comorbid active depression. In particular, the Not Distracting aspect of body awareness mediated the positive effect of MBCT on depressive symptoms. Furthermore, the MAIA appears to be a reliable instrument for measuring self-reported body awareness in this population. This finding is important because the reliability of this new and increasingly used instrument for measuring body awareness has so far not been assessed in a population of patients with chronic pain and comorbid depression. Our finding that MBCT can increase self-reported body awareness represents the first preliminary evidence in support of a causal link between a mindfulness-based intervention and increased body awareness (as measured with the MAIA) in this population. Finally, the fact that body awareness mediated the effect of MBCT on depressive symptoms provides preliminary first evidence for this long-hypothesized relationship and indicates that body awareness may have clinical relevance as an element of mindfulness approaches in the studied population. Author contributions {#s6} -------------------- All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication. Conflict of interest statement ------------------------------ The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a current collaboration with one of the authors WM on a Research Topic, though no other collaboration, and the handling editor states that the process nevertheless met the standards of a fair and objective review. This study was funded by an anonymous bequest to the DCRP, and the Mind and Life Francisco J. Varela (Grant 2010-01-010) to TG. We would like to thank Narayan Brach for his help with Red Cap, Jim Doorley, Rosemary Walker, and Thomas Calahan for logistics, Laurie Rhoades for teaching the MBCT program, Ronald Kulich, Karsten Kueppenbender, and Egilius Spiering for patient recruitment and Lee Baer, and Alisabet J. Clain, for assistance with data base conversion and general statistical questions, and Elizabeth Hoge, Willoughby Britton, Jonathan Alpert, and Maurizio Fava for advice. We also would like to thank all participants for their efforts. [^1]: Edited by: Olga Pollatos, University of Ulm, Germany [^2]: Reviewed by: Takamitsu Watanabe, University College London, UK; Karin Meissner, Ludwig Maximilians University of Munich, Germany [^3]: This article was submitted to Consciousness Research, a section of the journal Frontiers in Psychology [^4]: †These authors have contributed equally to this work.

Background ========== Although subjective threshold visual field testing and stereoscopic biomicroscopy of the disc and disc photos provide powerful clinical assessments of eye disorders that affect retinal ganglion cells, quantitative, objective measurement of NFL thickness has added an additional diagnostic indicator of such disorders \[[@B1],[@B2]\] and is routinely used in monitoring response to therapy. The mechanism by which NFL thinning develops, however, is poorly understood. Epidemiologic, clinical and laboratory evidence supports the contention that nerve fiber layer damage results from vascular compromise or dysregulation, probably within the lamina cribrosa \[[@B3]\], focussing attention on the highly specialized vasculature that supplies the nerve fiber layer \[[@B4],[@B5]\], known as the radial peripapillary capillaries (RPCs) \[[@B6]\]. Prior reports have suggested that these capillaries might be involved in the etiology of glaucoma \[[@B6]\], although others have pointed out that it would be difficult to establish cause and effect between nerve fiber layer thinning and disappearance of the NFL vasculature \[[@B7]\]. Although the RPCs are unlikely to represent a primary site of disease vulnerability, they may reflect NFL or contiguous disc capillary network compromise. A better understanding of these factors will be important in understanding NFL clinical and optical coherence tomographic (OCT) data, and may provide a supplementary indicator of ganglion cell loss \[[@B8]\]. Evaluation of RPCs in glaucoma and other eye diseases has been limited because of the difficulties of imaging these vessels using conventional fluorescein or indocyanine green angiography. However, as reported here, we can robustly image these vessels and determine their depth with high precision in macaque monkeys using *in vivo*adaptive optics fluorescent imaging. RPC vessels are thin and form arrays that are oriented parallel to the course of nerve fiber bundles \[[@B4],[@B6],[@B9]\]. As the axial focus of the imaging shifts toward greater retinal depth, the appearance of the vessels changes from parallel orientation to the polygonal and more random pattern that is typical of retinal vasculature \[[@B9]\]. Since the thickness of tissue supplied by RPCs is of great interest for evaluating the effect and potential participation in glaucomatous NFL changes, we compared *in vivo*to *ex vivo*thickness of the RPCs in the same eye and found strong correlation of RPC pattern within the NFL. Methods ======= Subjects -------- Two juvenile macaque monkeys (Macaca mulatta) 6.3 and 5.5 kg, served as subjects. All experimental protocols were approved by the University Committee of Animal Resources at the University of Rochester Medical Center, complied with the Public Health Service policy on Humane Care and Use of Laboratory Animals, and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Axial lengths of the imaged eyes were measured with an IOLMaster (Carl Zeiss Meditec, Jena, Germany) (5 determinations for each eye, standard deviation approximately 3% of mean) in the anesthetized monkey, and used to calculate transverse and axial retinal distances. Each monkey was fitted with a rigid, gas permeable contact lens to preserve the corneal epithelium and reduce astigmatism and spherical refractive error. Standard ophthalmic trial lenses were used to reduce residual astigmatism and spherical error to values that could be corrected with the AO deformable mirror. In-vivo imaging --------------- The fluorescence adaptive optics scanning laser ophthalmoscope (FAO-SLO) instrument, monkey imaging methods, and the post-processing methods have been previously described \[[@B10]\]. For *in-vivo*imaging, the monkeys were anesthetized with isoflurane at a dosage (typically 2%) sufficient to minimize large ocular movements and eliminate microsaccades. Vital signs and body temperature were continuously monitored. The monkey retinas were imaged *in-vivo*with the AO system using reflected light at 794 nm ± 17 nm to generate a high-resolution fundus photograph of the peripapillary region. Two regions of interest were selected (along the temporal arcuate NFL, see Figure [1](#F1){ref-type="fig"}), and 1000 frames were captured at each focal plane in a series of focus depths. We used a deformable mirror to focus through the retina at 0.1 D steps from the inner retinal surface, above the nerve fiber layer, to the pigmented epithelium. The Elmsley model eye was used to convert diopters of focus (F) to axial distance using the following formula: microns of focus = 4/3 \[1/\[power(D) +focus(D)\]-1/power(D)\]×10^6^, where P is the power of the eye in diopters, calculated by linearly scaling eye size by the measured axial length assuming an emmetropic eye. For the monkeys used in this study, 0.1D corresponded to approximately 26 μm. {#F1} ### Fluorescein Injections In each imaging session we used two 0.5 mL intravenous injections of sodium fluorescein (scaled to be equivalent to the standard human dose in ml/body weight) to enhance the contrast of the vasculature at the locations of interest. We obtained a reflectance video with 794 nm light and simultaneously used 488 nm laser excitation and recorded the emitted fluorescein fluorescence at 520 nm ± 35 nm. Each region of interest (ROI) was imaged at depths ranging from the NFL to the RPE in 0.1D steps. The detector gain was continually optimized to minimize the number of saturated pixels in the image. Because of the weak fluorescence signal from the retina, 1000 raw video frames were dual-registered and averaged using methods described previously \[[@B10]\]. (Although high signal/noise was present in single frames obtained in the first few seconds after fluorescein injection, registration and averaging allowed us to obtain high quality images up to 30 minutes after injections, despite the faint fluorescence present at this time). Single frame and registered/averaged frame results are similar in human subjects. Ex-vivo preparation ------------------- The monkeys were euthanized for histological analysis. Under deep anesthesia, both monkeys were perfused, initially with 2 liters of saline to flush blood from the vascular system and then with two liters of 4% paraformaldehyde, infused over a 1 hour period. The eyes were fixed further in the eyecup for an additional 40 minutes. The retinas were cut in half along the horizontal raphe, and relief cuts were made in each section. The two *in-vivo*imaging areas, located in the superior retina were preserved in one tissue block. The superior retinal ROI was simultaneously incubated with three primary antibodies, one which binds to neurofilaments (rabbit polyclonal neurofilament 200, Chemicon, Temecula, CA) and the other to blood vessel endothelium (mouse monoclonal CD31, and mouse monoclonal von Willebrandts factor, Lab Vision, Fremont, CA). Dye-coupled secondary antibodies were applied to label the neurofilaments with Alexa 555 (goat anti-rabbit (H&L)) and the vessels with Alexa 488 (goat anti-mouse (H&L)) (Invitrogen, Carlsbad, CA). Then the superior retinal tissue was removed from solution, placed as a flat mount on a slide, covered in Vectashield (Vector, Burlingame, CA) to minimize fading of fluorescence, and cover slipped. The inferior retinal tissue was embedded in agar and sectioned (transverse sections parallel to the horizontal raphe) at 60 μm thickness on a vibratome (Microm, Walldorf, Germany). Every third section was reacted with the same antibodies as above, followed by dye-coupled secondary antibodies to label the neurofilaments with Alexa 543 and the vessels with Alexa 488, as well as 4\', 6-diamino-2-phenylindole, dihydrochloride (DAPI) (Invitrogen, Carlsbad, CA) to identify nuclei of retinal neurons. The sections were covered in Vectashield and coverslipped. *Ex-vivo*imaging ---------------- *Ex-vivo*wholemount and transverse sections were imaged on a Zeiss 510 Meta confocal microscope using a 10× air objective with an NA of 0.3, an NA similar to that used to perform *in-vivo*FAO SLO. Six to ten 10× imaging fields were needed to visualize all areas of each section. The images were digitally combined to create one image per tissue section. The retinal location of the digitally assembled sections were registered against a color fundus photograph using vascular landmarks. *In-vivo*and *ex-vivo*measurements ---------------------------------- Figure [2](#F2){ref-type="fig"} illustrates the method that was used to measure the thickness of the nerve fiber and vascular layers in the retina at the locations indicated by large dots in Figure [1](#F1){ref-type="fig"}. Vessel caliber was also measured on these sections. Pairs of sections were dual-labeled for NF 200 and CD-31/Von Willenbrandt\'s factor. Because the fluorescence tags of the secondary antibodies (Alexa 543 and Alexa 488, respectively) differ in emission peak, we simultaneously recorded total fluorescence on a confocal microscope and separated fluorescence channels in post processing with ImageJ. Thus the images remain in registration throughout the analysis. Section A (Figure [2](#F2){ref-type="fig"}) portrays the neurofilament label, the intensely labeled NFL and other retinal layers with light labelling. Section B (Figure [2](#F2){ref-type="fig"}) portrays vessel labelling, showing the luminal border of retinal vascular endothelial cells. {#F2} a\. RPC thickness- *in-vivo*- Thickness of the RPC bed was measured fromadaptive optics images, some of which are illustrated in Figure [3](#F3){ref-type="fig"}. The inner border of the RPC bed was taken as the first image which showed RPCs clearly and the outer border as the first image which showed the vascular pattern typical of the ganglion cell layer. Uncertainty in this measurement reflects the depth sampling interval (approximately 26 μm) as well as the estimate of which vessels were near the center of focus. We also measured maximal RPC thickness in four quandrants of each image and averaged the four measures. {#F3} *ex-vivo*- RPC thickness was determined by measurements on transverse sections, as illustrated in Figure [2B](#F2){ref-type="fig"}, of the distance from the most superficial (vitread) NFL vessels to the outermost RPC boundary, which is determined as the inflection in pattern between the extremely long capillaries with thin walls, little variation in caliber and parallel-linked structure oriented parallel to the NFL axons, and the polygonal pattern typical of the ganglion layer and retinal vasculature. The outer (sclerad) border of the RPC bed was also correlated with the location of the most superficial ganglion cell dendrites, which were highly visible due to neurofilament labelleing. These patterns were easily distinguishable within *in-vivo*images as well as histologic transverse sections. This measurement is illustrated in Figure [2B](#F2){ref-type="fig"} as the distance between the two black arrows on a section close to the optic nerve head whose location is shown in Figure [1](#F1){ref-type="fig"}. Each *ex-vivo*result represents the average of five measurements over a 50 micron region centered at each data point. b\. NFL thickness - NFL thickness was determined by measuring neurofilament label in transverse sections as illustrated in Figure [2A](#F2){ref-type="fig"}. Each result represents the average of five measurements over a 50 micron region centered at each data point. NFL thickness could not be measured from *in-vivo*imaging data. c\. vessel pattern - the vessel pattern within versus beneath the RPC bed was determined by appearance (RPCs, figure [3a, b, c, d](#F3){ref-type="fig"},, and [3f](#F3){ref-type="fig"}) and (ganglion cell vasculature, figure [3g, h](#F3){ref-type="fig"}. and [3i](#F3){ref-type="fig"}). d\. vessel diameter - was measured at 20 randomly chosen locations on each of the 6 *in-vivo*AOSLO RPC images illustrated in Figure [3(a, b, c, d, e)](#F3){ref-type="fig"}, and [3f](#F3){ref-type="fig"} using the open-source NIH software package, ImageJ. Twenty (20) random x, y locations were generated on each image and a measurement was taken of the caliber of the nearest RPC vessel by drawing a line segment across the vessel, whose length was registered by the software. Similar measures were made at 20 randomly chosen locations on each digital image from 6 sections from the lower retina of monkey 1, 3 sections at each of the two locations that correspond to the *in-vivo*imaging of the upper retina. Results ======= Figure [1](#F1){ref-type="fig"} diagrammatically represents the relative locations in the retina of monkey 1 from which *in-vivo*FAO-SLO and *ex-vivo*confocal microscopy images of RPCs were taken, as well as the corresponding thickness measurements. The two heavy border squares in the superior retinal block represent the locations of *in-vivo*imaging in this monkey, and the respective measured average RPC thickness calculated from the *in-vivo*images is indicated within each box. RPC thickness ------------- *In-vivo*- From *in-vivo*FAO-SLO fluorescein angiography, the thickness of the superficial parallel-linked layer of RPC vasculature was measured as 104 μm near the optic disc and 78 μm at the location 5 mm along the arcade (heavy squares in superior retina) as shown in Figure [1](#F1){ref-type="fig"}. *Ex-vivo*- Thickness measurements of RPC bed were also made from transverse sections in the inferior retinal block, diagrammed in Figure [1](#F1){ref-type="fig"}, and are shown as the first value (before parentheses) adjacent to the marked location at which they were made (heavy dots). For comparison with the thickness measures made from *in-vivo*images, RPC network thickness measurements were also made (average of four points within each square) at the locations in inferior retina (light squares) that correspond in eccentricity to the *in-vivo*imaged locations. Thickness measurements are not reported for the imaged locations in the whole-mounted tissue because of tissue compression presumably caused by pressure of the coverslip. Comparison of *in-vivo*to *ex-vivo*measures - RPC thickness measured *in-vivo*was 104 and 78 μm at the locations shown in Figure [1](#F1){ref-type="fig"} closer to and more distant from the optic nerve. Comparison measures from comparable locations in inferior retina were 140 and 82 μm, indicating comparable values for the two methods on the same retinas. NFL thickness ------------- NFL thickness measures are shown in parentheses in Figure [1](#F1){ref-type="fig"} at each location indicated by dots at which RPC bed thickness was measured. In all cases, the NFL thickness was 10 to 20 μm thicker than the RPC bed, reflecting the absence of RPCs in the most superficial portion of the NFL. Vessel pattern -------------- Figure [3](#F3){ref-type="fig"} shows examples from a through-focus series of *in-vivo*adaptive optics images of radial peripapillary capillaries at two locations in monkey 1, near the maximum of nerve fiber layer thickness near the optic disc (A), and at a location about 5 mm from the optic disc (B), where the nerve fiber layer thickness is much narrower. The locations of these two imaged regions are shown to scale on the map of the retina of monkey 1 in Figure [1](#F1){ref-type="fig"} (heavy squares). An additional location near the optic disc is shown in Figure [3](#F3){ref-type="fig"} for the second monkey. These images illustrate the two important patterns of vessels identified by *in-vivo*imaging. First, there is an abrupt transition from a superficial parallel-linked architecture of extraordinarily long, uniformly narrow vessels (capillaries) oriented parallel to nerve fiber bundles, to a deeper polygonal architecture of more varied diameter vessels typical of the ganglion cell layer/retinal vasculature. The parallel-linked, long capillaries were found throughout the full thickness of the NFL (Figure [1, D, E, F](#F1){ref-type="fig"}) with the exception of the most superficial 10 - 20 μm. Second, in both the FAO-SLO images and in the microscopy superficial (inner) vessels show the highest contrast, while those deep (outer) in the retina show reduced contrast. The transverse separation of RPC vessels in *ex-vivo*measures was approximately 20 μm. The distinction between RPC and RGC vasculature can also be seen in Figure [4](#F4){ref-type="fig"}, which compares *in-vivo*FAO-SLO fluorescein-imaged vessels and *ex-vivo*antibody-imaged confocal microscopy of the retinal wholemounted tissue. There was precise correspondence of vessel pattern in the comparative images. The *in-vivo*imaging involves a larger depth of focus, explaining the simultaneous visibility of several vascular layers. We found a consistent gap between RPC vessels and large retinal vessels that was approximately 50 μm. (see figure [4](#F4){ref-type="fig"} lower right). {#F4} Vessel diameter --------------- *In-vivo*and *ex-vivo*vessel diameter measurements are shown in Table [1](#T1){ref-type="table"}. Mean *in-vivo*diameter was 5.12 and mean *ex-vivo*diameter was 5.03, not statistically different on an unpaired t test (P = 0.78). ###### RPC vessel diameter **in-vivo** **ex-vivo** -------- ------------- ------------- ------ ------ Figure Mean SD Mean SD 3a 5.9 1.0 5.9 1.0 3b 5.0 1.4 5.2 0.9 3c 5.0 1.0 5.0 1.4 3d 5.2 0.9 4.5 0.82 3e 4.5 0.82 5.1 1.0 3f 5.3 1.2 4.5 0.7 Discussion ========== This study demonstrates precise *in-vivo*quantification of the circulations of the NFL and retina, including features of the RPC vascular bed, which match *ex-vivo*images of the same tissue made by confocal microscopy. The inner or superficial RPCs have a distinctive and easily recognizable structure of parallel-linked, long uniform-diameter vessels that are oriented parallel to the nerve fiber bundles. The transition from RPCs to typical retinal/ganglion cell layer vasculature is abrupt and roughly corresponds to the bottom of the NFL (outer margin). The entire RPC network remains within the NFL, but its network thickness is consistently 10 to 20 μm thinner than the NFL. These results underscore the coupling of NFL and the RPCs. This relationship can now be imaged in human glaucoma patients to determine how RPCs and their networks may change with the development of glaucomatous NFL hemorrhages and thinning \[e.g. \[[@B11]\]\]. These findings also support the notion of a linked spatial and functional relationship between the NFL and the RPCs. Evaluation of disturbances of NFL/RPC density or distribution *in-vivo*are now possible for normal and diseased eyes. One clinically important, but poorly understood, phenomenon which could be functionally evaluated with this method is the development of disc hemorrhages in glaucoma. The superficial distribution, recurrent nature, and distance between hemorrhage and optic disc, suggest that RPCs are the source of the blood. However, without longitudinal (i.e. in vivo) evaluation of the association of disc hemorrhage with glaucoma progression will not be determined. Fidelity with which *in vivo*imaging captured the three dimensional structure of RPCs and their network ------------------------------------------------------------------------------------------------------- We have previously shown that the transverse resolution of reflectance AO is approximately 1.6 μm \[[@B12]\], thus it captures even subtle features of RPC vessels, which are approximately 5 μm in diameter. In comparing *in-vivo*FAO-SLO fluorescein angiography images to 10, 20 and 40× *ex-vivo*confocal images of wholemounts of the same regions we found that the *in-vivo*images could identify every feature and dimension of every vessel in the *ex-vivo*images. This was particularly useful when identifying portions of vessels that extended beneath other vessels. RPC bed thickness measured in-vivo and ex-vivo could be compared at the locations close to the optic disc (140 μm ex-vivo versus 104 μm in-vivo) and farther from the optic disc (82 μm ex-vivo versus 78 μm in-vivo) This fidelity reflected the confocal design of the AO system, whose resolution permits the determination of axial position with an accuracy of approximately 6.5 μm \[[@B12]\]. We found that RPC vessels were axially spaced on average approximately 20 μm apart, with no layering evident, and that each vessel was visible in the through-focus series. The thickness of the RPC layer could not be directly compared between *in-vivo*imaging and microscopy, because the wholemount preparation slightly decreased the thickness of RPC and NFL. However, comparison to the RPC network thicknesses measured from sections of the inferior retina at equal eccentricities showed close correspondence to *in-vivo*measurements. The major difference between the RPC networks at the two retinal regions of interest was network thickness. There was no apparent difference in the diameter or distance between RPCs. It is worth noting that the thinnest NFL was measured along the horizontal raphe. Even at this location, where NFL might be expected to be thin, the NFL exceeded 20 μm, consistent with the observation that upper and lower hemisphere fibers mix across the horizontal raphe \[[@B13]\]. Comparison to previous histological measures of RPC and NFL characteristics --------------------------------------------------------------------------- The characteristics of the RPC network measured in this study are in close agreement with the previous histological reports for the macaque monkey that used ink-fill to visualize RPCs \[[@B4],[@B5]\]. The NFL thickness measured here also agrees with previous histological \[[@B14]\] and OCT \[[@B15]\] measures. Macaque RPCs are very similar to human RPCs, whereas those of the cat and pig are distinctly different \[[@B4]\]. In precise agreement with the findings of the present study, macaque and human RPCs are most prominent in the Bjerrum region that is coincident with the thickest NFL, they are of small caliber, and they are straight and elongated superficially. The characteristic parallel distribution of RPC to nerve fibers, as well as a relative lack of anastomoses, reported by Henkind \[[@B6]\] can be seen in Figures [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"} of the present paper. Deeper images display the polygonal pattern that is typical of retinal vasculature. Figure [4](#F4){ref-type="fig"} demonstrates agreement with Henkind\'s \[[@B4]\] description that RPCs are normally supplied by retinal vessels from the ganglion cell layer that arch up abruptly to sustain the NFL. Our results clearly indicate that RPC bed thickness remains slightly less than NFL thickness, suggesting that RPC thickness in the macaque is less than that of humans and varies across the retina in parallel with NFL thickness \[[@B14],[@B16],[@B17]\]. This is a very consistent observation in all retinal sections and reflects the lack of RPCs in the superficial 10 - 20 μm of the NFL. *In-vivo*comparison of NFL and RPC bed thickness in macaques and humans would require simultaneous OCT and AO angiography. Potential use of RPC imaging in human eye disease ------------------------------------------------- In some cases RPCs develop into collateral (bypass) vessels following retinal vein occlusions. In addition, they have been implicated as the vessels occluded which lead to cotton-wool spots (superficial NFL infarctions), associated with numerous ocular and systemic disorders. Their primary or secondary involvement in glaucoma was postulated in 1968. \[[@B6]\] Although potential vascular changes within the lamina cribrosa is a more likely locus for the primary site of glaucomatous damage, no currently known modality can image this region or measure its functional state. Measurement of those vascular networks which are adjacent or functionally dependent upon this vasculature would be worthy of further evaluation. The RPCs are one such vascular network which is accessible. An important motivation for RPC imaging is to provide a better understanding and potential clinical indicator of early glaucomatous progression. As ganglion cells are lost in glaucoma the NFL progressively thins and this can be measured with high-resolution by OCT, making NFL thickness a valuable early indicator of glaucoma \[[@B18]\]. However, loss of NFL thickness does not provide a complete measure of glaucoma progression, since NFL thins less than the proportional loss of retinal ganglion cells \[[@B8]\]. One potential use of RPC imaging is to track changes in individual identified vessels, whose depth within the NFL is related to the retinal locus from which the axons originate. Thus, *in-vivo*imaging of RPCs and the surrogate blood flow indicator that they provide may complement the analysis of NFL changes during glaucomatous damage. Conclusion ========== We have demonstrated in this study that FAO-SLO techniques can deliver *in-vivo*images of RPCs and their network thicknesses with high fidelity. Application of this methodology to the measurable transverse NFL characteristics in retinal vascular or glaucoma patients is potentially feasible, since it is no more invasive than conventional angiography. One important concern is that the light exposure used in this imaging be completely safe, optimally more conservative than current ANSI safely limits for humans \[[@B19]\]. In other animal FAO-SLO imaging studies (data not shown) we have found that RPE damage may occur during imaging at relatively short wavelengths such as the 488 nm excitation source that is used to image fluorescein \[[@B20]\], but this damage can be avoided by adopting safety limits on light exposure that are more conservative than those previously used. Of additional concern is that a wider margin for exposure may be necessary, as RPE damage may occur with greater frequency or severity in the disease-affected eye \[[@B21]\]. Competing interests =================== This research was supported in part by Bausch and Lomb, and William Merigan and Robert Wolfe were partially supported by Bausch and Lomb. The University of Rochester holds a patent on the method for adaptive optics imaging of retina. Authors\' contributions ======================= DS carried out AO imaging, and collected and analyzed all confocal data, DCG was involved in the design of RPC imaging, JJH, RW, YG, and BDM carried out AO imaging, BPG was involved in planning the study and writing the manuscript, RTL helped coordinate imaging and histological measures, SR was involved in writing and revising the manuscript, DRW helped plan and carry out the AO imaging study, WHM conceived the study, coordinated its execution and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2415/9/9/prepub> Acknowledgements ================ This work was supported by a grant from Bausch and Lomb, Inc., NIH Grant BRP-EY014375, NIH Training Grant-EY07125, NIH Core Grant-EY001319, NSF Grant cfao-ast-9876783 and Grants from Research to Prevent Blindness.

Background {#Sec1} ========== In recent years, physical activity (PA) has become an important public health issue in both high- and low-income countries \[[@CR1], [@CR2]\]. Indeed, regular PA is well known to be beneficial for health and well-being \[[@CR3], [@CR4]\]. There is strong evidence that it reduces rates of all-cause mortality and a number of noncommunicable diseases (NCDs) \[[@CR5], [@CR6]\]. Evidence suggests that risk reduction routinely occurs in adults when at least 150 minutes of moderate- to vigorous-intensity activity are ensured per week \[[@CR7]--[@CR14]\]. Physical inactivity, on the other hand, is indicated as a major risk factor for morbidity and mortality in adults \[[@CR15], [@CR16]\]. It has identified as an independent and fourth-leading risk factor for global mortality (6.0% of global deaths) \[[@CR17]\]. Over the years global deaths due to physical inactivity have increased from 1.9 million in 2005 \[[@CR18]\] to 3.2 million in 2008 \[[@CR19]\] and 5.3 million in 2012 \[[@CR5]\]. Different studies suggest that the prevalence of physical inactivity is rising in both high and low-income countries \[[@CR1], [@CR5], [@CR19]--[@CR23]\]. NCDs are increasing in Bangladesh \[[@CR24]\]. This increase is concomitant to increasing sedentary lifestyle due to gradual mechanization of life in addition to the high prevalence of other major risk factors such as tobacco use \[[@CR25]\] and salt intake \[[@CR26]\]. Internationally comparable data on PA using standardized methods is still suboptimal in Bangladesh. Two studies \[[@CR27], [@CR28]\] reported the prevalence of physical inactivity 35.0% to 38.0% in Bangladeshi adults aged 25 years and older. However, earlier studies did not investigate the socio-demographic factors associated with insufficient PA, which is important information for planning of health-promoting interventions. Therefore, the objectives of this study were to measure PA levels and determine associated socio-demographic factors in Bangladeshi adults. Methods {#Sec2} ======= Study design and sampling {#Sec3} ------------------------- A population-based cross-sectional study was conducted among urban and rural Bangladeshi adults aged 25--64 years in 2011. A total of 806 individuals (equal numbers from the urban and rural area) were selected for interview by a two-stage systematic cluster sampling. Finally, data from 14 (1.7%) participants were identified as invalid during data cleaning, resulting in a final sample of 792 participants (urban 395, rural 397) for the current analysis. Data of 14 participants were removed if the value for at least one sub-domain (vigorous work, moderate work, transport, vigorous recreation, or moderate recreation activity) accounted more than 16 h; reported implausible values (eg. \> 7 days in any days column); had inconsistent answers (e.g. 0 days but values \> 0 in the corresponding time variables) as per GPAQ data cleaning guidelines \[[@CR29]\]. ### Urban sampling {#Sec4} We randomly selected three mahallas---*the lowest urban geographic unit having identifiable boundaries*---out of total 10 mahallas from two purposively selected wards of Dhaka City Corporation, consisting of 382 holding numbers. Each household was considered as a cluster and who were available in that cluster were approached to participate in this study. Eligibility criteria included Bangladesh nationals aged 25--64 years who stayed in the household the night before the day of the first visit. Individuals who were mentally challenged (inability to communicate) or remained bed-ridden were excluded. Finally, a total of 96 holding numbers starting from holding number 1 were visited to get the targeted sample size (403). ### Rural sampling {#Sec5} We purposively selected two villages of Tangail District---situated approximately 130 km northwest of the capital city, Dhaka. There were 467 households in these two villages. The same procedure was followed as in urban area for finding the eligible household in order to collect data. Finally, a total of 212 households were visited to get the targeted sample size (403). Data collection instrument {#Sec6} -------------------------- We used the Global Physical Activity Questionnaire version 2 (GPAQ-2) for measuring PA levels of this study \[[@CR29]\]. It was developed by the WHO for PA surveillance in developing countries. The questionnaire consists of 16 questions covering moderate- and vigorous-intensity PA participation in three settings---PA at work, commuting (travel to and from places), and recreational activities as well as sedentary behavior. Vigorous-intensity activities were defined as "activities that require hard physical effort and cause large increases in breathing or heart rate", and moderate-intensity activities were defined as "activities that require moderate physical effort and cause small increases in breathing or heart rate". The participants were asked whether they engaged in these types of activities for at least 10 min continuously and, if so, for how many days (frequency) they performed these activities in a typical or usual week, and for how much time (intensity) they spent on a typical day etc. \[[@CR29]\]. Question on sedentary behavior inquired only on time that they usually spent sitting or reclining on a typical day but did not include time spent sleeping \[[@CR29]\]. For this study, we translated GPAQ-2 questionnaire into Bengali. Firstly, the questionnaire was translated from English to Bengali by two independent experts and then back translation was done from Bengali to English by another two independent experts. It was then finalized through a consensus meeting among these four experts. We applied both forward and backward methodologies during translation in order to ensure the appropriate meaning of each item was retained. No changes were made to the original contents; however, local examples of types and intensity of activities were used to suit the Bangladeshi context. Field enumerators underwent a 2-day training before deployment. A list of moderate and vigorous activities for work and recreation domains with local examples were prepared and data collectors were adequately oriented with these activities including their operational definitions so that consistent findings could be obtained from both urban and rural settings. The GPAQ-2 analysis protocol was followed for data collection, processing, and analysis \[[@CR29]\]. ### Conversion of PA data to estimated energy expenditure {#Sec7} We converted inquired data on PA (Total time spent on work, commuting and leisure-time activities of each intensity) to METs (Metabolic Equivalent Tasks), weighted by GPAQ-assigned MET energy expenditure ratios per kilogram per hour of 4 for moderate, and 8 for vigorous intensity activities. MET is the unit used to express the intensity of physical activities. Detailed methods of calculating MET have been described elsewhere \[[@CR21], [@CR27], [@CR29]\]. ### Procedures for classifying PA levels {#Sec8} A person's normal level of PA was classified into the low, moderate, and high level as defined by the GPAQ-2; the criteria of these levels have been described elsewhere \[[@CR21], [@CR27], [@CR29]\]. We further categorized these three PA levels into **'sufficiently active'** or **'insufficiently active'** groups. The 'sufficiently active' group included participants who met the PA recommendation, therefore classified as being in the moderate or high level category (21, 29). ### Socio-demographic factors {#Sec9} Information on the location of residence, sex, age, occupational status, educational levels, and socio-economic status were obtained in order to assess the association of these socio-demographic factors with insufficient PA. In regard to level the socio-economic status, participants were asked to allocate themselves to one of three categories; under which category (poor, middle class, and rich) they fall in. Data analysis {#Sec10} ------------- Median (interquartile range) of METs for sexes and urban-rural areas were obtained. The prevalence of PA levels and other categorical variables are reported as percentages with 95% confidence intervals (CIs). Finally, the binary logistic regression model was used to estimate relationships between physical inactivity and socio-demographic characteristics. Variables included in the model were the location of residence, sex, age groups, occupational status, educational level, and socio-economic status. Data were analyzed using SPSS version 16.0. Ethical consideration {#Sec11} --------------------- Ethical clearance was obtained from the ethical review committee of Diabetic Association of Bangladesh (BADAS). International ethical guidelines for biomedical research involving human subjects were followed throughout the study \[[@CR30]\]. Written (or thumb impression if unable to write) consent was obtained from all participants. Total 48 (women, 33; men 15) participants (about 6%) were unable to write. Results {#Sec12} ======= Of total 792 (urban, 395; rural, 397) participants, 48.1% were women. The mean (standard deviation) age of the participants was 37.3 (10.4) years. One-fourth (23.4%) of the participants had no formal education and another one-third (35.2%) had completed any primary level education. Half of men (51.3%) were self-employed and one-third (37.5%) were salarymen both in public and nonpublic sectors. Of the women, 79.5% were homemakers. Further detail of socio-demographic information is given in Table [1](#Tab1){ref-type="table"}.Table 1Socio-demographic background (results in %) of the study participantsSocio-demographic factorsBoth sexesWomenMen*P* value(*n* = 792)(*n* = 381)(*n* = 411)(Chi-Square)Area of residence Urban49.946.253.30.47 Rural50.153.846.7Age groups (in years) 25--3444.64841.40.00 35--4428.532.325.1 45--5417.813.122.1 55--649.16.611.4Occupational status Employed26.615.037.50.00 Self-employed27.11.051.3 Student3.52.14.9 Housewife38.379.50.0 Unemployed4.42.46.3Educational levels No formal schooling23.428.918.20.00 Less than primary8.69.28.0 Primary completed26.631.821.9 Secondary completed16.716.017.3 Higher secondary completed11.47.115.3 Graduation degree and above13.47.119.2Socio-economic status^a^ Poor32.334.630.20.40 Middle Class51.649.953..3 Rich16.015.516.5^a^Self reported PA levels {#Sec13} --------- ### Distribution of total PA MET-minute {#Sec14} The overall median MET-minute of total PA in a typical week was 1280. It was almost double in the rural area (1720) than the urban area (960). Men also reported double (1680) compared to women (800). These differences were statistically significant (*p* \< 0.5). ### Composition of total PA {#Sec15} The compositions of total PA in both urban and rural areas were constituted mostly from work and commute domains. A little contribution was from leisure-time activity. Of total PA in urban, 40.0% was contributed by work-related activity followed by commuting (57.0%) and recreational activity (3.0%), whereas in rural, the composition was work-related activity (77.0%), commuting (21.0%) and recreational activity (2.0%) as shown in Fig. [1](#Fig1){ref-type="fig"}.Fig. 1Composition of total physical activity in urban and rural areas ### Prevalence of PA levels (low, moderate, and high) {#Sec16} According to the GPAQ-2 classification, the overall age-standardized prevalence of PA levels was low 50.3% (95% CI: 46.8--53.8), moderate 26.6% (23.5--29.7), and high 23.1% (20.6--26.0). The prevalence of low PA was significantly higher in urban areas (59.5%) than rural areas (41.9%). The prevalence of moderate and high level of PA in rural areas (31.8%, 26.3%) was comparatively higher than urban areas (21.3%, 19.2%) (Table [2](#Tab2){ref-type="table"}).Table 2Prevalence of physical activity levels in urban and rural area,%UrbanRuralOverallSexAge (years)nLowModerateHighnLowModerateHighnLowModerateHighMen25--3411031.831.836.46026.725.048.317030.029.440.635--445330.247.222.65020.028.052.010325.237.836.945--543357.618.224.25832.824.143.19141.822.036.355--642382.613.14.32470.829.20.04776.621.32.125--64 (Crude), (95% CI)22940.6 (34.2--47.0)31.5 (25.5--37.5)27.9 (22.1--33.7)19232.3 (25.7--38.9)26.0 (19.8--32.2)41.7 (34.7--48.7)41136.7 (32.0--41.4)29.0 (24.6--33.4)34.3 (29.7--38.9)25--64 (age standardized)^a^, (95% CI)22945.9 (39.4--52.4)29.8 (23.9--35.7)24.3 (18.7--29.9)19233.7 (27.0--40.4)26.3 (20.1--32.5)40.0 (33.1--46.9)41139.3 (34.6--44.0)28.6 24.2--33.0)32.1 (27.6--36.6)Women25--347660.513.226.310746.739.314.018352.528.419.135--446572.312.315.45827.655.217.212351.232.516.345--542692.37.70.02454.225.020.85074.016.010.055--64977.822.20.01693.86.20.02588.012.00.025--64 (Crude), (95% CI)17670.5 (63.8--77.2)12.5 (7.6--17.4)17.0 (11.5--22.5)20545.9 (39.1--52.2)39.5 (32.8--46.2)14.6 (9.8--19.4)38157.3 (52.3--63.3)27.0 (22.5--31.5)15.7 (12.0--19.4)25--64 (age standardized)^a^, (95% CI)17674.1 (67.6--80.6)13.2 (8.2--18.2)12.7 (7.8--17.6)20551.0 (44.2--52.7)34.8 (28.3--41.3)14.2 (9.4--19.0)38163.1 (58.3--67.9)23.9 (19.6--28.2)13.0 (9.6--16.4)Both25--3418643.524.232.316739.534.226.335341.628.929.535--4411853.428.018.610824.142.633.322639.435.025.645--545972.813.613.68239.024.436.614153.219.827.055--643281.315.63.14080.020.00.07280.518.11.425--64 (Crude), (95% CI)39553.9 (49.0--58.8)23.1 (18.9--27.3)23.0 (18.8--27.2)39739.3 (34.5--44.1)33.0 (28.4--37.6)27.7 (23.3--32.1)79246.6 (43.1--50.1)28.0 (24.9--31.1)25.4 (22.4--28.4)25--64 (age standardized)^a^, (95% CI)39559.5 (54.7--64.3)21.3 (17.3--25.3)19.2 (15.3--23.1)39741.9 (37.0--46.8)31.8 (27.2--36.4)26.3 (22.0--30.6)79250.3 (46.8--53.8)26.6 (23.5--29.7)23.1 (20.2--26.0)^a^Standardized to the age distribution of the new WHO world standard population (2000--2025) Socio-demographic factors associated with low PA level {#Sec17} ------------------------------------------------------ We also assessed whether PA associated with socio-demographic factors such as age, sex, occupation, education, and socio-economic status (Table [3](#Tab3){ref-type="table"}). Overall urban dwellers were positively associated (OR = 2.2, \[95% CI: 1.5--3.2\]) with insufficient PA compared to those residing in rural areas. Generally, women and housewives, in particular, were 2.1 and 3.8 times more likely than men, and other occupations respectively to have an insufficient PA. Although OR for having insufficient PA increased across the age groups, the oldest age group reported greatest insufficient PA compared to the youngest age group, especially in urban areas (30.8, 8.4--113.2). Compared with those having no formal education, insufficient PA was 8.6 times higher in individuals who completed graduation or more. Overall, individuals self-allocated to the higher socio-economic class category were 2.4 times more likely than those self-allocated to the poor category to report insufficient PA as shown in Table [3](#Tab3){ref-type="table"}.Table 3Odds ratio (95% confidence interval) of socio-demographic factors for insufficient physical activitySocio-demographic factorsBoth areasUrbanRural(*n* = 792)(*n* = 395)(*n* = 397)Sex MenRef.Ref.Ref. Women2.1 (1.4--3.9)3.6 (1.5--8.6)1.4 (0.4--5.3)Age groups (in years) 25--34Ref.Ref.Ref. 35--441.1 (0.7--1.6)1.9 (1.0--3.6)0.5 (0.3--1.0) 45--543.4 (2.0--5.6)9.4 (3.5--25.2)1.5 (0.8--2.8) 55--6415.6 (7.5--32.2)30.8 (8.4--113.2)10.6 (4.2--26.5)Occupational status EmployedRef.Ref.Ref. Self-employed1.1 (0.6--1.9)0.4 (0.2--1.0)0.9 (0.3--2.7) Student1.4 (0.5--3.3)0.6 (0.2--2.2)2.3 (0.5--10.8) Housewife3.8 (2.0--7.2)7.8 (3.2--18.7)1.9 (0.4--8.8) Unemployed1.7 (0.7--4.1)3.2 (0.9--11.6)0.7 (0.2--3.5)Educational levels No formal schoolingRef.Ref.Ref. Less than primary1.2 (0.6--2.3)2.2 (0.6--7.7)0.8 (0.3--1.9) Primary completed2.4 (0.8--3.8)3.4 (0.9--9.4)2.0 (0.8--3.6) Secondary completed1.2 (0.7--2.1)2.7 (0.9--8.0)0.7 (0.3--1.4) Higher secondary completed4.5 (2.3--9.0)17.0 (4.8--60.2)1.6 (0.6--4.3) Graduation degree and above8.6 (4.1--17.7)25.8 (7.7--86.4)5.2 (1.3--20.6)Socio-economic status^a^ PoorRef.Ref.Ref. Middle Class1.4 (0.9--2.0)2.1 (1.1--3.8)0.9 (0.5--1.5) Rich2.4 (1.4--4.2)6.7 (2.2--20.0)1.3 (0.7--2.7)^a^Self reported Discussion {#Sec18} ========== The paper reports the prevalence of PA and socio-demographic factors associated with low level of PA among adults living in urban versus rural settings in Bangladesh using GPAQ-2. Irrespective of age and gender, half (50.3%) of the adult population showed a 'low level' of PA. A few national studies done using GPAQ on adults with similar age groups showed the prevalence of low PA of 34.5% \[[@CR27]\], 38.0% \[[@CR28]\] and 49.2% \[[@CR31]\] while two other studies reported low PA which is not directly comparable because of using different approaches of leveling low PA (35.2%) \[[@CR32]\] and/or unable to provide point estimate \[[@CR33]\]. Results of these studies showed a wide variability with our estimate, which is a not uncommon phenomenon. A review demonstrated that PA estimates vary markedly even within a single country using different surveys in similar time periods \[[@CR34]\]. Although our estimate of low PA is much higher than the global estimate (31.0%) of adults aged 15 years and above \[[@CR35]\] it is similar to a recent prevalence estimate of our neighboring India (54.4%), whom population characteristics are similar to Bangladeshi population \[[@CR36]\]. Data of Behavioral Risk Factor Surveillance System showed that half of the U.S. adults (49.4%) were physically inactive which is also similar to our current estimate \[[@CR37]\]. Variability of low physical activity level between different regions of the world, among different countries and within the country is well documented \[[@CR22], [@CR34], [@CR38]--[@CR40]\]. This study also confirmed that urban adults are more physically inactive (or insufficiently active) than that in rural adults. It means that these people are not meeting the minimum recommendation of at least 30 minutes of moderate-intensity PA or walking for 5 or more days per week. This difference of physical inactivity between people living in the urban and rural area might be explained by poorly planned urbanization including mechanization of life, lack of play grounds, parks, walkable footpaths; unsafe roads for bicycles etc. \[[@CR25]\]. The existence of mechanized work appliance, sedentary life-style and less engagement in vigorous-intensity activities like agricultural work in urban area might also have limited overall participation in PA. In general, results of this study confirmed that urban dwellers, women, older age group, people with higher level of education, and higher socio-economic class are more likely to report insufficient PA. These observations are similar to the results observed in many low and middle incomes \[[@CR1], [@CR20]--[@CR22], [@CR25], [@CR40], [@CR41]\]. However, very few countries show dissimilarities in gender and age, for example, Croatia, Hungary, Slovakia, Kazakhstan, Ukraine, Argentina, Portugal and Saudi Arabia where men are more likely to report the low level of PA compared to women \[[@CR20], [@CR41]\]. Likewise, though there was a general decline across age groups, the rate of PA remained high in the older age group for some countries like New Zealand, China and Hong Kong \[[@CR20], [@CR41]\]. These differences can be explained by their unique lifestyle and workforce pattern. In this study, in general, men reported spending more time in work and transport-related physical activities compared to women in both urban and rural areas. The results of this study also showed that physical activities at work and commute domains are the main contributors to total PA among the study population. In addition, physical activities undertaken as part of recreational or leisure-time activity contributed very little (around 3.0%) to the total PA in this population. These results are in line with many low and middle-income countries where work and transport-related activities are the prime contributors to overall PA compared to leisure time activities \[[@CR21], [@CR22], [@CR38], [@CR42]\]. However, in high-income countries (such as Australia, Canada, New Zealand, USA), leisure-time PA is a major component of total PA undertaken by adults \[[@CR20], [@CR43], [@CR44]\]. This difference can be explained by a higher availability and accessibility to sports or recreational facilities and a history of long-term promotion of exercise in high-income countries. Moreover, PA through sports in leisure-time is a concept which is already well suited in high-income countries but not an established concept in many low-income countries like Bangladesh. This suggests that promoting leisure-time PA can be a strategy to increase overall PA at the population level in our country. Considering the growing burden of NCDs in our country, this potential risk factor that shows the higher prevalence in this study must need to address at national, community and individual level for combating this growing epidemic and improving the overall health of the population. Certain factors might influence the findings of the current study like the subjective judgment of vigorous and moderate PA might differ by the location, the level of education and gender. Generally, people, especially in the rural setting, do not habituate to measure the time they spend on certain activities in hours and minutes. This phenomenon is also true for certain urban people who are engaged in the informal sector. Therefore, people might have under or over-reporting their physical activities that cannot be over ruled with certainty. Potential bias related to the categorization of socio-economic status cannot be over ruled because it was completely subjective. However, efforts were given to overcome these challenges by preparing exhaustive check list and adequate training of data collectors. Age-specific prevalence of PA should be interpreted cautiously because of inadequate sample size for age groups. Finally, the cross-sectional nature of the study design itself is one of the limitations in terms of identifying the associated factors. It is noteworthy here that our results on the basis of education could be considered as a proxy of socio-economic status. Limitations aside, this study provides a valuable snapshot of domain specific (work, commute, and recreation) PA patterns for urban and rural adults of Bangladesh which will provide valuable information for public health intervention planning to promote PA at the population level to combat the growing epidemic NCDs in Bangladesh. Conclusion {#Sec19} ========== Insufficient PA is highly prevalent among Bangladeshi adult population. Interventions targeting women, oldest age group, people with higher education, and higher socio-economic class especially in the urban area are warranted. The results of this study will focus the necessity of primary prevention of NCDs through PA intervention at the population level and will provide the baseline information about the PA levels of adult population in Bangladesh which will help the policy-makers at the national level to develop the national guidelines for PA to promote overall PA level. CI : Confidence interval GPAQ-2 : Global Physical Activity Questionnaire version 2 MET : Metabolic Equivalent Task NCDs : Noncommunicable diseases PA : Physical activity SPSS : Statistical Package for the Social Sciences WHO : World Health Organization This article is based on a thesis which was submitted to Bangladesh Institute of Health Sciences (BIHS) as a partial fulfillment of MPhil degree in Noncommunicable Diseases under the University of Dhaka. Advice and supports from the faculties of BIHS (Prof. MA Hafiz, and Drs Pradip Sen Gupta, Shahanaz Chowdhury, Rabeya Yasmin, Farhana Zaman, Fahmida Binte Mezbah,) are gratefully acknowledged. The contribution of Dr Md Nazmul Karim to data analysis is deeply appreciated. Finally, participants who kindly agreed to participate in the thesis study and provided their valuable time for interviews are gratefully acknowledged. Funding {#FPar1} ======= This research work (MPhil thesis) was funded by the 'Norad's Programme for Master Studies (NOMA)' grant of the University of Oslo, Norway. Availability of data and materials {#FPar2} ================================== All data supporting our findings are contained in this article. The dataset is available from the corresponding author on request and with the permission of Bangladesh Institute of Health Sciences. Authors' contributions {#FPar3} ====================== MM designed the main thesis, trained field enumerators, analyzed data, interpreted data and drafted the manuscript; MSAMA oversaw the quality of the main thesis and reviewed the manuscript; MMZ interpreted results critically, conceptualized the manuscript and critically reviewed it. All authors have read and approved the manuscript. Competing interests {#FPar4} =================== The authors do not have any competing interest. The authors alone are responsible for views expressed in this article and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated. Consent for publication {#FPar5} ======================= Not Applicable Ethics approval and consent to participate {#FPar6} ========================================== Ethical clearance was obtained from the ethical review committee of Diabetic Association of Bangladesh. Written (or thumb impression if unable to write) consent was obtained from the participants.

Introduction {#sec1} ============ Polynuclear transition metal sites commonly comprise redox catalysts in biological and chemical settings. In Nature, multielectron redox transformations, such as O~2~ and H^+^ reduction,^[@ref1],[@ref2]^ H~2~O oxidation,^[@ref3]^ and N~2~ fixation,^[@ref4]^ are carried out at polynuclear metal cofactors.^[@ref5]^ In heterogeneous catalysis, high-nuclearity surface features, such as step edges and defects that are capable of interacting with substrates via multipoint binding, are often proposed as catalyst active sites.^[@ref6]−[@ref9]^ With the aim to harness the potential of multiple reaction centers in proximity to accomplish multielectron chemical transformations, synthetic chemists have targeted development of specific ligand-supported multinuclear catalyst sites.^[@ref10]−[@ref12]^ Accomplishing small molecule activation reactions relevant to energy conversion schemes mandates development of catalysis of multielectron, multiproton reactions. We have been particularly interested in development of HX-splitting photocatalysis, which, like H~2~O-splitting schemes, stores substantial energy but has the advantage that only two proton/electron equivalents need to be managed to accomplish a closed photocycle in contrast to the four implicit in H~2~O splitting.^[@ref3],[@ref13],[@ref15]^ In this context, we have developed a family of two-electron mixed-valent dirhodium HX-splitting photocatalysts (i.e., Rh~2~\[I,III\] complex **1**, Figure [1](#fig1){ref-type="fig"}) predicated on the hypothesis that two-electron mixed valency can engender molecular excited states with the proclivity to engage in two-electron redox transformations.^[@ref16],[@ref17]^ These complexes have proven to be adept at multielectron photoreactions; H~2~-evolving photocatalysis has been realized in which formal halogen elimination reactions close the catalytic cycle.^[@ref18],[@ref19]^ ![HX-splitting photocycle catalyzed by Rh~2~\[I,III\] complex **1** or Rh~2~\[II,II\] complex **2** involves both proton reduction and halide oxidation. The critical halide oxidation step is proposed to proceed from halide-bridged intermediate **3**.](ja-2014-08218v_0001){#fig1} The challenge in developing HX-splitting chemistry is the halogen elimination half reaction, which has been the bottleneck to development of HX-splitting photochemistry. Only recently has mechanistic information regarding this reaction from dirhodium complexes become available. Nanosecond-resolved transient absorption (TA) spectroscopy of halogen photoelimination reactions from a pair of valence isomers, two-electron mixed-valent complex **1** and valence-symmetric complex **2**, revealed photoelimination via a common intermediate. We proposed this intermediate to be Cl-bridged binuclear complex **3**, generated by photoextrusion of an isocyanide ligand (L, Figure [1](#fig1){ref-type="fig"}) and migration of a Cl ligand to a bridging position.^[@ref20]^ Intermediate **3** represents an intramolecular analogue of ligand-bridged intermediates proposed in binuclear elimination reactions.^[@ref21],[@ref22]^ We now utilize photocrystallography to provide direct information regarding structural changes associated with intermediates of consequence to halogen elimination. Together with solution-phase and solid-state transient absorption and DFT modeling, we establish that halide-bridged intermediates are the critical intermediates of halogen elimination from two-electron bimetallic cores. Complementary synthesis of a new suite of dirhodium complexes allowed isolation and independent evaluation of the chemistry of the halide-bridged bimetallic complexes. Direct observation of halide-bridged structures establishes binuclear reductive elimination as a critical pathway for metal-halide bond activation. Experimental Section {#sec2} ==================== General Considerations {#sec2.1} ---------------------- All reactions were carried out in an N~2~-filled glovebox. Anhydrous solvents were obtained from drying columns.^[@ref23]^ \[Rh(cod)Cl\]~2~ and \[Rh(CO)~2~Cl\]~2~ were obtained from Strem Chemicals and used without purification. Ligand tfepma,^[@ref24]^ Rh~2~\[I,III\] complex **1**,^[@ref20]^ and Rh~2~\[II,II\] complex **2**([@ref25]) were prepared as previously described. Physical Methods {#sec2.2} ---------------- NMR spectra were recorded at the Harvard University Department of Chemistry and Chemical Biology NMR facility on a Varian Mercury 400 spectrometer operating at 400 MHz for ^1^H acquisitions, 162 MHz for ^31^P acquisitions, and 375 MHz for ^19^F acquisitions. NMR chemical shifts are reported in ppm, with the residual solvent resonance as internal standard. UV--vis spectra were recorded at 293 K in quartz cuvettes on a Spectral Instruments 400 series diode array blanked against the appropriate solvent. IR spectra were recorded with powdered samples on a PerkinElmer Spectrum 400 FT-IR/FT-FIR spectrometer outfitted with a Pike Technologies GladiATR attenuated total reflectance accessory with a monolithic diamond stage and pressure clamp. Photochemistry {#sec2.3} -------------- Steady-state photochemical reactions were performed using a 1000 W high-pressure Hg/Xe arc lamp (Oriel), and the beam was passed through a water-jacketed filter holder containing the appropriate long-pass filter, an iris, and a collimating lens. Samples were photolyzed in a constant-temperature circulating water bath (23 °C). Nanosecond transient absorption (TA) measurements were made using a previously described home-built system.^[@ref26]^ Solution-phase TA measurements were performed on THF solutions of **1** and **2**, and solid-state TA measurements were performed on samples prepared by drop-casting solutions of **1** and **2** (from either CH~2~Cl~2~ or THF) on glass slides and allowing them to dry under ambient conditions for 1 h. All TA measurements were carried out at 23 °C. X-ray Crystallographic Details {#sec2.4} ------------------------------ X-ray structures of complexes **3** and **6**--**10** and variable-temperature (VT) X-ray data were collected on a Bruker three-circle platform goniometer equipped with an Apex II CCD and an Oxford cryostream cooling device operating between 100 and 275 K. Radiation was from a graphite fine focus sealed tube Mo Kα (0.71073 Å) source. Crystals were mounted on a glass fiber pin using Paratone N oil. Data was collected as a series of φ and/or ω scans. Data was integrated using SAINT and scaled with multiscan absorption correction using SADABS.^[@ref27]^ The structures were solved by intrinsic phasing using SHELXT (Apex2 program suite, v2014.1) and refined against *F*^2^ on all data by full matrix least-squares with SHELXL-97.^[@ref28]^ All non-hydrogen atoms were refined anisotropically. H atoms were placed at idealized positions and refined using a riding model. Crystal data and refinement statistics are summarized in Tables [1](#tbl1){ref-type="other"} and [S1](#notes-1){ref-type="notes"}, and thermal ellipsoid plots are collected in Figures [5](#fig5){ref-type="fig"}, [7](#fig7){ref-type="fig"}, and [S15](#notes-1){ref-type="notes"}. Powder diffraction experiments were carried out with a Bruker D2 Phaser using a Cu anode. ###### Crystal Data and Structure Refinement   **3** **6**·THF *trans*-**7** **8** **9**·Et~3~NHCl ---------------------------------------------- --------------------------------------- ---------------------------------------- ---------------------------------------- --------------------------------------- --------------------------------------- formula C~29~H~37~Cl~4~F~24~N~3~O~8~P~4~Rh~2~ C~24~H~28~Cl~2~F~24~N~2~O~11~P~4~Rh~2~ C~20~H~22~Cl~4~F~24~N~2~O~10~P~4~Rh~2~ C~19~H~22~Cl~4~F~24~N~2~O~9~P~4~Rh~2~ C~25~H~17~Cl~3~F~24~N~3~O~9~P~4~Rh~2~ CCDC no. 1008080 1006061 1006063 1006064 1006060 fw, g/mol 1483.12 1377.51 1377.90 1349.07 1415.05 temp, K 100 (2) 100 (2) 100 (2) 100 (2) 100 (2) cryst system triclinic monoclinic monoclinic triclinic tetragonal space group *P*1̅ *C*2/*m* *C*2/*c* *P*1̅ *I*4~1~*cd* color yellow orange orange orange orange *a*, Å 10.2286 (8) 32.468 (6) 23.308 (1) 12.2293 (7) 23.271 (1) *b*, Å 12.356 (1) 21.017 (4) 23.326 (1) 18.579 (1) 23.271 (1) *c*, Å 20.409 (2) 14.625 (3) 16.9357 (7) 19.257 (1) 36.041 (2) α, deg 94.387 (1) 90 90 89.993 (1) 90 β, deg 96.782 (1) 114.336 (2) 107.939 (1) 87.029 (1) 90 γ, deg 99.181 (1) 90 90 87.275 (1) 90 *V*, A^3^ 2516.5 (3) 9093 (3) 8760.2 (6) 4364.3 (5) 19517 (2) *Z* 4 12 8 6 24 R1[a](#t1fn1){ref-type="table-fn"} 0.059 0.087 0.070 0.061 0.033 wR2[b](#t1fn2){ref-type="table-fn"} 0.116 0.267 0.211 0.151 0.072 GOF[c](#t1fn3){ref-type="table-fn"} (*F*^2^) 1.00 1.07 1.03 1.00 1.04 *R*~int~ 0.095 0.094 0.066 0.118 0.100 R1 = ∑∥*F*~o~ -- \|*F*~c~∥/∑\|*F*~o~\|. wR2 = (∑(w(*F*~o~^2^ -- *F*~c~^2^)^2^)/∑(w(*F*~o~^2^)^2^))^1/2^. GOF = (∑w(*F*~o~^2^ -- *F*~c~^2^)^2^/(*n* -- *p*))^1/2^, where *n* is the number of data and *p* is the number of parameters refined. Photocrystallography data was collected using 0.41328 Å radiation at temperature of 15 K (Oxford Diffraction Helijet) on a vertical mounted Bruker D8 three-circle platform goniometer equipped with an Apex II CCD at ChemMatCARS located at the Advanced Photon Source (APS), Argonne National Laboratory (ANL). Illumination was provided by a Thorlabs 365 nm LED (M365L2) and was delivered to the sample via a 100 μm i.d. fiber optic. Dark structures were solved and refined as described above. For data sets obtained during irradiation, non-H atoms of the product were located in difference-Fourier maps, calculated with coefficients *F*~0~(irradiated) -- *F*~0~(dark), and then refined with constraints on the product molecule\'s atomic displacement parameters to the corresponding values of the reactant molecule (EADP instructions of SHELXL97). The percentage of the reactant in the crystal was treated as a variable in the refinements. Computational Details {#sec2.5} --------------------- B3LYP^[@ref29]−[@ref31]^ calculations were performed using the Gaussian 09, revision D.01, suite of software.^[@ref32]^ Model complexes in which adamantyl isocyanide ligand was replaced with a methyl isocyanide and the bridging tfepma ligands were replaced with bis(fluoromethoxyphosphino)methylamine (fmpma) ligands were used in all computations. These model structures are less severe truncations than have previously been employed in computational investigations of phosphazane bridged dirhodium complexes.^[@ref33]−[@ref35]^ Gas-phase geometry optimizations and TD-DFT calculations were carried out using an SDD basis set for Rh^[@ref36]^ and 6-31G\* for all other atoms.^[@ref37],[@ref38]^ Stationary points were characterized with frequency calculations. Single-point solvent corrections (THF) were carried out using a polarizable continuum model using the integral equation formalism variant. B3LYP geometries well reproduced experimental metrical parameters, obtained by X-ray crystallography (tabulated in [Tables S6 and S7](#notes-1){ref-type="notes"}), and provided similar structural parameters as optimizations carried out with either the M06^[@ref39],[@ref40]^ or M06-L^[@ref41]^ functional. Computed absorption spectra (line broadening 0.15 eV) reproduced experimental absorption spectra. NBO calculations^[@ref42]−[@ref45]^ were carried out using Gaussian NBO, version 3.1.^[@ref46]^ Rh~2~(tfepma)~2~(CO)~2~Cl~2~ (**6**) {#sec2.6} ------------------------------------ To a solution of \[Rh(CO)~2~Cl\]~2~ (38.0 mg, 9.77 × 10^--5^ mol, 1.00 equiv) in THF (3 mL) at 23 °C was added tfepma (95.2 mg, 1.95 × 10^--4^ mol, 2.00 equiv) dropwise as a THF (2 mL) solution. The reaction mixture was stirred at 23 °C for 1 h, during which time evolution of bubbles was observed and the color of the reaction mixture turned from orange to dark red. Complex **6** was not isolated as a solid because removal of solvent led to a mixture of Rh~2~(tfepma)~2~(CO)~2~Cl~2~ (**6**) and Rh~2~(tfepma)~2~(μ-CO)Cl~2~ (**9**) and thus solution characterization (^1^H NMR, ^19^F NMR, ^31^P NMR, and UV--vis) was carried out using the reaction solution without further purification.  On the basis of integration of the ^1^H NMR spectrum against hexamethylbenzene (internal standard), the yield of **6** was 98%. ^1^H NMR (THF-*d*~8~, 23 °C): δ 4.96--4.88 (m, 4H), 4.84--4.74 (m, 12H), 2.99 (pseudoquintet, *J* = 3.6 Hz, 6H). ^31^P NMR (THF-*d*~8~, 23 °C): δ 131.2--130.2 (m, 4P). ^19^F NMR (THF-*d*~8~, 23 °C): δ −75.5 (dt, *J* = 19.7 Hz, *J* = 9.2 Hz, 24F). IR: ν~CN~ = 2002 cm^--1^. Crystals suitable for single-crystal diffraction analysis were obtained from a THF solution layered with pentane at −30 °C. Satisfactory combustion analysis could not be obtained because **6** was not stable to evacuation of solvent. Rh~2~(tfepma)~2~(CO)~2~Cl~4~ (**7**) {#sec2.7} ------------------------------------ To a solution of \[Rh(CO)~2~Cl\]~2~ (44.0 mg, 1.13 × 10^--4^ mol, 1.00 equiv) in PhCH~3~ (5 mL) at 23 °C was added tfepma (110 mg, 2.26 × 10^--4^ mol, 2.00 equiv) dropwise as a PhCH~3~ (1 mL) solution. The reaction mixture was stirred at 23 °C for 1 h, during which time evolution of bubbles was observed and the color of the reaction mixture turned from orange to dark red. PhICl~2~ (46.6 mg, 1.70 × 10^--4^ mol, 1.50 equiv) was added as a solid. ^31^P NMR analysis of an aliquot of the reaction mixture showed the presence of two ^31^P NMR signals (multiplets at 105.3--104.5 and 102.2--101.5 ppm) in a 2:1 ratio, assigned as *trans*-**7** and *cis-***7**, respectively ([Figure S6](#notes-1){ref-type="notes"}). Heating the reaction mixture at 70 °C for 2 h resulted in the conversion of *trans*-**7** to *cis-***7**. The reaction mixture was cooled to −30 °C for 1 h, at which time a precipitate was observed. The solvent was decanted, and the residue washed with pentane and dried in vacuo to afford 123 mg of the title complex as a yellow solid in 79% yield. *trans*-**7**: ^1^H NMR (C~6~D~6~, 23 °C): δ 4.86--4.83 (m, 8H), 4.51--4.46 (m, 4H), 4.36--4.31 (m, 4H), 2.58 (pseudoquintet, *J* = 3.9 Hz, 6H). ^31^P NMR (C~6~D~6~, 23 °C): δ 105.3--104.5 (m, 4P). ^19^F NMR (C~6~D~6~, 23 °C): δ −74.68 (t, *J* = 7.9 Hz, 12F), −75.09 (t, *J* = 7.9 Hz, 12F). IR: ν~CN~ = 2082 cm^--1^. Crystals of *cis-***7** suitable for single-crystal diffraction analysis were obtained from a THF solution layered with pentane at −30 °C (see the [Supporting Information](#notes-1){ref-type="notes"}), and crystals of *trans-***7** suitable for single-crystal diffraction analysis were obtained from a CH~2~Cl~2~ solution layered with hexane at −30 °C. Rh~2~(tfepma)~2~(CO)(μ-Cl)Cl~3~ (**8**) {#sec2.8} --------------------------------------- A thin film of *trans*-**7** (47.0 mg, 3.41 × 10^--4^ mol, 1.00 equiv) was deposited on the wall of a two-chamber photoreaction vessel and dried in vacuo for 12 h. Broad-band photolysis of the thin film with a 1000 W Hg lamp was carried out for 4 h under dynamic vacuum (20 mTorr). The residue was taken up in CH~2~Cl~2~, layered with pentane, and cooled to −30 °C for 1 h, at which time the solvent was decanted. The residue was dried in vacuo to afford 40.1 mg of the title complex as a yellow solid in 87% yield. ^1^H NMR (C~6~D~6~, 23 °C): δ 5.02--4.92 (m, 2H), 4.72--4.64 (m, 2H), 4.56--4.46 (m, 4H), 4.45--4.39 (m, 2H), 4.29--4.15 (m, 6H), 2.61 (pseudoquintet, *J* = 3.9 Hz, 6H). ^31^P NMR (C~6~D~6~, 23 °C): δ 106.8--104.2 (m, 4P). ^19^F NMR (C~6~D~6~, 23 °C): δ −74.49 (t, *J* = 7.9 Hz, 6F), −74.90 (t, *J* = 7.9 Hz, 6F), −74.92 (t, *J* = 7.9 Hz, 6F), −75.08 (t, *J* = 7.9 Hz, 6F). IR: ν~CN~ = 2086 cm^--1^. Crystals of **8** suitable for single-crystal diffraction analysis were obtained from a THF solution layered with pentane at −30 °C. Rh~2~(tfepma)~2~(μ-CO)Cl~2~ (**9**) {#sec2.9} ----------------------------------- To a solution of \[Rh(CO)~2~Cl\]~2~ (63.7 mg, 1.64 × 10^--4^ mol, 1.00 equiv) in THF (5 mL) at 23 °C was added tfepma (160 mg, 3.28 × 10^--4^ mol, 2.00 equiv) dropwise as a THF (5 mL) solution. The reaction mixture was stirred at 23 °C for 1 h, during which time evolution of bubbles was observed and the color of the reaction mixture turned from orange to dark red. The solvent was removed in vacuo. The residue was dissolved in CH~2~Cl~2~ (5 mL), and the solvent was removed in vacuo. The redissolution/evaporation sequence was repeated four times, and the residue was triturated in pentane to afford 176 mg of complex **9** as an orange solid (84% yield). ^1^H NMR (CD~2~Cl~2~, 23 °C): δ 4.70--4.60 (m, 8H), 4.59--4.47 (m, 8H), 2.75 (pseudoquintet, *J* = 3.5 Hz, 6H). ^31^P NMR (CD~2~Cl~2~, 23 °C): δ 130.1--129.1 (m, 4P). ^19^F NMR (CD~2~Cl~2~, 23 °C): δ −75.3 (br s, 24F). IR: ν ~CN~ = 1812 cm^--1^. Combustion analysis, found (calcd): C, 17.95 (17.84); H, 1.96 (1.73); N, 2.17 (2.19). Crystals suitable for single-crystal diffraction analysis were obtained from a THF solution **9** and Et~3~NHCl layered with pentane at −30 °C. Rh~2~(tfepma)~2~(CO)(AdNC)Cl~4~ (**10**) {#sec2.10} ---------------------------------------- To a solution of Rh~2~(tfepma)~2~(μ-CO)Cl~2~ (**9**) (120 mg, 9.40 × 10^--5^ mol, 1.00 equiv) in THF (2 mL) at 23 °C was added AdNC (15.2 mg, 9.40 × 10^--5^ mol, 1.00 equiv) dropwise as a THF (1 mL) solution. The reaction mixture was stirred at 23 °C for 30 min, and the solvent was removed in vacuo. The residue was taken up in CH~2~Cl~2~ (2 mL), and PhICl~2~ (28.4 mg, 1.03 × 10^--4^ mol, 1.10 equiv) was added. Hexanes (15 mL) were added, and the reaction mixture was cooled to −30 °C for 12 h, at which time 75.3 mg of complex **10** was isolated as an orange crystalline solid (53% yield). ^1^H NMR (CD~2~Cl~2~, 23 °C): δ 5.22--5.14 (m, 4H), 4.83--4.68 (m, 8H), 4.65--4.55 (m, 4H), 3.02 (pseudoquintet, *J* = 3.9 Hz, 6H), 2.25 (br s, 3H), 2.18 (br s, 6H), 1.80 = 1.71 (m, 6H). ^31^P NMR (CD~2~Cl~2~, 23 °C): δ 113.6--111.1 (m, 2P), 108.4--106.7 (m, 2P). ^19^F NMR (CD~2~Cl~2~, 23 °C): δ −75.3 (t, *J* = 7.9 Hz, 6F), −75.4 (t, *J* = 7.9 Hz, 6F), −75.5 (t, *J* = 7.9 Hz, 6F), −75.8 (t, *J* = 7.8 Hz, 6F). IR: ν~CN~ = 2193 cm^--1^, 2093 cm^--1^. Crystals suitable for single-crystal diffraction analysis were obtained from a CH~2~Cl~2~ solution of **10** layered with hexanes at −30 °C. Rh~2~(tfepma)~2~AdNC(μ-Cl)Cl~3~ (**3**) {#sec2.11} --------------------------------------- A thin film of *trans*-**7** (52.0 mg, 3.44 × 10^--4^ mol, 1.00 equiv) was deposited on the wall of a two-chamber photoreaction vessel and dried in vacuo for 12 h. Broad-band photolysis of the thin film with a 1000 W Hg lamp was carried out for 4 h under dynamic vacuum (20 mTorr). The residue was taken up in CH~2~Cl~2~, layered with pentane, and cooled to −30 °C for 1 h, at which time the solvent was decanted. The residue was dried in vacuo to afford 48.0 mg of complex **3** as a yellow solid (94% yield). ^1^H NMR (CD~2~Cl~2~, 23 °C): δ 4.93--4.83 (m, 4H), 4.82--4.67 (m, 6H), 4.62--4.54 (m, 2H), 4.51--4.45 (m, 2H), 4.36--4.27 (m, 2H), 3.02 (pseudoquintet, *J* = 3.9 Hz, 6H), 2.13 (br s, 3H), 2.09 (s, 6H), 1.72--1.64 (m, 6H). ^31^P NMR (CD~2~Cl~2~, 23 °C): δ 116.5--114.4 (m, 2P), 113.2--111.1 (m, 2P). ^19^F NMR (C~6~D~6~, 23 °C): δ −74.64 (t, *J* = 7.9 Hz, 6F), −75.30 to −75.4 (m, 18F). IR: ν~CN~ = 2199 cm^--1^. Crystals of **3** suitable for single-crystal diffraction analysis were obtained by cooling a saturated CH~2~Cl~2~/hexanes solution of **3** to −30 °C. Results {#sec3} ======= Photocrystallography {#sec3.1} -------------------- Thin-film TA spectroscopy was used to establish the homology of solution-phase and solid-state photoreactions. Thin films of complexes **1** and **2**, drop-cast from either THF or CH~2~Cl~2~ solutions, showed UV--vis absorption spectra that overlaid with solution spectra of **1** and **2** ([Figure S24](#notes-1){ref-type="notes"}). The thin films are polycrystalline based on powder X-ray diffraction (**1**, [Figure S22](#notes-1){ref-type="notes"}; **2**, [Figure S23](#notes-1){ref-type="notes"}), but the crystal phase could not be established due to anisotropic orientation of crystallites, which is typical of polycrystalline thin films.^[@ref47]^ TA spectra (λ~exc~ = 355 nm) of these films were recorded at 1 μs delay and show similar features to those observed in solution-phase TA spectra of these complexes, confirming the formation of the same photointermediate in both solution and solid state (Figure [2](#fig2){ref-type="fig"}). ![UV--vis spectra (dotted black), solution-phase TA spectra (solid black), and thin-film TA spectra (red) of (a) Rh~2~\[I,III\] complex **1** and (b) Rh~2~\[II,II\] complex **2**. TA spectra were obtained by flash laser photolysis (355 nm) and recorded at a 1 μs delay.](ja-2014-08218v_0002){#fig2} Steady-state photocrystallography experiments were performed at the Advanced Photon Source (APS) housed at Argonne National Laboratory (ANL) using synchrotron radiation (0.41328 Å) and a 365 nm LED light source (5 mW power measured at the crystal). A photodifference map was generated by comparing diffraction data obtained for a single crystal of Rh~2~\[I,III\] complex **1** in the dark with diffraction data obtained for the same crystal during irradiation (Figure [3](#fig3){ref-type="fig"}a). The crystal was not moved between the acquisitions of the two data sets and thus the orientation matrix was unchanged during the experiment. The difference map showed the presence of a photoinduced structure populated at 5.7(9)% of the crystal. Examination of the structural perturbations in the photoinduced structure shows ligand reorganization that would be anticipated during a migration of one of the chloride ligands to a bridging position; key metrics include the Rh^1^--Rh^2^--Cl^3^ bond angle, which contracts from 91.05(5) to 83.2(2)° in the photoinduced structure. Concurrent with this, Rh^1^--Rh^2^--Cl^4^ expands from 90.97(5) to 92(2)°. Additionally, Rh^2^--Cl^3^, which is the bond vector participating in partial migration from terminal to bridging coordination mode, elongates from 2.362(2) to 2.66(4) Å (additional metrical parameters are provided in [Table S4](#notes-1){ref-type="notes"}). ![Thermal ellipsoid plots of photocrystallography results with photoinduced structures (solid) superimposed on dark structures (faded). (a) Rh~2~\[I,III\] plot; Rh^1^--Rh^2^--Cl^3^ 91.05(5)° (dark), 83.2(2)° (photoinduced). (b) Rh~2~\[II,II\] plot; Rh^1^--Rh^2^--Cl^3^ 91.15(5)° (dark), 78(2)° (photoinduced).](ja-2014-08218v_0003){#fig3} The extent of laser heating of the sample during data acquisition was examined by comparing the size of thermal ellipsoids of atoms not involved in the primary photoreaction (i.e., *N*-atoms in the phosphazane ligand) with the size of thermal ellipsoids for the same atoms as a function of temperature.^[@ref48]^ On the basis of this analysis, laser heating warmed the single crystal of **1** from 15 to ∼250 K ([Figure S18](#notes-1){ref-type="notes"}). The metrical parameters of **1** do not show significant temperature-dependent variation ([Table S2](#notes-1){ref-type="notes"}), confirming that the difference map arises from photochemical, not thermal, effects. The photoinduced structure derived from complex **1** was accessed rapidly during irradiation. Comparison of specific reflections from the structures obtained in the dark and under illumination revealed a set of reflections that showed substantial intensity changes upon irradiation. Examination of these particular reflections as the light was turned on showed that the intensity changes as a step function: the intensity increased rapidly relative to data collection and then reaches a steady-state value during irradiation ([Figure S19](#notes-1){ref-type="notes"}). A steady-state photocrystallography experiment was also performed using a single crystal of Rh~2~\[II,II\] complex **2** under identical conditions as those of the aforementioned experiment with **1** (15 K, 5 mW 365 nm irradiation, 0.41328 Å synchrotron radiation). A photoinduced structure was identified in the photodifference map in which the Rh^1^--Rh^2^--Cl^3^ angle contracts from 91.15(5) to 78(2)° upon irradiation (Figure [3](#fig3){ref-type="fig"}b). Partial migration of Cl^3^ toward the bridging position is accompanied by substantial elongation of the Rh--Cl bond (2.378(2) to 2.57(8) Å). The photoinduced geometry could be identified in the photodifference map generated from **2**, but full-molecule disorder in the available single crystals of **2** precluded determination of the population of the photoinduced structure. Variable-temperature crystallography experiments performed on a single crystal of **2** confirm that the observed structural effects are photoinduced, not thermally promoted. Similar to experiments carried out with complex **2**, the photoinduced structure is accessed rapidly relative to data acquisition. Upon cessation of irradiation, the photoinduced structure is no longer observed in the photodifference map, indicating that the photoinduced state is accessed only during steady-state irradiation. Computational Results {#sec3.2} --------------------- Natural bond order (NBO) calculations were undertaken to evaluate the electronic structure of the dirhodium complexes investigated in this study. Herein, computed structures are referred to by letter, not compound numbers (i.e., **A** is the computed structure of **1**). In each of **A**, **B**, and **C** (i.e., computed structures of **1**, **2**, and **3**, respectively), the highest-occupied NBO is Rh--Rh bonding. The Rh--Rh bond in **A** is polarized, as would be expected of a dative bond between a d^6^ and a d^8^ metal (Rh~a~/Rh~b~ = 38.5:61.5). For valence symmetric Rh~2~\[II,II\] structure **B**, the Rh--Rh bond is symmetrically shared by the two Rh atoms (Rh~a~/Rh~b~ = 51.3:48.7). Consistent with a two-electron mixed-valent formulation, the M--M bond of structure **C** is polarized to a similar extent as that of the M--M bond in **A** (Rh~a~/Rh~b~ = 42.6:57.4), consistent with the bonding predictions of the covalent bond classification for a dative covalent bond between the Rh centers.^[@ref49],[@ref50]^ Density functional theory (DFT) calculations, summarized in Figure [4](#fig4){ref-type="fig"}, address the structural manifestations of complete dissociation of one isocyanide ligand, as would be anticipated in the solution phase where free diffusion of the dissociated ligand is expected. Removal of one isocyanide ligand from either Rh~2~\[I,III\] structure **A** or Rh~2~\[II,II\] structure **B** results in site-vacant structures **A**~**vac**~ and **B**~**vac**~, respectively. Structures **A**~**vac**~ and **B**~**vac**~ were evaluated with single-point calculations because neither could be located as a stationary point; both **A**~**vac**~ and **B**~**vac**~ evolved to structure **C** without a barrier ([Figures S31 and S32](#notes-1){ref-type="notes"}). Reaction of photoextruded isocyanide ligand with chloride-bridged structure **C** could proceed at Rh~b~ to regenerate Rh~2~\[I,III\] complex **A** or at Rh~a~ to accomplish photoisomization to Rh~2~\[II,II\] complex **B**. The computed relative transition state energies for conversion of **C** to **A** and **C** to **B** are 10.8 and 11.7 kcal/mol, respectively. Consistent with these calculations, complex **1** displays wavelength-dependent photochemistry, undergoing both photoreduction (λ~exc~ \> 295 nm) and photoisomerization (λ~exc~ \> 380 nm) reactions. Both isomerization and photoreduction reactions proceed via a common intermediate; identical TA spectral line shapes were obtained when samples of Rh~2~ complex **1** underwent laser flash photolysis with either 355 or 385 nm incident light ([Figure S30](#notes-1){ref-type="notes"}). {#fig4} As opposed to solution photochemistry, free ligand dissociation during photocrystallography experiments is not possible because ligand motions are constrained by the crystal matrix.^[@ref51]^ To probe the impact of restricted isocyanide ligand dissociation, the geometry of Rh~2~\[I,III\] structure **A** was evaluated as a function of M--L bond length. The results are tabulated in Table [2](#tbl2){ref-type="other"} and show that small perturbations of the M--L bond length are sufficient to induce nascent migration of a proximal Cl ligand toward a bridging position. Similar M--L bond-length-dependent migrations were computed starting with Rh~2~\[II,II\] structure **B**. TD-DFT calculations of both **A** and **B** as a function of M--L bond length show that even partial migration of a Cl ligand toward a bridging site begins to give rise to the spectral features observed in thin-film TA measurements of both **1** and **2** ([Figure S39](#notes-1){ref-type="notes"}). Analogous results, both regarding free diffusion as would be encountered in solution and restricted diffusion as would be encountered in the solid state, have been obtained for homologous series of CO complexes (summarized in the [Supporting Information](#notes-1){ref-type="notes"}). ###### Computed Bond Metrics as a Function of M--L Distance {#fx1} Rh--L~a~ (Å) θ (deg) ϕ (deg) Rh--L~a~ (Å) θ (deg) ϕ (deg) -------------- --------- --------- -------------- --------- --------- 1.99 (**A**) 89.99 94.05 1.93 (**B**) 90.80 91.97 2.20 89.25 94.60 2.20 90.39 92.81 2.46 88.10 95.40 2.46 89.49 93.61 2.70 86.62 96.40 2.70 88.43 94.12 2.90 84.12 97.94 2.90 86.51 95.21 3.15 78.91 100.85 3.15 81.25 97.17 ∞ (**C**) 58.16 101.13 ∞ (**C**) 58.16 101.13 Synthesis and Photochemistry of Dirhodium Carbonyls {#sec3.3} --------------------------------------------------- To evaluate the hypothesis that complete ligand rearrangement to a Cl-bridged intermediate is impeded in the solid state, a new suite of complexes in which the bulky AdNC ligands of **1** and **2** are replaced with small volatile CO ligands was prepared. The synthetic chemistry and X-ray characterization of these complexes is summarized in Figure [5](#fig5){ref-type="fig"}. {#fig5} Entry into this suite of Rh~2~ carbonyl complexes was gained by treatment of \[Rh(CO)~2~Cl\]~2~ with tfepma in THF, which resulted in the formation of Rh~2~\[0,II\] carbonyl complex **6** in 98% yield (Figure [5](#fig5){ref-type="fig"}). Two features of **6** are notable. First, the complex displays rapid ligand fluxionality at 23 °C, as evidenced by observation of a single resonance in the ^31^P NMR spectrum of **6** at 23 °C. Variable-temperature (VT) ^31^P NMR of **6** between −97 and 23 °C shows that at temperatures below −65 °C two ^31^P NMR resonances are resolved, consistent with the two-electron mixed-valence formulation indicated by single-crystal X-ray diffraction. Second, one of the carbonyl ligands of **6** is labile. Evaporation of reaction solvent results in isolation of μ-CO--Rh~2~\[I,I\] monocarbonyl complex **9**, the product of CO dissociation. Treatment of PhCH~3~ solutions of **6** with PhICl~2~ results in a mixture of *cis*- and *trans-***7**. Heating this mixture of Rh~2~\[II,II\] complexes at 80 °C leads to isomerization of *cis*-**7** to *trans*-**7**, which was isolated in 79% yield. *trans*-**7** is an analogue of Rh~2~\[II,II\] complex **2** in which the AdNC ligands of **2** have been replaced by CO ligands. Our contention that complexes supported by volatile ligands would participate in solid-state photochemistry was probed by irradiation of a thin film of *trans*-**7** under vacuum (20 mTorr). Solid-state photolysis led to the expulsion of a CO ligand and the formation of Cl-bridged monocarbonyl complex **8** in 87% yield. Single-crystal X-ray diffraction of the photoproduct confirmed a μ-Cl-Rh~2~ geometry for **8**, similar to previously synthesized isocyanide supported Cl-bridged structures.^[@ref20]^ Solution-phase photolysis of *trans*-**7** in THF also led to the rapid conversion to **8**, as established by monitoring the reaction by UV--vis spectroscopy (Figure [6](#fig6){ref-type="fig"}a). ![Spectral evolution for the photolysis of *trans*-**7** in THF (λ~exc~ \> 305 nm). (a) During the first 120 s, *trans*-**7** is converted to Cl-bridged Rh~2~ complex **8**. (b) Subsequently, **8** is converted to Rh~2~\[I,I\] complex **9**, the product of halogen photoelimination.](ja-2014-08218v_0006){#fig6} Photolysis of monocarbonyl Rh~2~ complex **8** in THF results in the disappearance of the UV--vis features of **8** and the evolution of features attributable to complex **9**, the product of two-electron photoreduction (Figure [6](#fig6){ref-type="fig"}b). Complex **9** produced by photolysis of **8** displayed identical spectral features to those of an authentic sample prepared by removal of a CO ligand from complex **6** under vacuum. Monocarbonyl complex **9** is converted to dicarbonyl Rh~2~ complex **6** by exposure to 1 atm of CO in 99% yield. Access to Rh~2~ monocarbonyl complex **9** provided an avenue toward synthesis of AdNC-supported μ-Cl-Rh~2~ intermediate **3**. Treatment of monocarbonyl complex **9** with 1 equiv of AdNC, followed by oxidation with PhICl~2~ led to Rh~2~\[II,II\] complex **10**, supported by one AdNC and one CO ligand (Figure [7](#fig7){ref-type="fig"}). Solid-state photolysis of **10** under vacuum led to the expulsion of the CO ligand and isolation of μ-Cl-Rh~2~ complex **3**. Prepared from **10**, complex **3** was indefinitely stable and could be independently characterized. Subtraction of the extinction spectra of complexes **1** and **2** from the extinction spectrum measured for **3** confirmed that the photointermediate observed in the TA spectra of **1** and **2** is indeed Cl-bridged complex **3** (Figure [8](#fig8){ref-type="fig"}). {#fig7} {#fig8} Discussion {#sec4} ========== Photochemical HX splitting, in which H^+^ reduction to H~2~ is coupled to X^--^ oxidation to X~2~, offers a paradigm for the construction of a closed, carbon-neutral cycle for solar energy conversion. Halogen elimination accounts for the majority of the energy stored in HX-splitting cycles, and this half-reaction typically has been the roadblock to development of authentic HX-splitting photocatalysis.^[@ref17]^ When halogen elimination is achieved, chemical traps are frequently required to sequester the evolved halogen.^[@ref52]−[@ref56]^ The use of chemical traps provides a thermodynamic driving force for photoreduction and obviates significant energy storage. Trap-free halogen elimination chemistry has been realized in the solid state,^[@ref63]−[@ref65]^ but, as of yet, no system is available for which both proton reduction and solid-state halide oxidation are facile. Our group has developed dirhodium complexes on the hypothesis that mixed-valent complexes will give rise to the requisite multielectron photoreactions. Families of phosphazane-bridged complexes have been developed, and they display the targeted multielectron chemistry, but catalysts that accomplish authentic HX splitting to afford both H~2~ and X~2~ in the absence of chemical traps have proven to be elusive. Rational development of new HX-splitting platforms has been limited by a dearth of information regarding the mechanism of halogen elimination, thus establishing an imperative for an understanding of the critical steps preceding halogen elimination. To this end, the results reported herein provide direct insight into the nature of intermediates that promote halogen elimination from bimetallic centers. Halogen elimination proceeds from a common photointermediate for HX-splitting photocatalysis that is promoted by either Rh~2~\[I,III\] complex **1** or Rh~2~\[II,II\] complex **2**. Solution-phase, nanosecond-resolved TA spectroscopy has provided evidence for this photointermediate, but its structure has not been established.^[@ref20]^ We have employed photocrystallography to directly probe photoinduced structural changes associated with halogen photoelimination by X-ray diffraction.^[@ref58]−[@ref62]^ Such experiments are particularly attractive given that solid-state halogen elimination reactions provide inroads to authentic halogen elimination reactions.^[@ref63]−[@ref65]^ To use photocrystallography reliably to gain insight into the structures of reaction intermediates in solution, the same photointermediates must be accessed in the solid state. The thin-film, nanosecond-resolved TA measurements shown in Figure [2](#fig2){ref-type="fig"} establish the homology of solution-phase and solid-state intermediates; the same TA line shape is generated in solution-phase and solid-state experiments. The nature of this photoreduction intermediate that is common to complexes **1** and **2** is unveiled by the steady-state photocrystallography experiments summarized in Figure [3](#fig3){ref-type="fig"}. Both Rh~2~\[I,III\] complex **1** and Rh~2~\[II,II\] complex **2** display photoinduced structures characteristic of partial halide migration to a bridging position. DFT calculations lend support to the structural changes observed by photocrystallography. Specifically, in solution-phase experiments, photodissociated ligands can diffuse away from the transition metal fragment, whereas in the solid state, free diffusion is not possible. Our calculations support the contention that in solution complete dissociation of photoextruded ligands will lead to the formation of chloride-bridged structure **3** (Figure [4](#fig4){ref-type="fig"}). In the solid state, ligand diffusion is constrained and thus, while M--L bond elongation is achievable, complete dissociation is not possible. We modeled this scenario by evaluating the effect of M--L bond elongation of the geometry of the binuclear core and found that at small elongations of the M--L bond partial migration of a chloride ligand toward a bridging configuration was observed (Table [2](#tbl2){ref-type="other"}). The observation of incomplete ligand migration by photocrystallography emphasizes the rigorous demand of solid-state photoreactions, in which dissociation and diffusion of ligands are not typically possible; the presence of AdNC ligands prevents access to a complete rearrangement to a halide-bridged structure. Management of solid-state ligand inventory was achieved by replacing AdNC ligands with small, volatile CO ligands (Figure [5](#fig5){ref-type="fig"}). This strategy was predicated on the hypothesis that a small, volatile ligand such as CO could be removed as a gas during solid-state photochemistry. Photolysis of *trans*-**7**, an analogue of **2** in which the AdNC ligands are replaced by CO ligands, initially affords chloride-bridged complex **8**, which confirms that photoextrusion of an L-type ligand initially generates a halide-bridged complex (Figure [6](#fig6){ref-type="fig"}a). The halide-bridged complex appears as an intermediate in an interrupted halogen elimination reaction, as further irradiation of **8** led to the isolation of Rh\[I,I\] complex **9**, which arises from two-electron photoreduction of **8** (Figure [6](#fig6){ref-type="fig"}b). The two-step reaction sequence, ligand loss to generate a ligand-bridged intermediate followed by two-electron photoreduction, represents an intramolecular example of the two steps of canonical binuclear elimination mechanisms. Binuclear reductive elimination reactions, originally defined in the context of elimination of alkanes and aldehydes from binuclear metal alkyl or acyl complex,^[@ref21],[@ref22]^ are proposed to proceed via (1) generation of an open coordination site on the metal alkyl complex by either ligand dissociation or migratory insertion, (2) intermolecular reaction of the unsaturated fragment with a metal hydride to generate a hydride-bridged intermediate, and (3) migration of the alkyl or acyl group onto the bridging hydride to generate the observed organic products as well as binuclear transition metal complexes (eq [1](#eq1){ref-type="disp-formula"}).^[@ref68]−[@ref70]^ Such binuclear elimination has been proposed for complexes based on most of the transition metal series,^[@ref71]−[@ref75]^ and it is proposed to be operative during both Co- and Rh-catalyzed hydroformylation reactions under some conditions.^[@ref21],[@ref77]−[@ref84]^ We have previously proposed a binuclear reductive elimination mechanism for the H~2~ evolution step of HX-splitting catalysis with phosphazane-bridged Ir~2~ complexes.^[@ref85]^On the basis of the results shown in Figures [2](#fig2){ref-type="fig"}, [3](#fig3){ref-type="fig"}, and [5](#fig5){ref-type="fig"}, we now observe that a similar mechanism may be operative for halogen photoelimination. To firmly establish a binuclear elimination pathway for halogen photoelimination from AdNC-supported complexes **1** and **2**, we sought to directly prepare and characterize the proposed AdNC-supported chloride-bridged intermediate. While preparation by ligand extrusion from either **1** or **2** was judged to be implausible given the short lifetime measured for **3** under these conditions (∼15 μs),^[@ref20]^ we anticipated that the complex may be isolable if recombination of the photoextruded ligand with the Rh~2~ fragment could be avoided. Taking advantage of the solid-state ligand management enabled with CO ligands, we have prepared and isolated complex **3**, which is the exact transient intermediate that would be generated during halogen elimination from complexes **1** and **2**. We note that chloride-bridged complex **3** is furnished directly by the extrusion of one equivalent of CO upon the solid-state photolysis of **10**, an analogue of **2** in which the Rh~2~ core is supported by one AdNC and one CO ligand (Figure [7](#fig7){ref-type="fig"}). With the absorption spectrum of **3** in hand, Figure [8](#fig8){ref-type="fig"} shows that the TA spectra obtained by laser flash photolysis of **1** or **2** is closely replicated by computing the difference spectra expected based on the experimentally measured ground-state absorption spectra of **1**--**3**. The confirmed intermediacy of **3** in halogen elimination from **1** and **2** demonstrates that halogen elimination proceeds by a canonical binuclear elimination pathway in which both steps, ligand dissociation to afford a ligand-bridged intermediate and subsequent M--X bond activation, can be directly observed. To our knowledge, the chemistry reported herein is the first instance in which these two steps of binuclear elimination have been directly observed. Photoreduction of **3** may proceed by concerted or stepwise elimination. The results reported herein offer evidence that X~2~ elimination from Rh~2~ complexes proceeds via ligand-bridged intermediates, and, accordingly, they inform next-generation catalyst design. Phosphazane-bridged binuclear complexes have been proposed to facilitate ligand rearrangements to ligand-bridged intermediates owing to the ligand's ability to accommodate two-electron changes at the metal core with minimal reorganization energy.^[@ref85]^ The photocrystallography experiments described here support this contention by showing the prevalence of halide-bridged intermediates that preceded M--X bond activation. Although the solid state imposes constraints on the requisite ligand-bridged intermediates, we have leveraged the volatility of carbonyl ligands to allow for the isolation of halide-bridged structures during an interrupted binuclear elimination. This strategy has been exploited to characterize the absorption spectrum of the exact halide-bridged intermediate that is observed transiently (\<15 μs) during photocatalysis. Because the solid state offers a mechanism to prevent the back reaction of reactive volatile photoproducts such as X~2~, these results suggest that obstacles imposed on photochemical transformations in the solid state might be overcome by clever design of the ligand coordination sphere. Detailed experimental procedures, spectroscopic data for all new compounds, *XYZ* coordinates of computed structures, X-ray crystal data for complex *cis*-**7** and **10**, and complete ref ([@ref32]). This material is available free of charge via the Internet at <http://pubs.acs.org>. Supplementary Material ====================== ###### ja508218v_si_001.pdf ###### ja508218v_si_002.txt The authors declare no competing financial interest. We gratefully acknowledge the NSF for funding (CHE-1332783). D.C.P. is supported by a Ruth L. Kirchenstein National Research Service award (F32GM103211). Photocrystallography was carried out at ChemMatCARS, Sector 15, APS, which is principally supported by the NSF/DOE under grant no. NSF/CHE-1346572. Use of APS was supported by the U.S. DOE, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. Computations were carried out at the Supercomputing Facility and the Laboratory for Molecular Simulation at Texas A&M University.

INTRODUCTION {#s1} ============ Cellular senescence defined as an irreversible proliferation arrest promotes age-related decline in mammalian tissue homeostasis \[[@R1]\]. At present, investigation of this phenomenon becomes more and more widespread due to the following reasons: firstly, senescence is thought to contribute to a multiple age-related pathologies \[[@R2], [@R3]\], and secondly, senescence acts as a tumor suppressor mechanism that is able to block proliferation of incipient cancer cells \[[@R1], [@R4]\]. Similar to other types of normally proliferating cells that are characterized by a finite lifespan (Hayflick limit) human mesenchymal stem cells undergo the replicative senescence after a fixed number of cell divisions \[[@R5]\]. Moreover, the recent findings have revealed that human mesenchymal stem cells may respond to a variety of subcytotoxic stresses (UV-, γ-radiation, H~2~O~2~, histone deacetylase inhibitors, etc.) by induction of premature senescence \[[@R6], [@R7], [@R8]\]. Oxidative stress has been shown to play an important role for the development of aging and age-related diseases \[[@R9]\]. According to the free-radical theory of aging, reactive oxygen species (ROS), including the oxygen singlet, the superoxide anion (O2^.-^), the hydroxyl radical (OH^.^) and hydrogen peroxide (H~**2**~O~**2**~) might be the candidates, which are responsible for cellular senescence. Being membrane permeable and long-lived molecule, H~2~O~2~ can directly affect the cellular DNA, inducing both single- and double-strand breaks (SSBs and DSBs, respectively) \[[@R10]\]. DNA damage, in turn, triggers a specific DNA damage response (DDR), which involves the following events -- (1) activation of any sensor kinases (ATM, ATR, DNA-PK), (2) phosphorylation of adaptor protein 53BP1, and (3) formation of the discrete foci, containing phosphorylated histone H2A.X and p53BP1 \[[@R11]\]. Finally, DDR activation leads to cell cycle arrest via activation of p53/p21 \[[@R12]\] and/or p16/pRb pathways \[[@R13], [@R14]\]. Today it is generally accepted that both the replicative and stress-induced senescence are the outcome of DDR \[[@R15]\]. Conversion from proliferative arrest to irreversible senescence, a process named geroconversion, is driven in part by growth-promoting pathways in particular mammalian target of rapamycin (mTOR) which is mostly responsible for loss of RP (replicative/regene-rative potential) and hypertrophy \[[@R16], [@R17]\]. Inhibitors of mTOR such as rapamycin \[[@R18]\] and hypoxia \[[@R19]\] can suppress geroconversion, maintaining quiescence instead. Previously, the mTOR was the only pathway known to be involved in acquiring classic markers of a senescent phenotype, including cyclin D1 accumulation. Recent studies have been revealed an additional MEK/ERK pathway that is required for the acquisition of at least one hallmark of senescence: hyper-accumulation of cyclin D1 \[[@R20]\]. Furthermore, it was shown that p70S6K, a crucial substrate of mTOR, and MEK play different roles in geroconversion \[[@R21]\]. According to recent publications, a so-called feedback loop between the permanent DDR activation and the increased ROS production is necessary for development of senescence \[[@R22]\]. The main effectors of DDR, p53 and p21, were shown to be involved in regulation of ROS generation, leading to enhanced intracellular ROS during the establishing of senescence \[[@R23], [@R24]\]. In the senescent cells, elevated ROS can cause a direct DNA damage and the persistent DDR activation, thereby forming a feedback loop. It is well-established that the senescent cells are characterized by increased ROS levels. Taken into consideration that the overwhelming majority of intracellular ROS are of mitochondrial origin, it is reasonable to posit that the elevated ROS production might be caused by alteration in mitochondrial function during senescence. Mitochondria are the intracellular organelles responsible for ATP synthesis through the coupling of oxidative phosphorylation to respiration in mammalian cells. Currently, there are different points of view regarding age-related changes in mitochondrial physiology. Several authors considered cellular senescence to be accompanied by mitochondrial dysfunction defined by the decline of mitochondrial membrane potential (MMP). MMP decline leads to respiratory-chain defects and thus to enhanced ROS production \[[@R22], [@R25]\]. On the contrary, the others hypothesized the absence of defects either in electron transport system or in oxidative phosphorylation during senescence \[[@R26], [@R27]\]. In this case, ROS levels are elevated due to (1) the growing number of functional mitochondria and, (2) the age-related alterations in mitochondrial coupling that correlates with the increase in mitochondrial membrane potential. Importantly, both hypotheses manifested the involvement of mitochondria in the aging process via a relevant contribution to intracellular ROS generation. p38, a member of the family of mitogen-activated protein kinases (MAPKs) is activated by cellular stresses including ROS, UV- and γ-radiation, and proinflammatory cytokines \[[@R28]\]. In response to stress factors, p38 MAPK (hereafter p38) can rapidly phosphorylate and activate MAP kinase-activated protein kinases (MAPKAPKs), particularly MAPKAPK-2 (hereafter MK-2) that is a direct target of p38 \[[@R29]\]. The p38 effector kinase lies downstream of the MKK3/6 activator kinases, whose activity can be regulated by stress-sensitive apoptosis signal-regulating kinase-1 (ASK1) \[[@R28]\]. p38 plays an important causative role in cellular senescence induced by oxidative stress, radiation, genotoxic agents, Ras overexpression \[[@R30]\]. p38 has been demonstrated to participate in feedback relationships during senescence as, on the one hand, it can function as mediator of ROS signaling and, on the other hand, can directly phosphorylate p53 \[[@R22]\]. The significance of p38 MAPK pathway in stress-induced senescence was investigated predominantly in the cultures of either human fibroblasts \[[@R22], [@R31]\] or transformed cells \[[@R32]\]. However, there is no information available as to whether the functional p38 is required for premature senescence of human endometrium-derived mesenchymal stem cells (hMESCs). hMESCs are a relatively new source of adult stem cells intensively studied over the past decade. The fact that hMESCs isolation does not require invasive and traumatic procedures facilitates their use in regenerative medicine. So to date, promising results concerning the experimental and clinical application of these cells for treatment of heart failure, myocardial infarction, diabetes, stroke, Parkinson\'s disease, multiple sclerosis, Duchenne Muscular Dystrophy and infertility were obtained \[[@R33], [@R34], [@R35]\]. Despite the different nature of these disorders, namely oxidative stress is well known to play an essential role in their progression. The goal of the present study was to clarify the underlying mechanisms of both induction and further maintenance of premature senescence in hMESCs subjected to oxidative stress. RESULTS {#s2} ======= Recently, we have provided the reliable evidence that hMESCs undergo the premature senescence in response to the sublethal concentration of H~2~O~2~ \[[@R36]\]. H~2~O~2~-treated hMESCs were permanently arrested, lost Ki67 proliferative marker, and exhibited senescent phenotype, including cell hypertrophy and increased SA-β-Gal activity, indicating that the cells were driven into stress-induced premature senescence. However, the molecular mechanism of senescence induction in hMESCs under oxidative stress is far from being elucidated. DDR activation in response to a rapid accumulation of exogenous H~2~O~2~ in hMESCs {#s2_1} ---------------------------------------------------------------------------------- H~2~O~2~ by conventional diffusion may easily pass through the membrane into the intracellular space, causing damage to lipids, proteins, and DNA \[[@R37], [@R38]\]. To ascertain the dynamics of H~2~O~2~ penetration into the cells in our experimental conditions (200 µM H~2~O~2~, 1 h), in H~2~DCFDA-stained cells, the changes in H~2~O~2~ concentration were monitored for 60 min by either FACS or the confocal microscopy. Of note, the confocal microscopy enabled to estimate the individual changes of fluorescence occurring in each of the cells selected to test (Fig. [1 A, B](#F1){ref-type="fig"}), whereas FACS analysis evaluated the average fluorescence per cell (Fig. [1 C](#F1){ref-type="fig"}). Despite the apparent distinctions in fluorescence levels presented in Figures [1A and 1C](#F1){ref-type="fig"}, resulting from the behavior of individual cells in the heterogenous population, the dynamics of H~2~O~2~ diffusion evaluated by both methods were alike. H~2~O~2~ treatment of cells led to a rapid elevation of intracellular ROS levels, peaking at 15 min and returning progressively to the baseline by 30 min in majority of cells, indicating that exogenous H~2~O~2~ is almost completely utilized by cells during 1 h treatment. {#F1} Among intracellular ROS, which are able to induce DNA damage, H~2~O~2~ is known to provoke an appearance of both SSBs and DSBs that can trigger DDR \[[@R38]\]. Generally, DDR is characterized by activation of ataxia-telangiectasia mutated kinase (ATM) and formation of DNA-damage foci, containing γH2A.X and p53BP1 in chromatin, surrounding DSBs. Presently, it is generally accepted that γH2A.X, as well as p53BP1 are recognized as the reliable markers of DSBs \[[@R11]\]. To examine the possibility of DDR activation in H~2~O~2~-treated hMESCs, the functional status of key proteins involved in DDR was estimated. Immunofluorescent analysis with the use of specific antibodies against pATM, γH2A.X, p53BP1 revealed a rapid ATM phosphorylation (within 5 min after beginning of treatment) (Fig. [2 A](#F2){ref-type="fig"}) and further simultaneous phosphorylation of 53BP1 and H2A.X in 15 min (Fig. [2 B, C](#F2){ref-type="fig"}). Moreover, we observed co-localization of pATM with either γH2A.X or p53BP1 in 60 min after beginning of treatment (Fig. [3 A, B](#F3){ref-type="fig"}). The fast H~2~O~2~-induced ATM phosphorylation peaking at 30 min was confirmed by Western blot analysis (Fig. [4 A](#F4){ref-type="fig"}). Taken together, the results obtained demonstrate that the exogenous H~2~O~2~ was able to cause an immediate DNA damage followed by DDR activation. {#F2} {#F3} {#F4} The p53/p21/pRb pathway orchestrates both establishing and maintaining H~2~O~2~-induced senescence of hMESCs {#s2_2} ------------------------------------------------------------------------------------------------------------ In previous study, we have shown that an irreversible cell cycle arrest is the main marker of H~2~O~2~-induced premature senescence of hMESCs, however the signaling pathways providing the cell cycle arrest require the detail investigation. Senescence program is thought to be developed as result of DDR, leading to functional activation of the p53/p21 pathway, which can establish and maintain the growth arrest \[[@R12]\]. In order to determine whether cell cycle block may be realized via p53/p21 pathway in H~2~O~2~-treated hMESCs, we first investigated the functional status of p53 protein. Western blot analysis with using rabbit polyclonal antibodies against phospho-p53 at Ser15 revealed a rapid (within 10 min) p53 phosphorylation, gradually increasing during 60 min of H~2~O~2~ treatment (Fig. [4 B](#F4){ref-type="fig"}). At the same time, translocation of phospho-p53 into the nuclei and its partial colocalization with pATM was observed (Fig. [4 C](#F4){ref-type="fig"}). In H~2~O~2~-treated cells, enhanced p53 phosphorylation was also detected at 7 h post-treatment, however, over next 8 days it was dramatically diminished (Fig. [8 C](#F8){ref-type="fig"}). Remarkably, the decrease of the functional activity of p53 had no effect on elevated p21 induction during the entire observation period (Fig. [8 D](#F8){ref-type="fig"}). It is known that p53 activated acts as a transcription factor, inducing expression of p21 which may mediate the initiation of the cell cycle arrest by inhibiting various cyclin-dependent kinases (CDK) that contribute cell cycle phase progression. Therefore, we next examined mRNA and protein expression levels of p21. H~2~O~2~ promoted a significant elevation in mRNA and protein expression of p21 already at 7 h post-treatment (Fig. [4 D, E](#F4){ref-type="fig"}). An inducible expression of p21 was up-regulated, at least, during 7 days with following decline to insignificant, but not the control levels, which persisted up to 21 days. The elevated p21 expression was accompanied with the cell cycle arrest at the same time (data not shown). Retinoblastoma protein (pRb) whose activity is regulated by elevated p21 plays a crucial role for establishing the growth arrest. It is known that pRb in active hypophosphorylated state halts cell proliferation by suppressing the activity of E2F transcription factor that regulates cell cycle progression. To examine the functional status of pRb during establishing senescence, we performed monitoring the kinetics of pRb activation in H~2~O~2~-treated hMESCs. As expected, beginning 7 h post H~2~O~2~ treatment, no pRb phosphorylation was observed in the senescent cells, in contrast to the control proliferating cells, which displayed the high levels of pRb phosphorylation (Fig. [4 F](#F4){ref-type="fig"}). Collectively, our findings demonstrate that the p53/p21/pRb signaling pathway leading to the growth arrest is required to drive the premature senescence and apparently to maintain the long-term senescent state in hMESCs. An interplay between enhanced ROS levels and prolonged DDR activation {#s2_3} --------------------------------------------------------------------- As mentioned above, the exogenous H~2~O~2~ induced a strong increase in intracellular ROS levels within 1 h of cell treatment (Fig. [1 A, C](#F1){ref-type="fig"}) and accordingly triggered a premature senescence of hMESCs. To find out whether the intracellular ROS levels can be modulated during the senescence development, DCF fluorescence intensity was measured in H~2~O~2~-treated cells over the next 9 days. Surprisingly, on day 5 post-treatment, the senescent cells were characterized by strongly increased DCF fluorescence, consistent with higher levels of intracellular ROS that remained elevated further over 9 days (Fig [5A, B](#F5){ref-type="fig"}). These results were in agreement with the continuous elevated levels of intracellular peroxides measured by DHR123 in the senescent cells (Fig. [5 C, D](#F5){ref-type="fig"}). These findings clearly demonstrate that the process of H~2~O~2~-induced senescence of hMESCs is accompanied with the permanent generation of the intracellular ROS. {#F5} Previous studies have reported that there is the functional link between enhanced ROS production and DDR activation during the development and stabilization of senescence \[[@R22]\]. Therefore, we further characterized the functional status of DDR in the senescent cells by testing ATM, H2A.X and 53BP1 for their phosphorylation and an intracellular localization using the fluorescent microscopy. Remarkably, on 5 days post-treatment all of proteins tested remained in an active state and mostly co-localized in so-called senescence-associated DNA-damage foci (SDFs) (Fig. [5 E, F](#F5){ref-type="fig"}). It should be noted that, in the senescent cells, enhanced ROS production and DDR activation has been contemporized. Together, these observations allow us to suspect that enhanced intracellular ROS could be responsible for long-term DDR activation. An increase in mitochondrial activity in the senescent hMESCs {#s2_4} ------------------------------------------------------------- Although we have shown that permanently enhanced ROS are typical of the senescent state of hMESCs, nevertheless the actual reason of long-term ROS production remained unclear. We hypothesized that this phenomenon might be associated with the significant modulation of the mitochondrial function in time that was postulated to be one of the main contributory factors in senescence \[[@R39]\]. In order to examine this suggestion, the cells were assessed for cellular peroxide production, mitochondrial mass and mitochondrial membrane potential (MMP) by DHR123, NAO and Rho123 staining, respectively. Nonfluorescent dye DHR123 selectively accumulates in mitochondria, where it can be oxidized by mitochondria-derived ROS to a fluorescent rhodamine derivative. As seen in Fig. [5 C](#F5){ref-type="fig"}, at 24 h post H~2~O~2~ treatment the cellular peroxides levels were almost 2-fold higher than in the control cells and then gradually enhanced for 7 days, indicating that, in the senescent cells, there are permanently elevated ROS levels derived from mitochondria. Next, to determine whether H~2~O~2~ may cause the proliferation of mitochondria, NAO dye to monitor the mitochondrial mass was used. The relative NAO intensity of H~2~O~2~-treated cells gradually increased in time and, on 7 days post-treatment, it was found to be 2.5-fold higher than that of the control cells (Fig. [6 A, B](#F6){ref-type="fig"}). These results indicate that H~2~O~2~ can promote an increase in the number of mitochondria in hMESCs in a time-dependent manner. To analyze, whether the extra mitochondria in H~2~O~2~-treated cells were functional at the same time, MMP of cells was measured. In treated cells, the relative intensity of Rho123 fluorescence was substantially higher than that of the control cells (Fig. [6 C, D](#F6){ref-type="fig"}). Importantly, the increase in MMP correlated with corresponding increase in the mitochondrial mass over the entire observation period. In addition, both characteristics also correlated well with elevated cellular peroxide production. {#F6} Taken together, these results clearly indicate that oxidative stress induced by the sublethal H~2~O~2~ led to an increase in the amount of functional mitochondria. We assume that an increased mitochondrial activity may be responsible, at least in part, for long-term ROS production observed in the senescent hMESCs. The role of p38 in the regulation of H~2~O~2~-induced premature senescence of hMESCs {#s2_5} ------------------------------------------------------------------------------------ First, we examined whether p38 is activated by sublethal doses of H~2~O~2~ in hMESCs. As shown in Fig. [7 A](#F7){ref-type="fig"}, 200 µM H~2~O~2~ induced a significant increase in p38 phosphorylation within 1 h of treatment, whereas in untreated cells p38 phosphorylation was undetectable. Interestingly, H~2~O~2~ maintained the elevated p38 phosphorylation up to 8 days without affecting steady-state protein levels of p38 (Fig. [7 B](#F7){ref-type="fig"}). Phosphorylation of MK-2, a natural substrate of p38, was detected at the same time (Fig. [7 C](#F7){ref-type="fig"}). {#F7} To explore the role of p38 in regulation of premature hMESCs senescence, we employed SB203580 (hereafter SB), a specific inhibitor of the p38 MAPK pathway. SB is a small molecule that displaces ATP from the ATP-binding pocket of p38, thereby preventing from phosphorylation of p38 targets, in particular MK-2, without preventing p38 phosphorylation itself \[[@R40]\]. As expected, in H~2~O~2~-treated cells, p38 phosphorylation was unaffected by SB (Fig. [7 B](#F7){ref-type="fig"}), nonetheless, MK-2 phosphorylation was abolished throughout the 8-days period of the experiment (Fig. [7 C](#F7){ref-type="fig"}). Selective inhibition of p38 kinase activity with SB prevented the increase of the size of H~2~O~2~-treated cells (Fig. [8 B](#F8){ref-type="fig"}) but just slightly reduced their SA-β-Gal activity (data not shown), indicating some modulation of the senescence phenotype of H~2~O~2~-treated cells. Because the growth arrest was shown to be the major mechanism of the integral growth-inhibitory effect of H~2~O~2~ in hMESCs under our experimental conditions \[[@R36]\], we next checked the role of p38 in the regulation of cell proliferation. As presented in Fig. [8 A](#F8){ref-type="fig"}, blocking of p38 with SB led to marked increasing the number of proliferating cells compared with H~2~O~2~-stimulated cells. In order to relieve the proliferation block of the senescent cells to a great extent, in the separate experiments, SB was added at 24 or 48 h post H~2~O~2~ treatment unlike the routine immediate adding after H~2~O~2~ removal. Interestingly, in this case SB had no effect on the proliferative status of senescent cells, albeit was still able to reduce ROS levels (data not shown). Consequently, the p38/MK-2 inactivation could in part prevent the loss of the proliferative potential of pre-senescent hMESCs. In this case the senescence program has already been initiated however the proliferative arrest could yet be reversed. By contrast, suppression of p38/MK-2 activity in the senescent arrested cells was insufficient to resume the proliferation. These findings demonstrate that p38, acting as a negative regulator proliferation of hMESCs in response to oxidative stress, is required for establishing premature senescence, whereas its inhibition may at least in part rescue the cells from senescence induction. {#F8} Remarkably, in H~2~O~2~-treated hMESCs, p38 inhibition did not affect phosphorylation status of p53 (Fig. [8 C](#F8){ref-type="fig"}) and did not prevent p21 protein induction over the entire observation period (Fig. [8 D](#F8){ref-type="fig"}). These results suggest that p38/MK-2 and p53/p21 signaling pathways can act independently during establishing and maintaining of the premature senescence of hMESCs. On the other hand, suppression of p38/MK-2 in H~2~O~2~-treated cells noticeably elevated the pRb phosphorylation levels, indicating an inactivation pRb (Fig. [8 E](#F8){ref-type="fig"}). These findings correlate well with an increase in the proliferative potential of cells after common treatment with H~2~O~2~ and SB as compared with H~2~O~2~-treated cells (Fig. [8 A](#F8){ref-type="fig"}). To confirm that the effect of SB was specific to p38, we used another p38 inhibitor with an unrelated chemical structure, BIRB796 \[[@R41]\]. According to preliminary results, the observed effect of BIRB796 at concentration of 5µM was similar to the effect of SB, partially preventing both H~2~O~2~-induced growth inhibition and the increase in the size of H~2~O~2~-treated cells, thereby demonstrating that p38 participates in the establishing premature senescence in hMESCs induced by H~2~O~2~. p38 MAPK implication in feedback loop via ROS {#s2_6} --------------------------------------------- As described above, the senescent hMESCs are characterized by persistently elevated ROS levels. We presumed that long-term activation of p38 might be involved in regulation of ROS production. To test this idea, in the SB-treated and untreated senescent cells, the intracellular ROS production measured by H~2~DCFDA staining was evaluated. SB treatment of H~2~O~2~-stimulated cells led to a dramatic drop in the intracellular ROS levels in any of time points tested (Fig. [9 A](#F9){ref-type="fig"}). Taking into account the fact that increased mitochondria could mediate the elevation of ROS levels in the senescent hMESCs, we next examined whether p38 is able to affect the mitochondrial function. SB treatment of the senescent cells equally reduced the cellular peroxide levels and MMP compared with H~2~O~2~-treated cells as evaluated by DHR123 and Rho123 staining, respectively (Fig. [9 B, C](#F9){ref-type="fig"}). In addition, in SB-treated cells we observed the similar decrease of mitochondrial mass measured by NAO stain-ing (Fig. [9 D](#F9){ref-type="fig"}). Overall, the results obtained indicate p38 implication in continued ROS production mediated by in-creased mitochondrial function in the senescent hMESCs. {#F9} DISCUSSION {#s3} ========== In the current study, we have examined the molecular mechanism of premature senescence of hMESCs in response to oxidative stress. According to the modern concepts, the process of stress-induced senescence comprises two sequential stages -- establishing and maintaining (stabilization) that can be regulated by different mechanisms, depending upon the specific stimuli used, the cell context and other factors. Understanding of the interplay between various signaling pathways that provide the control either stage would have significant therapeutic implications and should be highly useful to determinate the strategy for the reversal of cellular senescence. DDR activation induced by ionizing radiation, chemotherapeutic drugs or oxidative stress has been investigated in different stem cell types, including bone marrow-derived hMSCs \[[@R8]\], hematopoietic stem cells (HSC) \[[@R14]\] and embryonic stem cells \[[@R42]\]. In the literature, the similar studies performed on hMESCs have not been described thus far. The results presented herein demonstrate that, in hMESCs subjected to sublethal oxidative stress, H~2~O~2~ generated a persistent DDR signaling associated with DNA double-strand breaks (DSBs) which are a signal for activation of ATM and downstream pathways, leading to cell cycle block, as well as the accumulation of DNA foci marked by γH2A.X and phospho-53BP1. Activation of DDR signaling can be a trigger for switching on senescence and it is essential for establishing and maintaining senescent phenotype of cells \[[@R15]\]. We have reported earlier \[[@R36]\] that, in hMESCs, the exogenous H~2~O~2~ caused an irreversible arrest of cell cycle with predominant accumulation of cells in G0/G1-phase. To find out the molecular mechanism, triggering the cell cycle arrest in H~2~O~2~-treated hMESCs, first of all we focused on p53-mediated signaling pathway which may lead to the cell cycle block. It is well documented that the p53/p21 pathway is critical for establishing the replicative senescence of human cells \[[@R12], [@R13]\], as well as the premature senescence of hMSCs \[[@R8]\] and HSC \[[@R14]\]. However, little is known about which signaling pathways are responsible for the induction of the premature senescence of hMESCs under oxidative stress. In agreement with a canonical p53/p21 model, our results demonstrate that DDR-activated p53 upregulated the CDK inhibitor p21 that, in turn, prevented the phosphorylation and inactivation of pRb. Besides, pRb activity may be controlled by another CDK inhibitor, p16 (INK4a). Previous studies suggested that p16 was crucial for long-term maintaining of senescence of both human fibroblasts \[[@R1], [@R13], [@R43]\] and HSC \[[@R14]\]. Interestingly, in these cases p16 was expressed much later than p21, forming a second barrier to prevent the cells from cell cycle re-entering. In contrast, our preliminary data indicate that up-regulation of p16 occurs solely at initial stage of senescence (within 1 h) but not at delayed time period. These findings support the possibility that p16/pRb pathway, in addition to p53/p21, is responsible for establishing the growth arrest, preventing entering the cells into S-phase. In consideration of strongly decreased p53 activity observed in senescent cells from 3 days, we can speculate that p53 is critical for rather establishing the senescence growth arrest than prolonged maintaining the senescent state of hMESCs. Unlike p53, an inducible p21 expression was persistently upregulated throughout experiment (up to day 21) and was accompanied with the cell cycle arrest \[[@R36]\]. Accordingly, the elevated p21 induction was indispensable to promote the senescence, as well as to maintain this state in hMESCs in response to H~2~O~2~. Previously, it was reported that the activated checkpoint kinase 2 (Chk2) can induce p21 transcription in the absence of functional p53 and that this contributes to Chk2-mediated senescence \[[@R44]\]. It was attractive to suppose that, in our experimental conditions, Chk2 permanently activated by ATM also is able to mediate a long-term p21 induction during senescence. Taken together, our findings definitely demonstrate that, in H~2~O~2~-treated hMESCs, the senescence program is triggered by DDR signaling, activation of which leads to an irreversible cell cycle arrest through p53/p21/pRb pathway. Further, we tested the role of stress-activated kinase p38 (in complex with MK-2, a direct downstream target of p38) as the most prominent mediator of stress-induced cellular senescence. MK-2 is known to be a negative regulator of cell cycle progression because it is directly responsible for phosphorylation-dependent inactivation of members of the Cdc25 family of phosphatases, which are positive regulators of Cyclin/CDK complexes. Finally, pRb was found to promote stress-induced growth arrest as a downstream molecule of p38 \[[@R45]\]. At present, MK-2 is recognized as a new member of the DNA damage checkpoint kinase family that functions in parallel with Chk1 and Chk2 to integrate DNA damage responses and cell cycle arrest \[[@R46]\]. Previous studies reported that the p38 pathway is implicated in H~2~O~2~-induced senescence of human fibroblasts however the data presented by various groups were controversial. Several authors observed in H~2~O~2~-treated cells the continuous p38 activation \[[@R45]\] while the others demonstrated that transiently elevated p38 kinase activity was reversible down-regulated after H~2~O~2~ removal \[[@R47]\]. Moreover, no activation of p38 was detected in H~2~O~2~-treated fibroblasts, regardless of endogenous p38 expression \[[@R48]\]. By contrast, our results indicate that elevated p38/MK-2 activation in response to H~2~O~2~ was persisted for a long time that suggests the importance of p38/MK-2 pathway in the control of both induction and a long-term maintaining of hMESCs senescence. The fact that the permanent p38/MK-2 activation was accompanied by pRb inactivation argues in favour of the p38/MK-2/pRb pathway that is likely to be mediated by Cdc25 family members. Interestingly, during treatment of hMESCs with H~2~O~2~, p38 activation was accompanied with increasing in phosphorylation of ASK1 at Thr845 (results not presented) that is correlated with ASK1 activity. Therefore, at this stage, we cannot exclude the possibility that ASK1 is involved in up-regulation of p38. It is noteworthy that inactivation of p38/MK-2 induced by SB did not affect the functional status of p53 and p21, suggesting that p38/MK-2 and p53/p21 pathways are uncoupled however can cooperate to induce an irreversible proliferative arrest, and further to maintain the premature senescence of hMESCs. Generally, this suggestion is supported by a recent report demonstrating that p38 participates in oxidative stress-induced senescence via an alternative ATM-independent pathway, implicating lamin B1 accumulation \[[@R49]\]. The pharmacological inhibition of p38 activation may be considered as a possible strategy for senescence prevention \[[@R45], [@R49]\]. According to our results, selective inhibition of p38 kinase activity with SB abrogated H~2~O~2~-induced cell enlargement and flattened morphology, but did not produce any significant effect on SA-β-Gal activity. At the same time, SB treatment of hMESCs allowed to avoid an irreversible cell cycle arrest in response to H~2~O~2~ however the recovery of proliferation was incomplete. This may point at the probable dissociation of hallmarks of senescence - senescent morphology, RP and SA-β-Gal staining. Likewise, elimination of cyclin D1 (a universal marker of cellular senescence) by specific inhibitors of the MEK/ERK pathway did not affect at least three classical hallmarks of senescence: loss of RP, senescent morphology and SA-β-Gal staining \[[@R20]\]. The findings that inhibition of p38 partially suppressed the H~2~O~2~-induced senescent phenotype of hMESCs, as well as prevented the proliferation arrest indicate that activation of p38 contributes to H~2~O~2~-induced cellular senescence. A plausible explanation for the partial effect of SB on growth arrest is that SB being a specific inhibitor of p38α and p38β cannot suppress the redundant γ and δ isoforms of p38 \[[@R50]\]. In addition, SB was reported to produce antiproliferative effect related to inhibition of pRb phosphorylation \[[@R51]\]. Thus, suppression of p38 kinase activity can at least in part rescue stressed hMESCs from the cell cycle arrest and entering premature senescence induced by H~2~O~2~. It is of noted, that the permanent growth arrest correlated with ROS accumulation during development of senescence. Our attempts to find out the possible reasons for maintaining the H~2~O~2~-induced senescence of hMESCs led to the observation that there is the interplay between permanently elevated ROS and the persistence of DDR signaling. In fact, the senescent cells displayed the persistent accumulation of DNA damage foci marked by p53BP1 and γH2A.X associated with pATM, as well as continuously increased levels of both intracellular ROS and mitochondrial peroxides. These findings are in line with previous study, presuming that the feedback loop between DDR and ROS production is necessary and sufficient to maintain senescent growth arrest during establishment of irreversible senescence \[[@R22]\]. Consistent with supposed mechanism, the senescent cells persistently accumulate senescence-associated DNA damage foci (SDFs), which contain proteins associated with DNA damage, particularly γH2A.X and p53BP1. The prolonged DDR activation results in up-regulation of p53 and p21 that may induce the increase in intracellular ROS levels. All together, the intracellular ROS are able directly to damage DNA and thus sustain DDR in an active state. To clarify the mechanism which may regulate the feedback loop, we utilized an inhibitor analysis of long-term ROS production. In senescent hMESCs, the specific inhibition of p38/MK-2 activity by SB had a pronounced negative effect on the intracellular (cytosolic) ROS production while the mitochondrial ROS production was diminished just in part. Similarly, mitochondrial mass and mitochondrial membrane potential were partially decreased. It is important to note that in senescent hMESCs preserving the metabolic activity we revealed the significant increase in the amount of functional mitochondria which might be responsible, at least in part, for long-term ROS production. These findings collectively allow speculation of p38/MK-2 involvement in modulation of intracellular ROS that are critical for maintaining feedback loop during senescence of hMESCs. Although our results suggest that the activated p38/MK-2 complex may regulate ROS generation via functional mitochondria, it is most likely that such regulation is indirect. Accumulating evidence pointed to an important role for TGFβ/TGFβ receptor and GADD45 in mitochondrial ROS production mediated by p38 \[[@R22], [@R28]\]. In conclusion, the present study is the first to elucidate the molecular mechanism of premature senescence of hMESCs under oxidative stress. The induction of senescence includes a prompt activation of response to DNA damage induced by H~2~O~2~ and following signal transduction through p53/p21 and p38/MK-2 pathways which are necessary and sufficient to establish the irreversible cell cycle arrest that is typical of senescence. We believe the prolonged induction of p21 as well as elevated activation of p38/MK-2 also might be indispensable to maintain persistent proliferative block in senescent cells. Additionally, p38 which may regulate both intracellular and mitochondrial ROS production is possibly involved in senescence stabilization via the feedback loop that provides sustained activation of DDR signaling (Fig. [10](#F10){ref-type="fig"}). {#F10} The main properties of hMESCs - the capacity for self-renewal, multilineage differentiation and noninvasive isolation procedures bring them to the cutting edge of regenerative medicine. Although hMESCs trans-plantation for treatment of heart failure, myocardial infarction, Duchenne Muscular Dystrophy has been successfully applied, it should be taken into account the possibility of premature senescence of these adult stem cells in stress conditions with following loss of the regenerative potential and, consequently, of the capability to regenerate the injured tissues. Understanding the mechanism of premature senescence of hMESCs induced by H~2~O~2~ should provide more effective strategies in transplantation of these cells into the recipients with age-related disorders inherently associated with increased levels of oxidative stress. METHODS {#s4} ======= Cell culture {#s4_1} ------------ Human mesenchymal stem cells isolated from desquamated endometrium in menstrual blood (hMESCs, line 2304) as described previously \[[@R52]\] were cultured in complete medium (DMEM/F12 (Gibco BRL, USA) supplemented with 10% FBS (HyClone, USA), 1% glutamax (Gibco BRL, USA), 1% gentamycin) at 37°C in humidified incubator, containing 5% CO~2~. hMESCs had a positive expression of CD73, CD90, CD105, CD13, CD29, and CD44 markers; expression of the hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130, and HLA-DR (class II) was absent. Besides, hMESCs partially (over 50%) expressed the pluripotency marker SSEA-4 but not Oct-4. Multipotency of isolated hMESCs was confirmed by their ability to differentiate into other mesodermal cell types, such as osteocytes and adipocytes. Immunofluorescent analysis revealed the expression of the neural precursor markers nestin and beta-III-tubulin that suggests a neural predisposition of hMESCs. Cells were characterized by high rate of cell proliferation (doubling time 22-23 h) and high cloning efficiency (about 60%). To avoid complications of replicative senescence, cells at early passages (between 5 and 9 passages) were used in all experiments. Cell treatments {#s4_2} --------------- Cells were harvested by trypsinization and plated at a density of 15\*10^3^ cells per cm^2^. For microscopy experiments, cells were grown on glass coverslips. H~2~O~2~ treatment was performed on sub-confluent cells to avoid variability of H~2~O~2~ toxicity. H~2~O~2~ stock solution in serum-free medium was prepared from 30% H~2~O~2~ (Sigma, USA) just before adding. Cells were treated with 200 µM H~2~O~2~ for 1 h, then washed twice with serum-free medium to remove H~2~O~2~, and either re-cultured in fresh complete medium for various durations as specified in individual experiments or, when indicated, treated with 5 µM SB203580 (Sigma, USA) for 40 min at 37°C. As rule, SB was added in the culture medium immediately after H~2~O~2~ treatment, and then it has been added daily to avoid the degradation of inhibitor. Measurments of ROS, cellular peroxides, mitochondrial membrane potential and mitochondrial mass by FACS and confocal microscopy {#s4_3} ------------------------------------------------------------------------------------------------------------------------------- Adherent cells treated with H~2~O~2~ were rinsed twice with PBS and probed with fluorescent dyes (all from Molecular Probes) prepared in serum-free medium. To detect intracellular ROS, cells were stained with 10 µM 2\',7\'-dichlorodihydrofluorescein diacetate (H~2~DCFDA) for 20 min at 37°C. Cellular peroxide levels were assessed by staining with 30 µM dihydrorhodamine 123 (DHR 123) for 30 min at 37°C. Mitochondrial membrane potential was measured after cell staining with 10 µM Rhodamine 123 (Rho123) for 30 min at 37°C. Mitochondrial mass was determined after cell staining with 10 µM 10-n-Nonyl-Acridine Orange (NAO) for 10 min at 37°C. After incubation, cells were analyzed either by FACS or confocal microscopy. For FACS cells were harvested by trypsinization and immediately analyzed on a Coulter EPICS XL Flow Cytometer (Backman Coulter, USA). All parameters were measured as FL1 fluorescence. For confocal scanning imaging, cells grown on coverslips were moved to the imaging system of scanning confocal microscope Leica TCS SP5 MP (Leica Microsystems Inc., Bannockburn, IL). Single focal plane images were merged and analyzed with standard Leica LAS AF Software (Leica Microsystems). Fluorescent cells were viewed with 20x objective (HCX APO CS20x/0.70; Leica Microsystems). For all dyes fluorescence was excited with 488 nm laser and observed in green spectral region (510-560 nm). Sampling frequency was set to 0.033 fps for fluorescent imaging. Fluorescent intensity was recorded in the regions of interest from 20-35 cells. After baseline collection (5 min), cells were treated with 200 µM H~2~O~2~ for 55 min. Next measurements were done at DIV5 (after cell incubation with H~2~O~2~ during 60 min at DIV1). Immunofluorescence {#s4_4} ------------------ H~2~O~2~-treated cells grown on coverslips were fixed with 4% formaldehyde (15 min), permeabilized with 0.1% Triton X-100 (10 min) and blocked with 1% BSA (1 h). Cells were incubated with primary antibodies -- a rabbit polyclonal antibodies against γH2A.X (Ser139) (Abcam), phospho-53BP1 (Ser1778) (Cell Signaling), phospho-p53 (Ser15) (Cell Signaling) and mouse polyclonal antibodies against phospho-ATM (Ser1981) (Thermo Scientific) overnight at 4°C and secondary antibodies -- Alexa Fluor 568 goat anti-rabbit or Alexa Fluor 488 goat anti-mouse (Invitrogen) for 1 h at room temperature after extensive washing with PBS/0.1% Tween 20 between each step. The slides were counterstained with 1 µg/ml DAPI (Sigma) and mounted using 2% propyl gallate. A Zeiss Axiovert 200M fluorescence microscope (Carl Zeiss) equipped with a digital camera DFC 420C (Leica) utilizing Adobe Photoshop software was used to view and acquire images. Western blotting {#s4_5} ---------------- Western analysis was performed as described previously \[[@R53]\]. Primary antibodies (all from Cell Signaling) against the following proteins were used: p21Waf1/Cip1, phospho-ATM (Ser1981), p38αMAPK, phospho-MAPKAPK-2 (Thr334), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, clone 14C10), phospho-p53 (Ser15), phospho-Rb (Ser807/811), phospho-p38MAPK (Thr180/Tyr182). RT-PCR assay {#s4_6} ------------ To analyze gene expression, total cellular RNA was isolated with RNesy Micro Kit (Qiagen) according to manufacturer\'s instructions. cDNA synthesis was performed with 1 μg of total RNA using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas) according to manufacturer\'s instructions. Specific genes were amplified by Taq DNA polymerase (Fermentas) with C1000 TouchThermal Cycler amplifier (Bio-Rad Laboratories). The program was described earlier \[[@R30]\]. Primers *p21Waf1/Cip1* and *beta-actin* were obtained from SYNTOL (Russia). The electrophoresis of amplified products was performed in 2% agarose gel with TAE buffer and ethidiumbromide. 100 kb DNA ladder (Fermentas) was used as molecular weight marker. Amplified products were visualized in UV-light (302 nm) with transilluminator and registered with a digital Canon camera. FACS analysis of cell viability and cell size {#s4_7} --------------------------------------------- Adherent cells were rinsed twice with PBS and harvested by trypsinization. Detached cells were pelleted by centrifugation. Finally, detached and adherent cells were pooled and resuspended in PBS. 50 µg/ml propidium iodide (PI) was added to each sample just before analysis and mixed gently. Samples were analyzed on a Coulter EPICS XL Flow Cytometer (Backman Coulter). The cell size was evaluated by cytometric light scattering of PI-stained cells with using Win MDI program version 2.8. To discriminate the live and dead cells, two-parameter histogram was used (FL4LOG vs. FSLOG). Analysis of each sample (at least 10,000 cells) was performed for 100 sec with high sample delivery. Statistics {#s4_8} ---------- All data are presented as the mean and standard deviation of the mean from at least three separate experiments performed. Statistical differences were calculated using the Student\'s t-test and considered significant at \*,§ p\< 0.05; \*\*,§§ p\< 0.005; \*\*\*,§§§ p\<0.001. This work was supported by Russian Foundation of Basic Research (№ 14-04-01720-a), MCB RAS Program Grant. Authors declare no conflict of interests.

M. Matveenko, S. Hackl, C. F. W. Becker, *ChemistryOpen* **2018**, *7*, 106. Expressed protein ligation (EPL) is a versatile method for protein semisynthesis that has tremendously expanded the range of achievable targets and enabled a multitude of discoveries regarding protein structure, function, localization, and other properties.[1](#open201700180-bib-0001){ref-type="ref"} This method relies on the native chemical ligation (NCL)[2](#open201700180-bib-0002){ref-type="ref"} reaction between two protein segments, either of which can be produced recombinantly or synthetically, one containing a C‐terminal thioester and the other containing an N‐terminal cysteine (Cys), and which react by means of transesterification and rearrange through an S‐to‐N acyl shift to form a native peptide bond. The initial requirement for N‐terminal cysteine residues, which occur at a low frequency of approximately 1.7 % in proteins,[3](#open201700180-bib-0003){ref-type="ref"} has subsequently been abrogated, largely by the development of the ligation--desulfurization strategy, which combines the use of introduced cysteines or non‐natural beta‐mercapto derivatives of amino acids with desulfurization to clip off the thiol moiety and reveal the native alanine or other proteinogenic amino acids at the ligation site.[4](#open201700180-bib-0004){ref-type="ref"} This strategy, pioneered by Yan and Dawson, who demonstrated the reductive desulfurization of Cys to Ala following NCL (Scheme [1](#open201700180-fig-5001){ref-type="fig"}),[4a](#open201700180-bib-0004a){ref-type="ref"} was made widely applicable by the development of mild, metal‐free radical desulfurization methods,[4b](#open201700180-bib-0004b){ref-type="ref"}, [5](#open201700180-bib-0005){ref-type="ref"} as well as the use of other β‐ or γ‐mercapto amino acids[4f](#open201700180-bib-0004f){ref-type="ref"}, [4g](#open201700180-bib-0004g){ref-type="ref"}, [5](#open201700180-bib-0005){ref-type="ref"} and selenocysteine.[6](#open201700180-bib-0006){ref-type="ref"} The ligation--desulfurization chemistry has greatly increased the flexibility of the semi‐ and total synthesis approaches toward proteins. The potential drawback of most of these methods, besides when using selective desulfurization of β‐mercapto aspartate[7](#open201700180-bib-0007){ref-type="ref"} or selenocysteine,[8](#open201700180-bib-0008){ref-type="ref"} is the concomitant desulfurization of any unprotected native cysteines, usually found in the larger recombinant segments of the protein of interest, as those in the synthetic part can be easily protected orthogonally during solid‐phase peptide synthesis (SPPS). Both the removal of any native cysteines as well as the incorporation of non‐native thiol‐containing amino acids is often undesirable, as these residues are capable of forming disulfide bridges that greatly affect the tertiary structure and moreover can themselves be functionally significant. {#open201700180-fig-5001} During our studies on the semisynthesis of human small heat shock protein, Hsp27, through the ligation--desulfurization strategy, the unwanted thiol removal of the single native cysteine residue (Cys137) was encountered (Scheme [2](#open201700180-fig-5002){ref-type="fig"}). The 205 amino acid protein was generated through NCL between the recombinant Hsp27(1--172) segment carrying a C‐terminal thioester and the smaller synthetic fragment Hsp27(173--205), wherein Ala173 had been replaced with a Cys residue, followed by desulfurization. Fortunately, the ensuing Cys137Ala modification had previously been investigated and rendered inconsequential in the context of our functional characterization of the protein.[9](#open201700180-bib-0009){ref-type="ref"} Nevertheless, we decided to solve the undesired desulfurization issue and, thus, to achieve a 'traceless' semisynthesis of Hsp27, keeping Cys137 intact. Two variants of this protein were pursued in accordance with our previous work: one carrying a non‐enzymatic posttranslational modification argpyrimidine (Apy) at residue 188, termed Hsp27^Apy^, and the control, non‐modified (NM) variant, referred to as Hsp27^NM^. {ref-type="ref"}](OPEN-7-106-g002){#open201700180-fig-5002} To this end, we searched for a suitable protecting group that would temporarily mask the native Cys137 of Hsp27 and satisfy several criteria. Firstly, this protecting group has to be selective for cysteine residues, as it needs to be appended following thiol‐mediated intein cleavage of the long, recombinantly produced protein segment containing many nucleophilic side chains. In addition, the conditions for this protection need to be mild, so as not to compromise the unstable C‐terminal thioester moiety. Protection prior to intein cleavage and concomitant thioester formation is not possible, owing to the requirement for the free thiol group of the critical cysteine residue in intein to be next to the cleavage site. Secondly, the resulting protected thiol has to be stable enough to withstand the NCL reaction followed by desulfurization of the ligation‐site cysteine, thus excluding any groups that are sensitive to reducing, basic, and radical conditions. Finally, it should be straightforward to remove such a protecting group by using reasonably mild reagents. Based on these criteria, two candidates were selected, trityl (Trt)[10](#open201700180-bib-0010){ref-type="ref"} and phenacyl (PAc)[11](#open201700180-bib-0011){ref-type="ref"} groups, both of which were attractive as they had previously been successfully used for similar purposes, although neither has been used to protect a thioester‐containing peptide.[10](#open201700180-bib-0010){ref-type="ref"}, [11c](#open201700180-bib-0011c){ref-type="ref"} Specifically, Trt was utilized by Mochizuki et al. to protect two internal Cys sulfhydryls within a 32‐amino‐acid‐long synthetic peptide, which was then deprotected at the N‐terminal cysteine and ligated to a C‐terminal thioester fragment, followed by desulfurization, Trt‐deprotection, oxidation, and purification, to obtain the desired product in 42 % overall yield from the Trt‐free peptide.[10](#open201700180-bib-0010){ref-type="ref"} Kawakami and co‐workers, on the other hand, used the PAc moiety, first applied in the protection of amino and thiol functions by Tang et al.,[11a](#open201700180-bib-0011a){ref-type="ref"} to mask two cysteines within a recombinant C‐terminal fragment of histone H3.1. After successful introduction of the PAc groups, the orthogonally protected N‐terminal cysteine was revealed and the resulting fragment underwent NCL, desulfurization, and PAc‐deprotection to afford semisynthetic H3.1 in 18 % yield over six steps from the protecting‐group‐free recombinant fragment.[11c](#open201700180-bib-0011c){ref-type="ref"} With these two promising candidates in mind, we decided to use wild‐type (WT) recombinant, HPLC‐purified Hsp27 (Hsp27^WT^) as a model system for the comparison of the protecting groups (Scheme [3](#open201700180-fig-5003){ref-type="fig"}). ![Overview of the process to find a suitable cysteine protecting group to enable the 'traceless' semisynthesis of Hsp27. Reagents and conditions: a) 6 [m]{.smallcaps} Gdn⋅HCl, 0.2 [m]{.smallcaps} NaPi, 0.25 [m]{.smallcaps} MESNA, 50 m[m]{.smallcaps} TCEP, pH 7.8, 37 °C, 48 h; b) 6 [m]{.smallcaps} Gdn⋅HCl, 0.2 [m]{.smallcaps} NaPi, 0.2 [m]{.smallcaps} TCEP, 50 m[m]{.smallcaps} MESNA, 16 m[m]{.smallcaps} V‐50, pH 6.7, 40 °C, 16 h; c) Zn, MPA (15 % v/v in 6 [m]{.smallcaps} Gdn⋅HCl).](OPEN-7-106-g003){#open201700180-fig-5003} Gratifyingly, both Trt and PAc protecting groups could be introduced easily onto Cys137 within the model system Hsp27^WT^ by alkylation with either Trt cation, generated from TrtOH in the presence of a moderately acidic hexafluoroisopropanol (HFIP), or PAcBr used in slight excess at a carefully controlled pH (Scheme [3](#open201700180-fig-5003){ref-type="fig"}) under the published reaction conditions.[10](#open201700180-bib-0010){ref-type="ref"}, [11c](#open201700180-bib-0011c){ref-type="ref"} The ESI‐MS spectra of the ensuing proteins (Figures S1 and S2) indicated a clean conversion, and thus the respective buffers were exchanged, without isolation of proteins, with that required for an NCL reaction. In previous work, we were able to conduct the ligation and the desulfurization steps in one pot by using sodium 2‐mercaptoethanesulfonate (MESNA) as the mediator for NCL, as this reagent is compatible with the subsequent radical‐mediated step.[9](#open201700180-bib-0009){ref-type="ref"}, [12](#open201700180-bib-0012){ref-type="ref"} Thus, the S‐protected proteins were submitted to these optimized conditions, consisting of 6 [m]{.smallcaps} Gdn⋅HCl, 50 m[m]{.smallcaps} tris(2‐carboxyethyl)phosphine (TCEP) and 250 m[m]{.smallcaps} MESNA at pH 7.8. Both protecting groups performed well under these conditions, and the corresponding protein samples remained unaffected even after 48 h (the typical reaction time from previous studies), as judged by their LC‐MS spectra (Figures S3 and S4). The more critical test was the subsequent radical desulfurization process, for which the reaction mixtures were diluted (4×) with a buffer containing additional TCEP to serve as the thiyl radical acceptor, and the water‐soluble radical initiator V‐50,[4b](#open201700180-bib-0004b){ref-type="ref"} whereas the remaining MESNA (ca. 50 m[m]{.smallcaps} final concentration) should function as the hydride source. After 16 h at 40 °C,[9](#open201700180-bib-0009){ref-type="ref"} the LC‐MS spectrum of the Trt‐protected protein (Figure S5) showed a mixture of products, including deprotected but not desulfurized proteins, indicating that the easily formed and stable trityl radical had potentially compromised the stability of the protecting group and the reaction itself. On the other hand, the spectrum of the PAc‐protected Hsp27^WT^ did not change, showing only the protected proteins (Figure S6) and indicating that only the PAc group was suitable for our purposes. In the last hurdle, this moiety was then smoothly cleaved by treatment with powdered Zn under acidic conditions \[mercaptopropionic acid (MPA) in 6 [m]{.smallcaps} Gdn⋅HCl, pH≈1, unadjusted\].[11c](#open201700180-bib-0011c){ref-type="ref"} After 30 min, the peak for the substrate was already undetectable in the LC‐MS spectrum of the reaction mixture, and the deprotected protein was isolated by HPLC (recovery \>60 % over 4 steps; see Figure S7 for characterization). Notably, Katayama and Hojo have demonstrated that such reducing conditions do not affect acetamidomethyl (Acm) or *p*‐methoxybenzyl groups,[11b](#open201700180-bib-0011b){ref-type="ref"} which are commonly used for Cys protection should such orthogonality be of use. In this way, the PAc group was selected and was then used to synthesize the non‐Apy‐modified (Hsp27^NM^) and Apy‐modified (Hsp27^Apy^) proteins that retain their native Cys137 residues (Scheme [4](#open201700180-fig-5004){ref-type="fig"}). PAcBr could then be smoothly reacted with Hsp27(1--172)‐thioester **1** to produce the corresponding Cys137‐protected variant **2**; the formation of which was verified by LC‐MS analysis of the reaction mixture (mass difference owing to PAc addition = +118 Da; Figure S8). The only inhomogeneity in the ensuing mass spectrum (Scheme [4](#open201700180-fig-5004){ref-type="fig"} A) arises from the presence of traces of the corresponding C‐terminal acid of **2** (mass deviation from desired product = −124 Da), formed by hydrolysis during the preceding MESNA‐mediated intein cleavage. On the other hand, the doubling of the main peak is the result of the inconsequential incomplete cleavage of the N‐terminal methionine by bacterial peptidases, which often occurs during expression in *E. coli*.[13](#open201700180-bib-0013){ref-type="ref"} Subsequently, buffer exchange to the NCL conditions followed by addition of the N‐terminal Cys‐carrying peptide **3** in a slight excess generated, after 48 h, ligation product **4**, as indicated by LC‐MS analysis of the crude reaction mixture and the ensuing mass spectrum (Scheme [4](#open201700180-fig-5004){ref-type="fig"} B), which also shows traces of unreacted **2** as well as the corresponding acid. Buffer exchange and submitting the crude **4** to radical desulfurization resulted in clean conversion into compound **5** (Scheme [4](#open201700180-fig-5004){ref-type="fig"} C), which was then deprotected as described above and purified by the single application of reverse‐phase HPLC to provide Hsp27^NM^ still containing Cys137 (**6**, 35 % from thioester **1**, 2.0 mg). The Apy‐modified variant **7** was generated by identical means, but using the Apy‐derivatized variant of **3** in 38 % yield (2.2 mg) over four steps. The proteins were obtained in good purity, as judged by analytical HPLC chromatograms and ESI‐MS spectra (Schemes [4](#open201700180-fig-5004){ref-type="fig"} D and 4 E), although both samples contained small amounts of the unreacted acid Hsp27(1--172)−OH, which also retained Cys137. The two samples were analyzed by far‐UV circular dichroism (CD) spectroscopy and analytical size‐exclusion chromatography (Figures S11 and S12, respectively). We did not observe any major differences between the properties of the two variants or, moreover, to those obtained previously and containing the Cys127Ala modification (Figures S11 and S12).[9](#open201700180-bib-0009){ref-type="ref"} ![Semisynthesis of Hsp27 variants through traceless one‐pot ligation--desulfurization by using the PAc protecting group. Reagents and conditions: a) \[**1**\]=0.5 m[m]{.smallcaps}, PAcBr (2.5 equiv), 6 [m]{.smallcaps} Gdn⋅HCl, 0.4 [m]{.smallcaps} NaPi, pH 7.15, 25 °C, 1 h; b) \[**2**\]=0.3 m[m]{.smallcaps}, **3** (3 equiv), 6 [m]{.smallcaps} Gdn⋅HCl, 0.2 [m]{.smallcaps} NaPi, 0.25 [m]{.smallcaps} MESNA, 50 m[m]{.smallcaps} TCEP, pH 7.8, 37 °C, 48 h; c) 6 [m]{.smallcaps} Gdn⋅HCl, 0.2 [m]{.smallcaps} NaPi, 0.15 [m]{.smallcaps} TCEP, 0.125 [m]{.smallcaps} MESNA, 13 m[m]{.smallcaps} V‐50, pH 6.6, 40 °C, 16 h; d) powdered Zn (37 mg mL^−1^), MPA (7.5 % v/v in 6 [m]{.smallcaps} Gdn⋅HCl), 25 °C, 1 h, 35 % over four steps. A--C) Mass spectra obtained by LC‐MS analysis of crude reaction mixtures of **2**, **4**, or **5**. D, E) Analytical HPLC traces (*λ*=214 nm) and ESI‐MS spectra of isolated **6**, **7**.](OPEN-7-106-g004){#open201700180-fig-5004} To probe the scope of the PAc‐protection approach, we chose another recombinant protein segment that had been utilized in the semisynthesis of a lipidated prion protein (PrP) variant in our laboratories. The C‐terminal thioester of N‐terminally truncated PrP (residues 90--231, T PrP‐SR, **8**; Scheme [5](#open201700180-fig-5005){ref-type="fig"}) contains two cysteines, Cys178 and Cys213, and was previously ligated to a synthetic C‐terminal peptide **9** that features a non‐native Cys232 residue at its N‐terminus for NCL, with two lysine‐bound palmitoyl groups to serve as a membrane anchor and a polymer tag to enhance solubility.[14](#open201700180-bib-0014){ref-type="ref"} The introduced ligation site Cys was left un‐desulfurized in the previous syntheses, which could potentially be problematic for the folding of the protein, as it involves the Cys178--Cys213 disulfide formation. In an attempt to achieve a 'traceless' semisynthesis of lipidated T PrP, we obtained the bis‐Cys‐protected product **10** (Scheme [5](#open201700180-fig-5005){ref-type="fig"} and Figure S13). Ligation of this thioester to peptide **9** (2.5 equiv) did not proceed with MESNA as a mediator, and the more reactive thioester‐forming mercaptophenylacetic acid (MPAA) had to be utilized, which resulted in rapid and efficient ligation (Figure S14). Unfortunately, this aromatic thiol was not compatible with the radical desulfurization reaction, owing to its radical‐quenching properties and, moreover, it could not be removed sufficiently by using precipitation or buffer‐exchange methods. Thus, the ligation product was isolated by reverse‐phase HPLC (Figure S15), and then submitted to the optimized desulfurization conditions. The readily formed Cys232Ala product (Figure S16) was then treated with the PAc‐deprotection mixture used previously. Unfortunately, the reaction was very sluggish in this case, producing insoluble material and unable to reach completion. Although even worse results were observed with Mg as the metal, using Zn in AcOH (45 % v/v in 6 [m]{.smallcaps} Gdn⋅HCl)[11b](#open201700180-bib-0011b){ref-type="ref"} allowed the desired product to be formed with full conversion in 60 min, as indicated by LC‐MS. After centrifugation and isolation by reverse‐phase HPLC, the desired lipidated T PrP variant **11** was isolated in 65--90 % yield over two steps and characterized by ESI‐MS and analytical reverse‐phase HPLC (Scheme [5](#open201700180-fig-5005){ref-type="fig"} A). The semisynthetic PrP samples were folded, as described previously,[14a](#open201700180-bib-0014a){ref-type="ref"} and analyzed by CD spectroscopy (Scheme [5](#open201700180-fig-5005){ref-type="fig"} B), which showed that they adopt a secondary structure similar to that observed for the samples containing the additional Cys232 residue (Figure S17 and Table S1), corroborating the previous results. ![Traceless semisynthesis and characterization of lipidated T PrP variant. Reagents and conditions: a) \[**8**\]=0.4 m[m]{.smallcaps}, PAcBr (5 equiv), 6 [m]{.smallcaps} Gdn⋅HCl, 0.4 [m]{.smallcaps} NaPi, pH 7.15, 20 °C, 1 h; b) \[**10**\]=0.6 m[m]{.smallcaps}, **9** (2.5 equiv), 6 [m]{.smallcaps} Gdn⋅HCl, 0.2 [m]{.smallcaps} NaPi, 30 m[m]{.smallcaps} MPAA, 20 m[m]{.smallcaps} TCEP, pH 7.2, 37 °C, 6 h, ca. 30 % over two steps; c) 6 [m]{.smallcaps} Gdn⋅HCl, 0.2 [m]{.smallcaps} NaPi, 0.25 [m]{.smallcaps} TCEP, 0.1 [m]{.smallcaps} MESNA, 20 m[m]{.smallcaps} V‐50, pH 6.7, 40 °C, 16 h; d) Zn (180 mg mL^−1^), AcOH (45 % v/v in 6 [m]{.smallcaps} Gdn⋅HCl), 25 °C, 1 h, 65--90 % over 2 steps. A) Analytical HPLC trace (baseline corrected) and ESI‐MS spectrum of isolated **11**. B) Far‐UV CD spectrum of folded **11**.](OPEN-7-106-g005){#open201700180-fig-5005} Overall, the PAc moiety proved to be a useful cysteine protecting group that can be appended onto recombinantly produced protein segments containing a sensitive thioester moiety. The group remains unaffected during the NCL and radical desulfurization reactions as well as reverse‐phase HPLC, and can be cleanly removed under reductive, acidic conditions that are orthogonal to the deprotection of common cysteine protecting groups. Therefore, this methodology may help to achieve even more precise semi‐ and total synthesis approaches for proteins in the future. Experimental Section {#open201700180-sec-0002} ==================== Procedures and characterization data can be found in the Supporting Information. Conflict of interest {#open201700180-sec-0004} ==================== *The authors declare no conflict of interest*. Supporting information ====================== As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. ###### Supplementary ###### Click here for additional data file. *We gratefully acknowledge Gilyana Kücükkaplan and Gerhard Niederacher for excellent technical assistance and the Austrian Academy of Sciences for financial support (APART fellowship to M.M.)*.

Introduction {#s1} ============ Past influenza pandemics and the recent H1N1 pandemic alert people the unpredictability and potentially overwhelming impacts of influenza outbreaks. While it is certain that the next pandemic will arrive in human societies, it is almost impossible to predict the virus type, transmission manner, and attack and mortality rates etc. Such unpredictability seriously challenges the public health system. Supplies of vaccine and pharmaceuticals may not be available or may be in shortage for a few months or even longer while a substantial number of infected cases has been reported. Under such critical circumstances, non-pharmaceutical interventions are usually considered in the first place, aiming at mitigating the spread and lowering the attack rate and fatality. Workplaces and schools are both crucial community structures in epidemic control and mitigation planning. High contact rate and long contact duration in workplaces and schools may promote the transmission among workforce and school population. However, closure of workplaces causes significant disruption to economic activities and social functioning. Therefore a large-scale of workplace closure has seldom been implemented in the history of infectious disease control. In order to reduce contacts in workplace during epidemic, policy makers may seek alternative interventions, such as workforce shift. In workforce shift intervention, a portion (work team) of workforce is scheduled away from workplaces for a certain time span and then return by shifting with others. Workforce shift has been planned in real-life epidemic control. UK influenza contingency plan suggests 25% of employees taking 5--8 days off to enhance social distancing [@pone.0032203-Newman1]; in the Singapore guideline of infectious disease control for workplace, dividing employees into work teams with minimum contacts between teams by shift system is suggested [@pone.0032203-Ministry1]. To our best knowledge, there are no studies on evaluating policies similar to workforce shift for influenza mitigation. We therefore investigate how effective team-based rotational workforce shift is. Compared to workplace closure, school closure had been practiced more frequently and also widely evaluated in epidemic and pandemic control [@pone.0032203-Milne1]--[@pone.0032203-Lee1]. In a recent article [@pone.0032203-Cauchemez1], multiple aspects of school closure were reviewed, and it concluded that there are still many uncertainties on mitigation benefits of school closure as a public health policy. Historical school closure implementation data in real-world epidemic mitigation also showed contradictory conclusions, e.g., the encouraging results achieved in Israel [@pone.0032203-Heymann1] and the less encouraging ones in Hong Kong [@pone.0032203-Cowling1]. As workforce shift and school closure target different portions of the population, the combination of the two strategies may achieve better mitigation for influenza epidemic. On the other hand, however, as mass social distancing strategies, they may cause considerable economic and social costs. Any decision on intervention combination should be cautiously deliberated. This calls for quantitative evaluations on the effectiveness of combined intervention strategies. Combined interventions for influenza epidemic have been evaluated widely in the literature. Germann et al [@pone.0032203-Germann1], Carret et al [@pone.0032203-Carrat1] and Milne et al [@pone.0032203-Milne1] assumed that combined interventions are implemented before the outbreak of epidemic and lasted until the end. Halder et al. [@pone.0032203-Halder1] evaluated combined interventions with limited durations. Longni et al [@pone.0032203-Longini1] and Ferguson et al [@pone.0032203-Ferguson1] studied how different effectiveness levels and coverage of interventions could impact the attack rate and peak incidence. Halloran et al [@pone.0032203-Halloran1] and Rizzo et al [@pone.0032203-RIZZO1] simulated the epidemic by implementing multiple strategies simultaneously at different time points with their own fixed durations. Duerr et al [@pone.0032203-Duerr1] tested the combination of two interventions in which one starts at the beginning of epidemic and the other may start at different time but always last until the end of the epidemic. In this study, we evaluate a series of scenarios under workforce shift and its combination with school closure, with different trigger thresholds and durations. To our best knowledge, this is the first study evaluating combination effects of workforce shift and school closure for influenza mitigation. In comparison with the timing configuration in other studies, our study is different: 1) trigger thresholds of individual interventions can be configured in the combination independently; and 2) the duration of the combined interventions can be varied. Through simulation evaluations, we aim to provide a more comprehensive view on the impacts of temporal factors on social distancing interventions for influenza epidemic, helping to answer three key questions: a) *do combined interventions always outperform single interventions?* b) *how do trigger threshold and duration affect the effectiveness of combined interventions?* c) *does the implementation sequence in a combined intervention make a difference in its effectiveness?* Methods {#s2} ======= Considering the importance of social structure in infectious disease spread, network-based models [@pone.0032203-Newman1], [@pone.0032203-DelValle1], [@pone.0032203-Meyers1] have been commonly used for exploring the effectiveness of interventions in a heterogeneous-structured population for assisting policy makers to make proper decisions. In this work, we use a contact-network-based simulation model to carry out the evaluations based on Singapore\'s social structure. Specifically, we adopt an agent-based simulation model running on top of a social contact network. The network represents the statistical properties of interpersonal contacts which may lead to disease transmission in the specific community structure in Singapore. We evaluate workforce shift and its combination with school closure respectively, via extensive simulations with different trigger thresholds and implementation durations. Contact Network Construction {#s2a} ---------------------------- To address infectious disease spread with the consideration of the heterogeneity in social interactions, the most expressive approach is to form a structure of "network" by taking all individuals as vertices (or nodes) and their social connections as edges. We can further specify that an individual\'s social connections are the set of people with whom the individual may contact during the period when he or she is infectious. Thus, the disease transmission among the population can be simulated as the probabilistic propagation of viruses via the connecting edges in the contact network. Generating a contact network representing for all individuals\' contacts is complicated. To simplify the problem, we adopt a divide-and-conquer approach based on community structures of a typical society since social contacts most extensively take place in such community structures. For example, students contact with their peers at the schools; working adults contact with their colleagues at the workplaces; patients contact with healthcare workers and other patients at the hospitals, etc. We firstly determine the six types of community structures that are commonly reviewed in the literature [@pone.0032203-Milne1], [@pone.0032203-Meyers1], [@pone.0032203-Zhang1] - *households*, *hospitals*, *schools*, *workplaces*, *shopping places* and *public transport.* Then we generate the community structures according to the statistics of them. To lower the computational cost, the contact network is only comprised of 10% of Singapore population: age structure, household size distribution, characteristics of the modeled community structures have been retained proportionally in the simulated population to keep the epidemic trend consistent with that in the whole population. The sizes of communities in the network are obtained proportionally to the statistical numbers in the whole Singapore society. Specifically, a list of households is firstly generated based on the household size distribution. Subsequently, 35 schools are created proportionally according to the total number of students and school size distribution. Then each school is sub-divided into classes based on class size distribution. After that, students are assigned to schools and classes following the "enrollment in the nearby schools" policy, i.e., the students living nearby have a higher chance to be enrolled into the same school or class. Students in the same class may have more contacts (class contacts) and than those between students from the same school but different classes (school contacts), as shown in [Figure 1](#pone-0032203-g001){ref-type="fig"}. Similarly, 3 hospitals are constructed with sub-divisions -- "wards" (i.e., sections in a hospital for accommodating hospitalized patients) based on hospital and ward size distribution (in term of number of beds) and bed occupation rate. Furthermore, ∼5,300 workplaces (equivalent to companies) are constructed based on the number of working adults, number of companies, and company size distribution, with no further sub-divisions; 10 shopping malls are created according to the survey data including the population size going for shopping, shopping frequency and daily traffic of malls, with no sub-divisions. Finally, a single structure of public transport is created as a single-layer giant component which includes all the commuters in the population. {#pone-0032203-g001} Once the above community structures are constructed, individuals selected from the population pool are filled into each structure. The selection criteria are a set of rules defining the eligibility for a community structure. For example, age-based criteria can be used to define the enrollment to schools. Note that while an individual typically can be selected to join multiple different communities, some community enrollments are exclusive to each other. For example, an individual selected to be a patient staying in hospital should not participate in any of the school, workplace, shopping mall and public transport communities. However, his/her contacts within household may still remain as the visits from family members maintain such contacts. After assigning the subpopulation to a community structure, a local contact network is created by connecting the individuals of the subpopulation with the interpersonal contacts following a Poisson degree distribution [@pone.0032203-Meyers1] where the mean contact degrees are obtained from our social contact survey [@pone.0032203-Zhang1]. All the local contact networks are finally integrated into a global network. The whole network generated in this study is comprised of ∼480000 vertices and ∼7.6 mil edges from 100,000 simulated households. Within the population, 11% are students, 61% are working adults, 0.2% stay in hospital, 22% visit shopping malls regularly and 34% use public transport on daily basis [@pone.0032203-Department1]. The social contact survey among the public of Singapore was conducted in 2008 with a survey form containing 45 questions. There are totally 1040 pieces of valid survey data collected. The extracted average numbers of contacts in different social locations are summarized in [Figure 1](#pone-0032203-g001){ref-type="fig"} with the assumption that every household is fully connected [@pone.0032203-Zhang1]. Note that the contact network constructed in this study is unweighted. According to Newman [@pone.0032203-Newman1], a disease will propagate equivalently in the population as a whole if all individual transmission probabilities are equal to the average transmission probability. By using the average transmission probability to replace each individual one, we simplify the transmission function incurred during the infection. Intervention Policies {#s2b} --------------------- Intervention polices are implemented to mitigate the transmission of disease. There are two categories of intervention: pharmaceutical and non-pharmaceutical interventions. Pharmaceutical interventions are mainly associated with vaccines and anti-viral drugs; and non-pharmaceutical interventions include isolation/quarantine, social distancing, etc. As vaccine production and anti-viral stockpiling often require substantial time after a pandemic occurs, non-pharmaceutical interventions are necessary to delay and dampen the pandemic before pharmaceuticals become available [@pone.0032203-U1]. Workforce shift and school closure are the examples of social distancing interventions and will be evaluated in our work. ### 1) Workforce Shift {#s2b1} In many countries, working adults occupy the largest portion of the population, and make close contacts with their co-workers in their daily activities. Closing workplaces has significant economic and social costs; so it is one of the least favorable choices that policy makers may consider. Another social distancing measure is workplace non-attendance, in which each worker has a 50% chance each day to choose either staying at home or attending to work. This policy is hard to implement as random and voluntary attendance of workers may cause chaos in the workplace. Although workplace closure is seldom implemented in practice, policy makers do consider and suggest alternative workplace control, like workforce shift, for mitigating disease spread when necessary. In this study, we evaluate the workforce shift policy. Specifically, we assume that 1) each company or institution splits its employees into two work teams and implements 7-day rotation among the teams; 2) workforce shift is implemented immediately after the trigger threshold is reached; 3) for home-staying team members, all their contacts taking place in work places are removed from the contact network during the shifting period; and 4) workforce shift operation does not increase the contacts in other community structures. ### 2) School Closure {#s2b2} School closure is a typical social distancing policy for mitigating the spread of infectious diseases among the student population. Generally, there are different types of school closure: 1) class closure, i.e., a class is closed if there are diagnosed cases; 2) individual school closure, i.e., a school is closed if there is a certain number of diagnosed cases, and 3) all-school closure, i.e., all schools are closed simultaneously if a threshold number of cases are diagnosed. All three types of school closure had been implemented in the real-world interventions in countries like Australia, UK, USA, and Japan to mitigate the spread of pandemic influenza [@pone.0032203-Newman1], [@pone.0032203-DelValle1], [@pone.0032203-Meyers1]. In a previous study [@pone.0032203-Zhang1], all-school closure had been evaluated based on the same Singapore society setting with the consideration of different trigger thresholds and implementation durations. It was found that, in a cost-cautious situation where short intervention is preferred, school closure of 2-week should be implemented at a higher threshold (a later time); if reducing the epidemic size is the top priority, it is wise to implement a longer school closure (more than 6 weeks) as early as reasonable. In this paper, we evaluate combined workforce shift and school closure strategy. Models for Disease Spread and Intervention {#s2c} ------------------------------------------ [Figure 2](#pone-0032203-g002){ref-type="fig"} describes the host progression in the process of infectivity development of influenza illness within the host person. Any susceptible person has a chance (transmission probability) to be infected for every infectious contact s/he has. If the person (denoted as p) is infected, p is exposed but has no infectivity or any symptom yet. After the latent period, p becomes infectious (incubation period is assumed to be equal to latent period in the model). Specifically, p has a chance (symptomatic rate) to develop the clinical symptoms of influenza and turn into symptomatic infectious, or turn into asymptomatic infectious if without any symptoms. After the infectious period, p is finally removed, i.e. either recovered from influenza or dead. {#pone-0032203-g002} Note that in our model, the probability of becoming infected goes up when a person is in contact with more infectious people, despite of the locations where the contacts occur. In lack of data about the infectivity regarding contact duration, we have assumed an unweighted contact network that propagates the disease with the average transmission probability along every edge of the network. The focus of this study is on investigating the effectiveness of intervention polices under different scenarios. Specifically, we parameterize an intervention policy by six parameters: *trigger threshold, duration, target, control level, compliance rate and shift length*: - *Trigger threshold* is a percentage of diagnosed (symptomatic) cases in the overall population, which is used to determine the starting time of intervention. For example, trigger = 0.1% means that an intervention will be implemented when 0.1% of the population is diagnosed as symptomatic cases of influenza. - *Duration* refers to how long an intervention will be implemented. - *Target* specifies what type of contacts is targeted by an intervention, such as school contacts, workplace contacts etc. - *Control level* is used to differentiate the interventions performed at the different levels of a community structure, e.g. school-level closure and class-level closure. - *Compliance rate* refers to the percentage of contacts that is removed by an intervention. As compliance rate is often affected by other interventions (e.g. workplace absenteeism may improve compliance rate during the school closure as adults will stay at home to take care of their children), we assume the 100% compliance rate for all-school closure to simplify our simulation scenarios. - *Shift length* refers to the time span between team rotations. Results {#s3} ======= The evaluation results of uncontrolled epidemic in the contact network serve as the baseline results. Different mitigation scenarios with different trigger thresholds and implementation durations are simulated based on the individual-based contact network simulation model. We then evaluate and compare the impacts of different temporal factors on the effectiveness of mitigation methods. Experiment Settings {#s3a} ------------------- The basic reproductive number, *R* ~0~, is defined as the average number of secondary infections produced by a randomly selected infected person in a fully susceptible population [@pone.0032203-Diekmann1]. Previous estimates of *R* ~0~ in the past pandemic influenza were in the range of 1.5--2.3 [@pone.0032203-Ferguson1], [@pone.0032203-Fraser1]--[@pone.0032203-Mills1]. Unless otherwise specified, we assume *R* ~0~ = 1.9 in our simulations, and adopt 66.7% symptomatic rate [@pone.0032203-Novel1], 1-day latent period and 1.5-day mean infectious period [@pone.0032203-Fraser1], which are the same as those in a previous study [@pone.0032203-Zhang1]. By using Longini\'s approach [@pone.0032203-Longini1], we approximate *R* ~0~ = 1.9 empirically by tuning the base transmission probability. Specifically, we assume a scenario in which only a single individual is randomly infected where everyone else is susceptible yet not able to further transmit the disease, and count the number of secondary infections. The process is repeated for 10,000 times and *R* ~0~ 1.9 is then obtained as the average number of secondary infections. We found when the base transmission probability is 0.04, the empirical tests give the best approximation to *R* ~0~ 1.9 (95% Confident Interval (CI) 1.871--1.924), which yields the mean generation time of 2.5 days (95% CI, 2.489--2.508). The transmission probability is doubled to be 0.08 if the person is symptomatic infectious and meanwhile, half of his/her contacts are randomly removed due to self-isolation or self-shielding. Note that, in case of a new strain of influenza pandemic with unknown *R* ~0~, the transmission probability in the network simulations can be tuned with assumed latent and infectious periods and symptomatic rate to get estimation of the new *R* ~0~ by fitting to the reported epidemic curve. In this study, we focus on examining the impacts of trigger threshold and duration length of interventions on the effectiveness of mitigating the influenza epidemic. The test scenarios are tabulated in [Table 1](#pone-0032203-t001){ref-type="table"}. Each of those scenarios, including the baseline case, is simulated for 200 days and iterated for 100 times. All the results described in the following section are the average values of 100 simulation runs. 10.1371/journal.pone.0032203.t001 ###### Intervention scenario description. {#pone-0032203-t001-1} Parameters School Closure Workforce Shift --------------------- ------------------------ ------------------------ *Trigger Threshold* 0.02%, 0.25%, 1.5%, 5% 0.02%, 0.25%, 1.5%, 5% *Duration* 2,4,6,8,10 weeks 2,4,6,8,10 weeks *Target* school contacts workplace contacts *Control Level* Schools workplaces *Compliance Rate* 100% 100% *Shift length* NA 7 days Every simulation starts at *day* 0 with 10 infectious persons seeded into a susceptible population without prior immunity to the influenza virus. In our experiments, there are four trigger thresholds and five implementation durations available to choose for an intervention scenario. Hence there are totally 100 scenarios: 20 scenarios for workforce shift and 80 scenarios for the combined workforce shift and school closure (We assume that the individual interventions in each combination scenario share the same length of implementation duration.). The effectiveness of interventions is examined by evaluating attack rate (AR), peak incidence (PI), and peak day (PD). Attack rate refers to the cumulative proportion of symptomatic cases of influenza infection in the overall population; peak incidence refers to the highest number of the daily incidence of symptomatic cases; peak day refers to the day when the peak incidence happens. In the public health perspective, attack rate indicates the size of epidemic and the overall burden on the public health system due to an epidemic; and peak incidence and peak day display the challenge to an effective response to patient surges in public health system. Influenza Spread without Intervention {#s3b} ------------------------------------- [Figure 3](#pone-0032203-g003){ref-type="fig"} shows the average epidemic curves of 100 simulation runs for the case with no intervention. The epidemic reaches its peak at *day* 26 and fades out on *day* 73. The total attack rate *(AR)* is 44.47% (95% CI, 44.45%--44.48%); peak incidence (*PI*) is 42.45 per 1000 people (95% CI, 41.72--43.17). This result is comparable with 43.5% attack rate found in [@pone.0032203-Germann1]. It is noted that the trigger thresholds {0.02%, 0.25%, 1.5%, 5%} are reached at *day* {7, 13, 17, 20} respectively. {#pone-0032203-g003} Impact of Workforce Shift {#s3c} ------------------------- As shown in [Figure 4](#pone-0032203-g004){ref-type="fig"}, the attack rates under workforce shift are in range from 36.51% to 44.21%, a 0.59% to 17.90% reduction compared to the baseline. The lowest attack rate takes place when the 10-week workforce shift is triggered at 0.02%. Consistent with the observation in school closure\'s results [@pone.0032203-Zhang1], the difference of attack rates at different thresholds but the same duration declines when the threshold increases. But the magnitude of the difference is larger for workforce shift compared to that for school closure. An extra 8.58% of the overall population can be saved from infections by choosing the appropriate trigger threshold for 2-week workforce shift, in comparison to 2.33% for 2-week school closure [@pone.0032203-Zhang1]. {#pone-0032203-g004} [Figure 5](#pone-0032203-g005){ref-type="fig"} shows that workforce shift has the remarkable impact of suppressing the peak incidence of influenza epidemic. The peak incidences under workforce shift range from 29.87 to 42.27 per 1000 people, a 0.04% to 29.63% reduction compared to the baseline. The lowest peak incidence occurs when the 2-week workforce shift is triggered at 1.5%. It is noted that 4 weeks are sufficiently long for reducing the peak incidence as no additional reduction is gained by extending the intervention. {#pone-0032203-g005} [Figure 6](#pone-0032203-g006){ref-type="fig"} shows that workforce shift has a mixed impact on peak day. Consistent with peak day results for school closure, varying duration makes no effect on peak day; and trigger threshold is the dominant factor deciding peak day. When trigger threshold rises from 0.02% to 5%, a consistent decline of peak days is observed. It could be explained that when workforce shift is implemented at a higher threshold, a larger number of the population has been infected and more potential transmissions will be blocked. Therefore, it sooner reaches the cutoff point at which the disease is unable to sustain the growth trend of incidences, so the peak would occur earlier. On the other hand, when workforce shift is implemented at a lower threshold, there are fewer infectious cases within the population and the amount of susceptible contacts left is still tolerable to maintain the chain of infections. Therefore, the daily incidence could be still growing but at a lower pace, consequently leading to a later peak day. [Figure 7](#pone-0032203-g007){ref-type="fig"} shows divergent impact of workforce shift on peak day. 6-week workforce shift triggered at 5% advances the peak incidence by 1 day compared to the baseline; on the other hand, 6-week workforce shift triggered at 0.02% reaches the peak incidence 1 day later than the baseline. {#pone-0032203-g006} {#pone-0032203-g007} Impact of Combined Workforce Shift and School Closure {#s3d} ----------------------------------------------------- We then examine the combined intervention of workforce shift together with all-school closure. We are interested in the effectiveness of the combined intervention as well as the impact of the temporal sequence of individual interventions in a combination. [Figure 8 A--E](#pone-0032203-g008){ref-type="fig"} show that the lowest attack rate (*AR*) under the combined intervention is 31.17%, achieved when workforce shift and school closure are both triggered at 0.25% and lasted for 10 weeks. In the single interventions, the lowest *AR* is 40.42% for all-school closure and 36.51% for workforce shift, both happen at 10-week duration and 0.02% trigger threshold. 8.01% of population can be further saved from the infection by applying the combined intervention compared to the single interventions. {#pone-0032203-g008} [Figure 8 F--J](#pone-0032203-g008){ref-type="fig"} show that the lowest peak incidence (*PI*) occurs when 10-week workforce shift and school closure are triggered at 5% and 0.02% respectively. Compared with the lowest *PI* from single interventions (30.75 from school closure and 29.87 from workforce shift), the combined intervention is able to further reduce *PI* to 14.27. [Figure 8 K--O](#pone-0032203-g008){ref-type="fig"} show that the combined intervention can delay the peak day (*PD*) by 14 days compared to the baseline. It is much longer than *PD* delay in individual interventions, i.e. 5-day delay by school closure and 2-day delay by workforce shift. In the followings, we summarize our results in an attempt to answer the three questions asked in the earlier section: ### (a) Do combined interventions always outperform single interventions? {#s3d1} It is commonly believed that combined interventions will outperform single interventions. But we notice some cases in which combined interventions lead to higher attack rates than single interventions at the same trigger threshold and duration. The worst case is observed when the 4-week workforce shift and school closure are both triggered at 0.02%. If we apply only workforce shift at 0.02% threshold with a 4-week duration (Scenario A -- single intervention), the AR is 38.25%; on the other hand, AR from the combined intervention (Scenario B -- Combined intervention) is 43.12%, which is 4.87% higher. [Figure 9](#pone-0032203-g009){ref-type="fig"} further describes what happens in *Scenarios A* and *B*. On *day* 7, the trigger threshold (*t* = 0.02%) is reached and the epidemic curve of the combined intervention grows much slower than the single intervention because more contacts have been removed and chance of infection is lower. On *day* 35, the interventions in both scenarios end and the removed contacts are restored. Because the growth of infected cases is much slower in *Scenario B*, there is more susceptible left in the population. Specifically, on *day* 35, 49.65% and 85.88% of population are susceptible in *Scenarios A* and *B* respectively. This nearly doubled size of susceptible population allows more disease-causing contacts and higher chance of infection in *Scenario B* compared to those in *Scenario A*, leading to the divergent developments of the epidemic after *day* 35 -- the incidence continues to decline and gradually fades out in *Scenario A*; and oppositely in *Scenario B*, the incidence number grows exponentially until *day* 40 and a large number of infections take place after the intervention. {#pone-0032203-g009} It is observed that 11 out of 16 combined scenarios of 2-week intervention underperform 2-week single interventions; 7 of 16 scenarios of 4-week interventions and 1 out of 16 scenarios in 6-week interventions lead to similar observation. Apparently combined interventions with a longer duration (\> = 6 weeks) are less prone to underperform, meaning that combined interventions have to be maintained long enough to prevent the rapid spread of influenza after the intervention period. ### b) How do trigger and duration affect the effectiveness of combined interventions? {#s3d2} The performance of combined interventions can be affected by both trigger and duration. When the duration increases, AR and PI decline consistently. When trigger threshold rises, AR and PI drop if the duration is shorter than d weeks (d = 8 for AR; d = 4 for PI); if the duration is longer than d weeks, AR and PI increase instead. In [Figure 8 E and G](#pone-0032203-g008){ref-type="fig"}, convex curves clearly show the existence of the above trends. For the peak incidence time, the PD drops when the triggers rises with d\> = 4weeks. It also shows that a longer duration of intervention (\>4 weeks) does not bring in any further delay of the peak incidence time. ### c) Does the implementation sequence in a combined intervention make a difference in its effectiveness? {#s3d3} The temporal implementation sequence of individual interventions within the combined strategy may also affect the outcome of intervention. The maximal differences of the attack rates among sixteen threshold combinations are {6.13%, 8.24%, 3.47%, 3.21%, 2.59%} with {2, 4, 6, 8, 10}-week durations respectively. When duration is less than or equal to 6 weeks, the performance of the synchronized interventions (two individual interventions start from the same threshold) improves when the trigger rises. With longer control durations, the trend is not retained anymore. Comparing to the asynchronized combinations (individual interventions start at different thresholds) with the same duration, the relative performance of synchronized interventions turns from "underperformance" to "outperformance" when their triggers rise from 0.025% to 5% subject to the condition that the duration is within 8 weeks. When the duration is longer than 8 weeks, synchronized interventions underperform in most of the scenarios and hence it is wise to start them at different thresholds in the implementation. For asynchronized combinations, the sequential order of implementing single interventions can affect the *AR* as well. We term two combined strategies with swapped trigger thresholds of the two individual interventions as a pair of symmetric strategies. The maximal differences in attack rates between a pair of symmetric strategies are {2.13%, 1.31%, 1.55%, 2.73%, 1.66%} for {2, 4, 6, 8, 10}-week durations respectively. It is observed that school closure should be implemented later when duration is less than 4 weeks; and workforce shift should start later when duration is longer than 4 weeks. Sensitivity Test on Values of R~0~ {#s3e} ---------------------------------- The results of temporal effects in the combined interventions of school closure and workforce shift are based on *R* ~0~ = 1.9. To examine if our conclusions hold for other *R* ~0~ values, we tested on different cases where *R* ~0~ = 1.5 and 2.3. Similar to [Figure 9](#pone-0032203-g009){ref-type="fig"}, [Figures 10](#pone-0032203-g010){ref-type="fig"} and [11](#pone-0032203-g011){ref-type="fig"} show the effectiveness of the combined interventions at different pairs of thresholds and durations for *R* ~0~ = 1.5 and 2.3 respectively. {#pone-0032203-g010} {#pone-0032203-g011} The results are consistent with our findings based on *R* ~0~ = 1.9. Specifically, the worst combination happens when both school closure and workforce shift are implemented at 0.02% for 2 weeks. It yields 36.61% attack rate, 25.71 peak incidence (per 1000 people) on day 28 for *R* ~0~ = 1.5; 48.52% attack rate, 53.88 peak incidence (per 1000 people) on day 28 for *R* ~0~ = 2.3. The majority of single interventions, either school closure or workforce shift, show significant impact, except for 2-week school closure or workplace shift at 0.02% threshold. In [Figure 10](#pone-0032203-g010){ref-type="fig"} & [11](#pone-0032203-g011){ref-type="fig"}, we also can observe the significant impact by adjusting temporal settings of the combined interventions. When *R* ~0~ = 1.5, attach rate ranges from 36.90% down to 22.97% (37.8% reduction); peak incidence (per 1000 people) ranges from 27.20 to 6.12 (77.5% reduction); and peak day varies from 28 days to 76 days (171.4% increase). When *R* ~0~ = 2.3, attach rate is in range from 48.67% down to 37.21% (23.5% reduction), peak incidence from 55.43 down to 27.73 (50.0% reduction), and peak day from 22 days to 31 days (40.9% increase). The observations suggest stronger impact of temporal factors for a lower value of *R* ~0~. For asynchronized combinations, the maximal differences in attack rates between a pair of symmetric strategies are {2.88%, 2.26%, 4.03%, 4.68%, 4.86%} for {2, 4, 6, 8, 10}-week durations where *R* ~0~ = 1.5, and {3.49%, 1.69%, 2.14%, 0.85%, 0.9%} where *R* ~0~ = 2.3. Again the observation is that when *R* ~0~ is lower, switching the order in a combined intervention could make more significant difference. It is also interesting that the difference is particularly significant when duration is short (2 weeks) for all the three values of *R* ~0~. Study on Weekend Effect {#s3f} ----------------------- So far we have been adopting only the contact patterns during weekdays in our study. In urban life, however, social contact patterns may be significantly different during weekends. For example, the contacts in shopping malls may increase while contacts within workplace/schools may decrease. Such changes are terms as weekend effect in the context, which recurs for 2 days (Saturday and Sunday) in every week. We conduct simulation to evaluate the impact of weekend effect. Specifically, we assume that school contacts are reduced by 50% and workplace contacts by 70% during the weekends compared to those during weekdays, and meanwhile shopping mall contacts are increased by 35.79% according to our survey data. Numerical experiments are then repeated at *R~0~* = 1.9 with the same configurations as listed in [Table 1](#pone-0032203-t001){ref-type="table"} for evaluating combined workforce shift and school closure. Our simulations are assumed to start on Monday; and when workforce shift intervention or school closure intervention is exercised, the population involved in the intervention will follow intervention arrangement regardless of weekday or weekend. [Figure 12](#pone-0032203-g012){ref-type="fig"} shows the spread dynamic after introducing weekend effect. Compared to the experiments shown in [Figure 8](#pone-0032203-g008){ref-type="fig"} without considering weekend effect, there exist similar patterns while varying thresholds and durations of the combined interventions. Meanwhile, however, we can observe the impact of weekend effect: the baseline attack rate under the weekend effect falls by 3.21% compared to the original one; peak incidence is only of a 0.18% difference; and peak day postpones by 1 day. Such results may be interpreted: the total removal of contacts from schools and workplaces is more than the contacts increased in shopping malls in the weekends. {#pone-0032203-g012} When comparing the individual scenarios of the combined interventions, we find that the impact of weekend effect diminishes gradually with the increase in duration of interventions. This may be due to the enforcement on the control effect by the interventions from weekdays to weekends, i.e., weekend effect may be overridden by the control. For example, a part of weekend effect -- 50% removal of school contacts in weekends may be overridden by school closure intervention and the 100% removal would happen during the whole period of school control. Therefore, the shorter the inventions are, the more notable the weekend effect is. The most notable decline in attack rate under the weekend effect is spotted in the 2-week intervention scenarios with the average reduction of 3.44% compared to the baseline of weekend effect. Discussion {#s4} ========== Using an individual-based simulation model based on the social community structure of Singapore, we investigate the effectiveness of workforce shift and its combination with school closure as means to mitigate the spread of influenza. Specifically, the impacts of interventions have been investigated through evaluating the total attack rate and daily incidence as well as the delay of peak incidence time quantitatively. Both workforce shift and school closure are social distancing measures that aim to reduce disease-causing contacts between individuals so as to reduce consequent secondary infections. As the production of vaccine and stockpiling of anti-viral drugs usually take considerable time, the shortage of pharmaceuticals has ever been the challenge in the preparedness planning for pandemic influenza and might not be ready at the time of influenza outbreak. Our simulation results show that both workforce shift and school closure are able to lower attack rate and daily incidence as well as delaying the epidemic in most intervention scenarios. Such social distancing through enforcement from administration is necessary to mitigate the diffusion of influenza virus among the communities, especially when a large number of asymptomatic cases exist. Our experiments provide guidance on choices of trigger threshold and length of duration for implementing school closure, workforce shift and their combination intervention measures. These results will be relevant to future contingency plan for influenza pandemic, which is estimated to be more pathogenic and might have higher case fatality rates than that shown in 2009 H1N1 pandemic flu [@pone.0032203-Halder1]. We find that the durations of 8 weeks and 6 weeks are sufficiently long for workforce shift and school closure respectively. Short interventions should be implemented after a longer delay since outbreak; in contrast, long interventions should start as early as reasonable. The cutoff values between long and short duration are 6-week for school closure and 4-week for workforce shift, if lowering the attack rate is the priority. Comparing the effect of workforce shift with school closure, we observe that workforce shift is generally more impactful. One of the main reasons is because of the difference in the number of people that can be affected by school and workplace interventions. In our contact network, school closure removes the school contacts from ∼53,000 people; workforce shift affects ∼148,000 people at any time during the intervention. So there is around 2.8 times more population controlled in the workforce shift. Furthermore, we examine combined interventions as temporal combinations of single policies. We fix the duration shared by single policies in combination for simplicity; but allow different trigger thresholds so that the two policies may be implemented either one after another or at the same time. Our results show that the combined interventions do not always outperform the single interventions while varying trigger threshold and duration. It is shown that short closures (less than 6 weeks) are more prone to underperformance compared to that of the workforce shift only. Secondly, we observe that switching the order of single policies in combination can make a difference in the effect of intervention. Planning multiple interventions in the appropriate order is able to strengthen the mitigation to the spread of epidemic without significant additional cost. Among all choices of combined interventions examined, the near-optimal policy happens when all workforce shift and school closure are both implemented at the 0.25% trigger threshold and lasted for 10 weeks (31.17% attack rate; peak incidence of 17.42 per 1,000 people at *day* 33). Enforcing a social distancing policy always associates with considerable cost, on both economic and social aspects. For example, the major cost of school closure comes from absenteeism of working parents who have to stay home to take care of their children. A UK study [@pone.0032203-Sadique1] estimated 16% of UK workforce as the main carers of dependent children and likely to be absent due to school closure. This percentage could further climb to 30% if counting healthcare workers only, meaning more absenteeism could happen in public healthcare system which has been already stressful during an epidemic. Besides, there are also problems about social justice, ethical issues etc as the social consequence of school closure [@pone.0032203-Cauchemez1]. On the other hand, workplace distancing measures like workforce closure might lead to an abrupt shortage of manpower, lower productivity and inevitable economic loss. As an alternative to workplace closure and uncontrolled absenteeism, workforce shift might be an option for disease containments. Nowadays, accessible infrastructure for telecommunication is widely available at many workplaces and homes. Tele-working has become feasible and can be equipped in advance along with the planned workforce shift. It makes workforce shift with longer duration more acceptable. The planned workforce shift would help companies and other institutions to minimize the impact of mass absenteeism and sustain the usual business and production as much as possible. In lack of information about the compliance rate of school closure, we have assumed a 100% compliance rate in all relevant intervention scenarios in this study. However, in a real-world school closure, the student compliance rate for social distancing may be at a lower value. The compliance rate of students for social distancing would be increased when implementing workforce shift together with school closure. We ignore the variation of compliance rate for not complicating the analysis on the combined interventions. A higher compliance rate is definitely preferred in real-world interventions and needs the coordination among education agency, health agency and communities to achieve. Considering the network dynamics in weekends, we study the disease spread under the weekend effect. As schools and many workplaces are closed during the weekends, the contacts between schoolmates or between colleagues may be partially removed (Schoolmates or colleagues may hang out together during the off days) [@pone.0032203-Cauchemez2] but the shopping mall contacts may increase. In our experiments of the weekend dynamics, we find that the weekend effect does not bring significant variation to the baseline epidemic curve compared to that of the original setting without considering the weekend effect. It is worth noting however that due to the lack of real-world data, we have made assumptions on the reduction degrees of school/workplace contacts during weekends. In our future work, we will keep collecting real-world dynamic contact parameters of Singapore, and further evaluate the temporal effect of social distancing in dynamic settings. The evaluation of intervention scenarios in this study is based on Singapore\'s social structure. The results presented here should be interpreted with the following caveats in mind. First, the Singapore community is not a closed system. There are millions of visitors arriving in Singapore (e.g., a peak of 10 million visitors in 2007). Singapore has a population size of around 4.9 million. The large volumes of visitors flowing into the country implicitly indicate that the influence of imported cases should be considered when planning intervention strategies. However, the influence of visitors is not considered in our research as we focus on investigating and comparing the effectiveness of the individual and combined intervention scenarios. We note that it is desirable to further analyze the influence of visitors on the disease spread in the community for combating future pandemic. Further, Singapore is a highly urbanized city and its population density is among the top in the world, which will definitely lead to high contact numbers in different community structures. The best intervention scenario in terms of control timing may vary when the social structure is drastically different from the one studied in this paper, as the heterogeneity of social structure is a significant factor affecting disease spread and consequently affecting mitigation planning strategies as well. Conclusion {#s4a} ---------- Though the combined intervention strategy outperforms its individual strategies in most cases, it is found that combined intervention strategies underperform its individual intervention strategies under inappropriate timing configurations. Our results suggest that trigger threshold and duration are critical to the effectiveness of the combined intervention, specifically, for lowering attack rate and daily incidence as well as having a longer peak delay. Our studies also show that the implementation order of individual interventions in the combination could affect the effectiveness of combined interventions as well. Exploring correct timing configuration is therefore crucial to achieving optimal or near optimal effect of mitigation for influenza epidemic. Such an evaluation is recommended for assisting policy makers in influenza preparedness planning with their specific situation and constraints. **Competing Interests:**The authors have declared that no competing interests exist. **Funding:**This work was supported in part by Biomedical Research Council of Singapore A-STAR (<http://www.a-star.edu.sg/AboutASTAR/BiomedicalResearchCouncil/tabid/64/Default.aspx>) under Grant 06/1/21/19/457. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [^1]: Conceived and designed the experiments: XF SM TZ. Performed the experiments: TZ XF. Analyzed the data: TZ XF. Contributed reagents/materials/analysis tools: TZ XF SM GX LW CKK ML GKKL TH. Wrote the paper: XF TZ GX LW.

Introduction ============ Nasopharyngeal cavity is a somewhat cuboid space, which communicates anteriorly with the nasal cavities through the choanae and inferiorly with the oropharynx. Sphenoid body limits it superiorly, the first two vertebrae posteriorly, and the soft palate inferiorly\[[@B1]\]. To treat nasopharyngeal cancer, a combination of radiotherapy and chemotherapy are used. In radiotherapy, cancer can be removed or prevented from spreading using photons (in the form of external radiotherapy or brachytherapy). Typically, in nasopharyngeal radiotherapy, 18 Gy in 6 fractions are prescribed to the tumor through brachytherapy and 46-60 Gy by external irradiation\[[@B2]\]. Regarding the source high dose rate and tumor location, accuracy of calculating TPS is very important. There are three main ways to assess the accuracy of treatment planning systems in radiotherapy: 1. Practical and laboratory procedures using radiation detectors 2. Computational methods, using formulas, tables and radiation laws 3. Computer simulation methods\[[@B3]\]. For some reason, using laboratory and practical methods is limited: 1. In the field of internal dosimetry in radiotherapy, using practical and laboratory methods to determine the dose is often impossible. 2. Using this method can result in high measurement error, which is due to issues related to radiation detection devices. 3. Reaching the final result of measurement by some means of radiation measurement may take a long time. 4. Another limitation is its cost. Hence, using computer simulations can be considered as a powerful tool with high capabilities to investigate the dosimetry inside and outside the body\[[@B4]\]. One of the computer methods that its use is growing in dosimetric calculations in radiotherapy is Monte Carlo method. The method is based on the statistical processes like nucleon interaction probability during the transversal of a path in the substance\[[@B5]\]. EGSnrc code is a Monte Carlo multipurpose simulation package of photon and electron collisions in the arbitrary geometry. Particle energy in code ranges from 1 Kev to 10 Gev. Of course this energy range depends on the target substance atomic number\[[@B6]\]. EGSnrc is the best code for calculations related to medical physics and calculations of absorbed dose in radiotherapy as compared to other Monte Carlo codes, it is more convenient to use complex geometries and actual patient anatomy can be converted into a Voxel-Based Phantom\[[@B6]\]. In EGSnrc creating CT phantoms from CT data is done using ctcreate independent code. Considering high dose rate and tumor location, calculation accuracy of treatment planning system is of particular importance. In this study, Iridium source (Ir -192) will move in applicator and stand in several places to deliver appropriate dose to the treatment volume. The final goal is to calculate the dose delivered to the treatment volume due to source dwelling in different places using DOSXYZnrc. These calculations will lead to plotting isodose diagrams and dose-volume histograms and comparing them with treatment planning systems. Materials and Methods ===================== To create virtual phantom, 51 CT images of a 42-year-old man in DICOM format and dimensions 512×512 with Rotterdam applicator were used. The prescribed dose was 3 Gy. This operation was performed in radiotherapy ward of Mahdieh M.R.I Center using Flexitron produced by Microselectron®. CT images were converted to virtual phantom using ctcreate. Virtual phantom dimensions were 33×33×30 and each voxel size was determined as 0.2×0.2×0.2 cm^3^. Simulation was carried out using DOSXYZnrc which is a part of BEAM project. ECUT and PCUT were selected 0.513MeV and 0.01MeV, respectively. In simulating the source mHDR-v2r, source number 6 was used (rectangular isotropic parallelepiped source). Source geometry was estimated using a cube with dimensions 0.1 cm and Ir-192 Microselectron spectra. (Result of reliability of this estimation is presented in section 3.1). In afterloaders, shielded source is connected to the end of a cable. After determining CTV and prescribed dose by oncologist, TPS calculates the source dwell position and dwell time for delivering prescribed dose to CTV. Each dwell position has its particular dwell time. In this study, source had 56 dwell positions in applicator, and voxels absorbed doses of each dwell position and dwell time were gathered and eventually a 3D dose profile (3D dose file) was obtained. In addition to CT phantom, a water phantom was created in DOSXYZnrc similar to dimensions and size of the virtual phantom, and treatment plan was performed on it. This phantom does not contain applicator and heterogeneity, and it is only created to compare Oncentra™ TPS and EGSnrc Monte Carlo Code in water phantom. Dosxyz_Show Program was used to display the geometry and dose profiles in EGSnrc. By extracting absorbed doses of CTV voxels, dose volume histogram (DVH) was calculated and plotted. As previously mentioned, voxels size contains cubes with 2 mm side. In calculating dose volume histogram, voxels that more than half of which are in CTV were used in DVH calculations. Results ======= Results of Source 6 DOSXYZnrc Validation ---------------------------------------- Validation result includes comparison parameters of Ir-192 source mHDR-v2 model and source 6 DOSXYZnrc. It must be said that this validation is only done to compare two source parameters to explain the differences in dose volume histogram and dose profile of TPS and Monte Carlo Code. The aim of this study was not determining the accuracy of these parameters though. Results of comparison of dose rate constant Ir-192 source and source 6 DOSXYZnrc are presented in [Table 1](#T1){ref-type="table"}. ###### Dose Rate Constant Comparison -------------------------------------------------- -------------------------------------- Ir-192  Dose Rate Constant Model  mHDR-v2r \[7\] Λ = 1.108 ± 13% *cGy.h* ^-1^.*U*^-1^ Source 6 DOSXYZnrc Dose Rate Constant Λ = 1.05 ± 21% *cGy.h* ^-1^.*U*^-1^ -------------------------------------------------- -------------------------------------- Data of radial dose function for Ir-192 model mHDR-v2 was taken from Papagianis et al.\[[@B7]\], and was compared with source 6 DOSXYZnrc radial dose function. Results indicate a good agreement of radial dose functions for these two sources in low distance as given in [Figure 1](#F1){ref-type="fig"}. {#F1} Due to small volume of treatment area (2.6cm^3^), simulation error of Ir-192 source version mHDR-v2 with source 6 DOSXYZnrc with 0.1 cm side in treatment area is less than 3%. Comparison of Dose Volume Histogram Functions --------------------------------------------- [Figure 2](#F2){ref-type="fig"} displays a comparison of cumulative dose volume histograms of Monte Carlo method and TPS. As seen below, for a specific volume ratio of CTV, TPS estimates more doses than MC method. Also the diagram calculated by Monte Carlo drops in lower doses than TPS. This sharp drop means that difference in voxels absorbed dose in Monte Carlo method is higher. {#F2} [Figure 3](#F3){ref-type="fig"} shows the comparison between histogram of differential dose volume between MC and TPS. These diagrams are obtained by deriving cumulative dose volume histogram and normalizing the numbers. {#F3} Dose Profiles ------------- In [figures 4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}, dose profile comparison is done in two 0.25 Gy and 1Gy doses in two sets of 27 and 37 slices. It can be seen that in water phantom, the results of comparing doses shows good agreement. So, dose difference in CT phantom is due to heterogeneities of tissue and applicator attenuation. {#F4} {#F5} Discussion ========== The purpose of this study is to evaluate the dosimetric accuracy for Oncentra™ treatment planning system. In nasopharynx brachytherapy, treatment area consists of three matters, soft tissue, bone and air. TPS, in dose calculations, considers all the materials as water. Here, we made patient-specific phantom using patient CT images and we involved inhomogeneity. As can be seen in [figures 4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}, considering actual heterogeneities, a difference occurs between absorbed doses in voxels. In areas that voxel constituent is air, the difference reaches its maximum value, because the attenuation coefficient and density of air and water are very different. Due to the fact that simulations results of water phantom in DOSXYZnrc and TPS have shown good agreement, dose differences observed in voxels of CT phantom can all be attributed to heterogeneities and applicator attenuation.  DVHs suggest that in the presence of tissue heterogeneity, greater heterogeneity is observed in voxels absorbed dose. The difference is due to the fact that in the presence of heterogeneity and ir-192 low energy photons, the photoelectric effect dominates and bones absorbed dose is more than air and soft tissue. Cumulative dose-volume histograms showed a sharper drop in MC method. Although, due to the rarity of nasopharyngeal cancer, no study is available on nasopharyngeal brachytherapy QA, by the agreement between TPS and MC simulation in water phantom, we can ensure the accuracy of our calculations in CT phantom. Today, most treatment planning systems use TG-43 protocol. This protocol may results in errors such as neglecting tissue heterogeneity, scattered radiation as well as applicator attenuation. Due to these errors, AAPM emphasized departure from TG-43 protocol and approaching new brachytherapy protocol TG-186 in which patient-specific phantom is used and heterogeneities are affected in dosimetry. Author would like to thank Dr. A. Sedighi and the staff of Mahdieh M.R.I Center in Hamedan, Iran for sincere cooperation to conduct this study. This work is extracted from M.S. thesis NO.900329. **Conflict of Interest:**None

Background ========== Cardiac involvement is common in hereditary transthyretin -- related amyloidosis (ATTR), but there is a significant phenotypic heterogeneity depending on the mutation. Patients and methods ==================== We evaluated forty consecutive ATTR patients with Glu89Gln mutation, focusing on cardiac involvement - 18 male, 22 female at a mean age of 57.6±6,7 years. A clinical examination, 12--channel ECG, conventional 2D, Doppler and tissue Doppler echocardiography were performed. The patients were followed for 36 months in the range from 2 to 78 months. Results ======= Median age of symptoms development was 52, 3±6, 4 years. Cardiac onset was found in 5 (12,5%) patients. Cardiomyopathy and peripheral polyneuropathy were evident at diagnosis in all patients. Echocardiography revealed a significant increase in wall thickness of both left and right ventricles (septum -- 18,6±3,4 mm; posterior wall -- 17,5±2,5 mm; RV free wall -- 8,4±2,0 mm). Varying degrees of LV diastolic dysfunction were found -- Grade 1 in 11 (27,5%) patients, Grade 2 in 12 (30%) and Grade 3 in 17 (42,5%) patients. A reduced LV ejection fraction was found in 9 (22,5%) patients. A common finding were significantly reduced mitral annulаr systolic velocities (s´septum-5,4±2,0 cm/s, s lat.-5,7±1,9 cm/s), registered in all the evaluated patients, pointing to an impaired LV longitudinal systolic function. The systolic myocardial velocities of the tricuspid annulus and TAPSE values were reduced respectively 6,9±2,1cm/s and 12,8±3 mm in 14 of the patients (35%). Pericardial effusion was found in 13 (32,5%) patients. Pathological ECG was present in 35(87,5%) of the evaluated patients. Atrial fibrillation was registered in 4 (10%) patients, A-V block first degree in 8 (20%), low voltage in 15 (37,5%), left bundle branch block in 3 (7,5%), left anterior fascicular block in 9 (22,5%), pathological Q wave in 14 (35%), right bundle branch block in 2 (5%), and pace-maker rhythm in 2 (5%). Rhythm and conduction disturbances on ECG were found in 24 patients (60%). The following events occurred during the follow-up period: two deaths (5,4%) (one patient due to ischemic stroke; and another due to heart failure). Two other patients suffered from ischemic strokes. 24-hour Holter ECG revealed short periods of atrial fibrillation and an oral anticoagulant was initiated. A sinus pause \> 3 s was observed in one of the patients and a permanent pace-maker was implanted. Four new cases (10%) with symptomatic heart failure, requiring diuretic treatment were observed. In 15 patients a worsening of the symptoms from the peripheral neuropathy were found. Conclusion ========== Our study confirms that ATTR associated with the Glu89Gln mutation has a mixed phenotype -- neurological and cardiac and an unfavorable prognosis. Our findings imply that patients and carriers of Glu89Gln require close multidisciplinary (both cardiological and neurological) follow-up in order to initiate treatment in time.

1. Introduction {#sec1} =============== Understanding the evolutionary relationships between groups of organisms has become increasingly reliant on phylogenetic analysis. Phylogenies are usually presented as tree diagrams, known as phylogenetic trees. These trees are constructed from genetic similarities and differences between different organisms. Comparative sequence analysis is a useful method by which one can identify gene, infer the function of a gene\'s product, and identify novel functional elements. By comparing several sequences along their entire length, researchers can find conserved residues that are likely preserved by natural selection. Reconstructing ancestral sequences can reveal the timing and directionality of mutations. These comparative analyses rely on the phylogenetic tree construct. A reading frame is a set of consecutive, nonoverlapping triplets of three consecutive nucleotides. A codon is a triplet equating to an amino acid or stop signal during translation. An open reading frame (ORF) is the section of reading frame containing no stop codons. A protein cannot be made if RNA transcription ceases prior to reaching the stop codon. Therefore, to ensure that the stop codon is translated at the correct position, the transcription termination pause site is located after the ORF. The ORFs can identify translated regions in DNA sequences. Long ORFs indicate candidate protein coding regions in a DNA sequence. ORFs also have been utilized to classify various virus families \[[@B1]--[@B3]\], including members of *Norovirus* \[[@B3], [@B4]\]. The Open Reading Frame Finder (ORF Finder) \[[@B5]\] is a graphical analysis tool that searches for open reading frames in DNA sequences. The ORF Investigator \[[@B6]\] program provides information on the coding and noncoding sequences and performs pairwise alignment of different DNA regions. This tool efficiently identifies ORFs and converts them to amino acid codes, declaring their respective positions in the sequence. Pairwise alignment also detects mutations, including single-nucleotide polymorphisms between sequences. StarORF \[[@B7]\] facilitates identification of the protein(s) encoded within a DNA sequence. First, the DNA sequence is transcribed into RNA, and all potential ORFs are identified. These ORFs are encoded within each of the six translation frames (3 in the forward direction and 3 in the reverse direction), so that users can identify the translation frame yielding the longest protein coding sequence. Several biological organizations have implemented bioinformatics tools on websites. The National Center for Biotechnology Information (NCBI) \[[@B8]\] provides many tools for comparing database-stored nucleotide or protein sequences, including the well-known BLAST algorithms. NCBI also provides several databases, such as GenBank and SNP, in which biologists can seek homology or specific functions. The European Molecular Biology Laboratory (EMBL) \[[@B9]\] provides freely available data and online bioinformatics tools to all facets of the scientific community. These data and tools are indispensable in medical and biology studies. Most of these services are accessed via the Internet and utilized online. Cloud computing is a recently developed concept that delivers computing resources, either hardware or software, over the Internet. Many types of cloud computing have been proposed, such as infrastructure as a service (IaaS), platform as a service (PaaS), software as a service (SaaS), network as a service (NaaS), and storage as a service (STaaS). Most of these services rely on virtualization technology---the creation of virtual hardware platforms, operating systems, storage devices, and network resources. Cloud computing is welcomed for its user friendliness, virtualization, Internet-centric focus, resource variety, automatic adaptation, scalability, resource optimization, pay-per-use, service SLAs (Service-Level Agreements), and infrastructure SLAs \[[@B10]\]. Many cloud computing vendors distribute these resources on demand from large resource pools installed in data centers. Amazon EC2 \[[@B11]\] supplies an infrastructure service, while Google App Engine \[[@B12]\] and Microsoft\'s Azure Services Platform \[[@B13]\] supply platform services. In academia, numerous cloud computing projects are under construction or fully operational \[[@B14]--[@B17]\]. Cloud computing is essentially a distribution system that enables parallel computing. Hadoop \[[@B18]\] is an open-source software framework that supports data-intensive distributed computation. Under Hadoop, applications can be implemented on large clusters of commodity computers. The Hadoop cluster includes a single master and multiple slave nodes. The master node assigns jobs to slave nodes, which complete the assigned tasks. Hadoop provides the MapReduce programming model for parallel processing of large datasets. The computational task is divided into many small tasks, each of which may be executed or reexecuted on a compute node in the Hadoop cluster. MapReduce also provides a distributed file system, the Hadoop Distributed File System (HDFS), that stores the data on compute nodes \[[@B19]\], enabling a very high aggregate bandwidth across the cluster. Both map/reduce and the distributed file system are robust against failure. Several sequence analysis tools have been redeveloped as cloud tools based on the Hadoop architecture, such as CloudBlast \[[@B20]\] and CrossBow \[[@B21]\]. Therefore, standard online tools can be ported to the cloud architecture. Such importing of preexisting tools constitutes the main goal of bioinformatics as a service (BaaS). In this paper, we develop a high-availability, large-scale ORF phylogenetic analysis cloud service based on virtualization technology and Hadoop. This service provides phylogenetic analyses from ORFs based on Hadoop clusters to support multiple requests. The essence of the cloud computing environment is virtualization. The physical computing power is regarded as a user-pays utility that users can request as desired. The utility is also known as a virtual machine. Each node in a Hadoop cluster is a virtual machine. Users can upload their sequence data or files through the master node (web portal) and then submit a job. The job is assigned to the slave node containing the uploaded data, and the slave node completes the job. Since ORF comparisons have unambiguously established the homology of *Norovirus* \[[@B22]\], we here adopt *Norovirus* as a case study. The results show that the proposed cloud-based analysis tool, by virtue of virtualization technology and Hadoop framework, can readily facilitate BaaS. The proposed cloud-based ORF phylogenetic tool is available at <http://bioinfo.cs.pu.edu.tw/CloudORF/>. 2. Methods {#sec2} ========== In this paper, we propose a cloud-based ORF phylogenetic analysis service combining Hadoop framework, virtualization technology, phylogenetic tree tool, and diversity analysis. As mentioned previously, the cloud platform is constructed from virtualization and Hadoop framework. Hadoop is performed on the VMs created by virtualization technology such as Kernel-based Virtual Machine (KVM). Hadoop performs the phylogenetic analysis in a distributed computing manner. The underlying architecture ensures elasticity, scalability, and availability of the proposed cloud-based service. 2.1. Phylogenetic Analysis {#sec2.1} -------------------------- The proposed cloud service integrates the ORF finding process, phylogenetic tree contractions, and ORF diversity analysis to generate a complete phylogenetic analysis. The procedure of the analysis is outlined below and shown in [Figure 1](#fig1){ref-type="fig"}. *Step  1: Detecting Open Reading Frames*. Functional ORFs are extracted from sequences. Although many ORFs exist in a protein sequence, most are insignificant. The ORF finder locates all open reading frames of a specified minimum size in a sequence. In this study, the ORF Finder commonly used on the NCBI tools website was adopted. This tool identifies all open reading frames using the standard or alternative genetic codes. *Step  2: Constructing Phylogenetic Tree Based on Open Reading Frames*. A phylogenetic tree (or evolutionary tree) is a branching (tree) diagram showing the inferred evolutionary relationships between biological species or other entities based on similarities and differences in their physical and/or genetic characteristics. The taxa clustered together in the tree are presumably descended from a common ancestor. Phylogenetic analysis usually aligns whole-length sequences. However, different ORFs might yield different phylogenetic trees. Virus ORF alignments might reveal a common viral ancestor or an ORF that is common to all viruses. Such a discovery would greatly assist viral drug design. The phylogenetic tree is computed using ClustalW \[[@B23]\]. This algorithm builds two phylogenetic trees; one based on full sequences and the other for ORFs only, thereby revealing the variance between the two trees. *Step  3: Diversity Analysis among Open Reading Frames*. Diversity usually depicts the number of different identities in a group. In this paper, diversity demonstrates species variance at a specific position in the protein sequence. Small diversity value at a position implies that protein sequences are very similar at that position. By contrast, a high diversity value denotes low similarity at that position. A frame with high variance also indicates that this frame mutates easily. Such high-variance frames can be used to observe protein structural differences and to aid vaccine development. In this paper, diversity is calculated from the entropy as follows: $$\begin{matrix} {H\left( i \right) = - {\sum_{}{p\left( x_{i} \right)\text{lo}\text{g}_{2}p\left( x_{i} \right),}}\quad x_{i}{\,\,} = \left\{ {G,A,I,V,\ldots} \right\},} \\ \end{matrix}$$ where *H*(*i*) is the value of entropy and *p*(*x* ~*i*~) is the probability of finding a specified amino acid at position *i*. To find the significant position, entropy values under a certain threshold are filtered out. In this study, the threshold was set at 1.4. 2.2. Cloud Platform Based on Virtaulization and Hadoop Framework {#sec2.2} ---------------------------------------------------------------- The cloud platform for proposed phylogenetic analysis tool is constructed on two important technologies: virtualization and the Hadoop framework. Hadoop is a highly scalable and available distributed system. The scalability and availability are guaranteed by HDFS, a self-healing distributed storage system and MapReduce, a specific fault-tolerant distributed processing algorithm \[[@B24]\]. The architecture of a Hadoop cluster is shown in [Figure 2](#fig2){ref-type="fig"}. The Hadoop cluster constitutes a single master and multiple slave nodes. The master node consists of a job tracker, task tracker, name node, and data node. A slave node, or computing node, comprises a data node and a task tracker. The job tracker assigns map/reduce tasks to specific nodes within the cluster, ideally those already containing the data or at least within the same rack. A task-tracker node accepts map, reduce, and shuffle operations from a job-tracker. The map/reduce operation is shown in [Figure 3](#fig3){ref-type="fig"}. HDFS is the primary distribution file system used by the Hadoop framework. Each input file is split into data blocks that are distributed to data nodes. Hadoop also creates multiple replicas of data blocks and distributes them to data nodes throughout a cluster to enable reliable, extremely rapid computations. The name node serves as both a directory namespace manager and a node metadata manager for the HDFS. The HDFS architecture contains a single name node. One desirable characteristics of Hadoop is its high fault tolerance. The HDFS allows the data to spread across hundreds or thousands of nodes or machines, and the tasks are computed on data-holding nodes. Hadoop replicates data, so that if one replica is lost, backup copies exist. When a node fails during computation, Hadoop restarts the halted task on another node containing replicate data. In the Hadoop framework, node failures are detected using the heartbeat mechanism, by which individual task nodes (task trackers) constantly communicate with the job tracker. If a task tracker fails to communicate with the job tracker for a period of time, the job tracker will assume that the task tracker has crashed \[[@B25]\]. The job tracker knows which task trackers (data nodes) contain replicate data, and it issues a restart task. In this paper, the proposed cloud service was implemented by combining Hadoop cluster distribution with a management model. In our cloud server, a submitted job is computed in a data node. Rather than processing parallel data, jobs themselves are parallelized. Therefore, submitted data are distributed to a data node by the HDFS, while the computing process is delivered to the task tracker and copied with the submitted data. Virtualization is a critical component of the cloud computing environment. The physical computing power is essentially a utility that users can purchase as required. The usual goal of virtualization is to improve scalability and overall hardware-resource utilization. Virtualization permits the parallel running of several operating systems on a single physical computer. While a physical computer in the classical sense constitutes a complete and actual machine, a virtual machine (VM) is a completely isolated machine running a guest operating system within the physical computer. To ensure scalability and efficiency, all components---job tracker, task tracker, name node, and data node---in our cloud service operate as virtual machines. [Figure 4](#fig4){ref-type="fig"} shows the VM architecture of our proposed service. 2.3. Cloud-Based ORF Phylogenetic Analysis Service {#sec2.3} -------------------------------------------------- Cloud-based ORF phylogenetic analysis service was developed on a virtualization platform with the Hadoop framework as described above. The procedure of the proposed service is shown in [Figure 5](#fig5){ref-type="fig"}. The master node (name node) and slave node (data node) are the master VM and slave VM, respectively. When a phylogenetic analysis request is submitted, it is saved in a job queue. The master node periodically extracts the jobs from the job queue and assigns them to slave nodes (or mappers), which perform the task. At the completion of all jobs, the reducer collects the results and saves them in the Network File System storage (NFS). A single comparison result of a phylogenetic job is saved in a single file of NFS. As shown in [Figure 5](#fig5){ref-type="fig"}, a data node running in VM2 performs a phylogenetic analysis and a name node runs in VM1. The reducer, running in VM~*n*+2~, collates the results from the data nodes executing the phylogenetic analyses. In this service, the user uploads protein sequences and submits a phylogenetic analysis request on the website portal. All submitted analysis jobs are gathered in the job queue and sequence data are stored in different hosts by HDFS. Phylogenetic analyses are assigned to the data nodes already containing sequence data. The analysis results are sent to both data node and reducer to produce the final result stored in NFS. The user retrieves the final result by logging into the website. The service is implemented as follows. *Step  1: Job Submission.* Users submit their job online through the web portal of the proposed cloud service. Users either enter the comparative DNA/RNA sequences on the web portal or upload a file containing comparative RNA sequences from a web portal. *Step  2: Sequence Translation*. To detect the ORF regions, all input RNA sequences are translated to protein sequences based on the genetic code. The genetic code is the set of rules by which RNA sequence information is translated into proteins. Each codon in an RNA sequence usually represents a single amino acid specified by the corresponding genetic code. The code specifies the amino acid to be added next during protein synthesis. The genetic codes are displayed in [Table 1](#tab1){ref-type="table"}. *Step  3: Phylogenetic Analysis*. This step identifies the functional ORFs, recall that significant ORFs are rare. In our service, the user can provide the length of ORF that he/she regards as meaningful. The service then locates the significant ORFs. An example of ORFs is shown in [Figure 6](#fig6){ref-type="fig"}. In this example, the first ORF (denoted as AB447445_1) extends from positions 3 to 5099 in the sequence AB447445. In this step, two types of phylogenetic trees are built, one using the full sequence length and the other using ORFs only. From the three ORF regions identified in the analysis, three ORF phylogenetic trees are built. These trees are recorded in *ph* format and are then transferred to and stored in the portal. Meanwhile, the diversity value of each position in the sequence is calculated. These values are saved in a file. *Step  4: Report Result*. In this step, the *ph* formatted trees are drawn as three diagrams and displayed on the portal. The user observes these diagrams online or downloads them from the website. Similarly, a bar graph of aggregate diversity appears on the website. 3. Experiment {#sec3} ============= The proposed cloud service for virus analysis was performed on four IBM blade servers. Each server was equipped with two Quad-Core Intel Xeon 2.26 GHz CPUs, 24 GB RAM, and 296 GB hard disk, running under the Ubuntu operating system version 10.4, with 8 virtual machines on each server. Hadoop version 0.2 MapReduce platform was installed on each server. One VM constituted the job tracker and name node; the others are task trackers and data nodes. The job tracker is also the portal of our cloud service. The portal is depicted in [Figure 7](#fig7){ref-type="fig"}. Our current cloud environment permits eight virtual machines. Two of these VMs are name node and data node running the Reducer; the remaining six are responsible for map operation. For the experiment, we randomly produced three datasets, each containing 20 sequences of different lengths (300, 400, and 600 nucleotides). All sequences in each dataset were compared by phylogenetic analysis methods. ClustalW and the proposed service were applied three times, for simulating three ORF phylogenetic analyses. The computation time of the proposed service illustrated in [Figure 8](#fig8){ref-type="fig"} is proportional to the number of mappers. The execution time is considerably reduced when six mappers are used, relative to two mappers. [Figure 9](#fig9){ref-type="fig"} compares the performance between sequential phylogenetic analysis methods such as ClustalW and the proposed service with six mappers, for different sequence lengths. Clearly, the proposed service in the Hadoop framework achieves better performance than standard sequential phylogenetic analysis. 4. Case Study {#sec4} ============= *Norovirus* (NoV) is an important etiological agent of acute gastroenteritis worldwide. It causes diarrhea in all ages, especially in Taiwan. The NoV genome is a single-stranded, positive sense, polyadenylated RNA encoding three open reading frames, ORF1, ORF2, and ORF3 \[[@B26]\]. ORF1 encodes a long polypeptide that is cleaved intracellularly into six proteins by the viral proteinase \[[@B27]\]. These proteins enable NoV to replicate in host cells \[[@B28]\]. ORF2 encodes a viral capsid protein, VP1, while ORF3 encodes a VP2 protein that is regarded as a minor structural component of virus particles \[[@B29]\], apparently responsible for the expression and stabilization of VP1 \[[@B30]\]. Like the majority of RNA viruses, NoV is genetically and antigenically diverse \[[@B31]--[@B33]\]. The virus is tentatively divided into five genogroups and more than 25 genotypes, based on similarities between ORF2 sequences \[[@B33], [@B34]\]. Therefore, the homology of this type of virus may be identified from ORF similarities. Identifying this homology will assist in viral drug and vaccine design. Therefore, NoV was selected as a case study in our experiments. We selected fifteen NoV that have been discovered in Taiwan. These NoV sequences can be downloaded from NCBI. The phylogenetic trees constructed from full length sequences and three ORFs are shown in [Figure 10](#fig10){ref-type="fig"}. Obviously, these trees differ from each other. The tree constructed from the full length sequences ([Figure 10(a)](#fig10){ref-type="fig"}) demonstrates an evolutionary relationship between the viruses. However, different ORFs yield distinctly different trees (Figures [10(b)](#fig10){ref-type="fig"}--[10(d)](#fig10){ref-type="fig"}), suggesting that viruses can copy ORFs from other viruses and alter their function by integrating them into their own sequences. Therefore, by establishing evolutionary relationships for each ORF, virologists can analyze the diseases caused by specific ORFs. [Figure 11](#fig11){ref-type="fig"} shows the diversity bar graph generated by the platform. The residue position of high entropy is provided in [Figure 12](#fig12){ref-type="fig"}, which shows four phylogenetic trees and the diversity bar graph. The positions (also the amino acids) of high diversity are shown in the box. 5. Conclusion {#sec5} ============= Cloud computing is the online delivering of computing resources, such as hardware and software. Users can access cloud-based applications through a web browser or via applications on mobile devices. Although many bioinformatics tools have been developed as web applications, these are typically deployed in a server, which has limited computing power. Currently, some tools have been redeveloped as distributed computing tools based on the Hadoop framework. These tools are readily deployed on a cluster provided by a cloud computing vendor such as Amazon EC2. Deployment of preexisting tools to the cloud environment is the current trend of bioinformatics as a service. In this paper, we propose a high-scale, available cloud-based open reading frame phylogenetic analysis service based on a Hadoop cluster using virtualization technology. Virtualization enables the proposed service to copy large quantities of jobs. Because Hadoop is strongly buffered against faults, the proposed cloud service guarantees that submitted jobs are recovered by task reassignment, ensuring a high-availability cloud service. Our case study demonstrated that our service can construct different phylogenetic trees from comparisons of different ORFs. These relationships can significantly assist biologists to observe sequence evolutions in different ORFs. The proposed service can also assist researches to develop novel drugs against pathogenic viruses. There are no competing interests for this paper. Part of this work was supported by the National Science Council under Grants NSC-99-2632-E-126-001-MY3 and NSC-100-2221-E-126-007-MY3. {#fig1} {#fig2} {#fig3} {#fig4} {#fig5} {#fig6} {#fig7} {#fig8} {#fig9} {#fig10} {#fig11} {#fig12} ###### The genetic code: nucleotides to amino acids.   2nd base ------------------------------- ------------------------- ------------------------- ------------------------- -------------------- 1st base          U UUU Phenylalanine (Phe) UCU Serine (Ser) UAU Tyrosine (Tyr) UGU Cysteine (Cys) UUC Phe UCC Ser UAC Tyr UGC Cys UUA Leucine (Leu) UCA Ser UAA STOP UGA STOP UUG Leu UCG Ser UAG STOP UGG Tryptophan (Trp)  C CUU Leucine (Leu) CCU Proline (Pro) CAU Histidine (His) CGU Arginine (Arg) CUC Leu CCC Pro CAC His CGC Arg CUA Leu CCA Pro CAA Glutamine (Gln) CGA Arg CUG Leu CCG Pro CAG Gln CGG Arg  A AUU Isoleucine (Ile) ACU Threonine (Thr) AAU Asparagine (Asn) AGU Serine (Ser) AUC Ile ACC Thr AAC Asn AGC Ser AUA Ile ACA Thr AAA Lysine (Lys) AGA Arginine (Arg) AUG Methionine (Met) or START ACG Thr AAG Lys AGG Arg  G GUU Valine Val GCU Alanine (Ala) GAU Aspartic acid (Asp) GGU Glycine (Gly) GUC (Val) GCC Ala GAC Asp GGC Gly GUA Val GCA Ala GAA Glutamic acid (Glu) GGA Gly GUG Val GCG Ala GAG Glu GGG Gly [^1]: Academic Editor: Chuan Yi Tang

Obtaining an orthopaedic residency is extremely competitive and is one of the most applied to surgical subspecialties. This has led to increasing competition for an orthopaedic residency position. Thus, as expected, the average United States Medical Licensing Examination (USMLE) Step 1 and 2 scores continue to increase for this cohort, as does the proportion of those achieving Alpha Omega Alpha (AOA) status during their medical school tenure^[@R1]^. There is increasingly more preparation that must be done by the applicant including research involvement early in medical school and making the decision to apply to more programs in the fear of not matching. In the 2019 National Resident Matching Program (NRMP) Applicant Survey Report, matched applicants had applied for a median number of 84 programs, whereas unmatched applicants had applied to 92 programs and matched applicants attended a median of 13 interviews, whereas unmatched applicants attended 6 interviews^[@R2]^. Similarly, successfully matched applicants in 2018 were able to rank 12.5 programs, whereas unmatched applicants ranked 6.6 programs^[@R2]^. Hence, the "magic number" of interviews yearly is approximately 13 to have a greater than 90% chance of matching. However, this must be viewed in light of the fact that more attractive candidates are often offered more opportunities to interview. In 2018, matched applicants had higher Step 1 scores (248 vs. 240 points), Step 2 scores (255 vs. 246 points), more research projects (11.5 vs. 6.7 projects), and were more likely to have AOA status (40.4% vs. 15.9%)^[@R2]^. However, because the number of orthopaedic applicants increases yearly, there is a disproportionately lagging increase in the number of residency positions available. In the past 3 years of the NRMP, there were 987 total applicants for 742 allopathic orthopaedic residency positions in 2018 compared with 1,037 applicants for 755 positions in 2019. With the recent allopathic-osteopathic merger, the most recent data from the NRMP for 2020 demonstrate 1,192 total applicants for 849 spots. This has led to approximately 25% to 30% of orthopaedic applicants not matching yearly with usually no unfilled positions in orthopaedic residency programs. Furthermore, these are mostly US graduates. The total number of US orthopaedic applicants in 2018 were 839, which represents 85% of all orthopaedic applicants; 149 of these applicants did not match, leading to an unmatched rate of 18%, which is the lowest match rate among all surgical specialties^[@R2]^. Multiple studies exist regarding factors important for orthopaedic resident selection and subsequent performance for first-time applicants^[@R3]-[@R5]^. However, for these unmatched applicants who are reapplying, the new application cycle is a perplexing and disconcerting period, where unusual decisions and tailored strategies must be constructed by the applicant. Were there weaknesses or flaws in the application that could be addressed to improve their chance at matching next year? How persistent or passionate are these applicants about orthopaedic surgery that they are willing to wait an entire year to reapply for the orthopaedic residency match? Can these individuals realistically "give up" another year and accrue more loans in the interim? Should they settle for another career option in the postmatch scramble known as the Supplemental Offer and Acceptance Program? What should they do in the interim year before reapplying, if they choose to reapply? Although many of these questions are personal and specific to each individual applicant, for those who want to pursue orthopaedic surgery residency, there is currently sparse literature on how they will fare as unmatched applicants reapplying for the match. A study by Amin et al.^[@R6]^ is the first study that addressed the unmatched applicant and consisted of orthopaedic program director responses indicating that most programs recommend an unmatched applicant do a surgery internship for 1 year to increase their chances of matching. However, this was based on survey results and not on objective match statistics on this specific population. A study by Rivero et al.^[@R7]^ attempted to determine more objective answers; however, the authors examined an older period and used third-party websites to obtain their information on applicants because of the retrospective nature of their study. Thus, the aims of our study were to investigate the risk factors and outcomes of unmatched orthopaedic applicants through anonymous questionnaires immediately after each Match Day annually from 2016 to 2019. Furthermore, we present a discussion regarding measures to prevent an applicant from not matching and next steps for unmatched applicants who are still interested in pursuing a residency in orthopaedic surgery. Methods ======= A retrospective survey-based questionnaire ([Appendix 1](#app1){ref-type="sec"}) was administered to medical students applying into orthopaedic surgery residency in the United States annually from 2016 to 2019 immediately after Match Day. In total, there were 3,194 recipients of the survey; 934 anonymous orthopaedic applicants completed the survey (29.2% response rate), of which 81 of these applicants identified themselves as unmatched from the previous year and had reapplied for the subsequent cycle. The questionnaire was administered anonymously through a secure, web-based electronic survey platform. Thus, the study was blinded to the study personnel regarding applicant name, institution, and certain demographics, including race and location of residence. Variables collected in the questionnaire included demographics (age, gender, geographic location, domestic versus international medical graduate, and medical school ranking according to U. S. News that year), USMLE scores (Step 1 and Step 2 clinical knowledge \[CK\] scores), Electronic Residency Application Service (ERAS) application characteristics (AOA status, clerkship scores, the number of residency applications submitted, number of interviews offered, and number of publications as defined by ERAS), interim year pursuits (research year, surgical internship, etc.), and changes made to their application during the subsequent cycle including new letters of recommendation or personal statements. Univariate analyses were performed with the use of Student *t*-test for continuous variables and Fisher exact test for categorical variables. All statistical analyses were performed using IBM SPSS version 23.0 statistical software (SPSS). An alpha level of 0.05 was used to determine the statistical significance in all aforementioned instances. Results ======= Overall, 58.0% (47/81) of unmatched applicants subsequently matched into an orthopaedic residency after reapplying the subsequent year. Of the 47 successful reapplicants, 43 matched when they entered the match a second time, 3 matched on their third attempt, and one matched after more than 5 attempts. After not matching, applicants most commonly pursued either a research year or surgical internship, whereas few applicants had other pursuits. Applicants who pursued a research year (n = 48) or surgical internship (n = 25) after initially not matching had a subsequent match rate of 52.1% (25/48) and 64.0% (16/25), respectively (p = 0.46, Fig. [1](#F1){ref-type="fig"}). Furthermore, 29.2% (14/48) of applicants who reapplied were subsequently matched at the institution where they pursued their research year or surgical internship. Applicants were more likely to be interviewed at different programs than their first application because they were only offered on average 1.7 interviews from same programs as their first attempt. {#F1} Although unmatched students reapplied to more programs on their subsequent attempt (84.6 first attempt vs. 107.2 second attempt, p \< 0.001), there was no increase in the number of interviews (8.1 vs. 8.0, p = 0.93). Unmatched applicants who subsequently matched had more interviews when reapplying compared with those who did not match (9.3 vs 6.0, p = 0.03). There were 40/81 applicants who were interviewed by their home program; 65% of these applicants (26/40) then subsequently matched into orthopaedic surgery. For the 41 applicants who were not interviewed by their home program, 21 (51.2%) of them subsequently matched into orthopaedic surgery; this was not statistically different from those who were interviewed by their home program (p = 0.26). Of those who subsequently matched, 19.1% (9/47) were AOA compared with 2.9% (1/34) in the unmatched group (p = 0.04); thus, applicants with AOA status had a 90.0% match rate (9/10) during their subsequent cycle, which was significantly higher than those without AOA status (38/71 \[53.5%\], p = 0.04). When stratified by sex, 83.3% of women (15/18) subsequently matched compared with 50.8% of men (32/63, p = 0.02, Fig. [1](#F1){ref-type="fig"}). There were no differences in Step 1 USMLE scores (242.5 vs. 240.7, p = 0.60), Step 2 CK scores (248.3 vs. 244.5, p = 0.60), or number of publications (15.6 vs. 10.9, p = 0.25) between applicants who subsequently matched or did not match, respectively. However, both groups had an increased number of publications compared with their first application cycle (15.6 vs 6.0 publications for subsequently matched applicants, p \< 0.01; 10.9 vs 4.8 publications for unmatched applicants, p = 0.04). When reapplying, 96.3% (78/81) of applicants used at least one new letter of recommendation. By the same token, 92.6% (75/81) changed their personal statement when reapplying, 65.4% (53/81) of applicants mentioned "not matching" in their personal statement, and 62.3% (33/53) of these applicants subsequently matched into orthopaedic surgery. 91.4% (74/81) applicants mentioned "not matching" during their interviews and 62.2% (46/74) of these applicants subsequently matched, whereas all applicants who did not mention this during their interviews did not match. Discussion ========== This is the first study on the fate of unmatched orthopaedic surgery applicants through applicant-reported responses. The aim of this study was to ultimately provide an objective guide for unmatched applicants as they navigate their paths post-Match Day. We examined applicant factors and their association with the match outcome for each applicant. Overall, although the odds of matching in a subsequent cycle were lower than matching in the initial cycle, there was still greater than 50% chance of matching, which may be more than enough odds for some applicants to pursue a second attempt at matching in orthopaedic surgery. Most unmatched applicants will either pursue a surgical internship year or a dedicated research year. What may be reassuring from this study is that there was no statistical difference in match outcome rates regardless of either pursuit. Therefore, based off of our results, the applicant should choose a pursuit that they are interested in and aim to excel during that year. Furthermore, based on a recent study on unmatched orthopaedic residency applicants^[@R7]^, there was no observed difference in the match success rate between pursuing a research year versus surgical internship. The lack of a statistical difference between both of these pursuits suggests that they are similarly beneficial to the match outcome. Interestingly, our study also showed that nearly 30% of subsequently matched applicants matched at the institution where they pursued their research year or surgical internship. This may demonstrate that familiarity with the applicant and observation of their performance during this interim year plays an important role in achieving a successful match outcome. However, this further raises ethical questions regarding programs interviewing numerous applicants despite planning to highly rank students who have spent a year with them, whether on a clinical service or in research. Because orthopaedic residency continues to be more competitive, this factor can certainly affect future matches and the potential for new rules needed for the match process. Applicants should plan to apply to more programs on their subsequent attempt because they are more likely to be interviewed at different programs than their first application. Unfortunately, applicant-specific factors that increased the odds of matching were nonmodifiable: AOA status and female sex. Although the reason remains unclear from the data, female sex may have improved an applicant\'s chances in matching to an orthopaedic surgery residency because this has been a traditionally male-dominated specialty; with increased demands for diversity in programs, this could be one beneficial factor for female applicants. There were no differences in Step 1 or 2 CK scores or research publications between those who subsequently matched and those who did not. Applicant candidness regarding their unmatched status in their personal statement or during interviews interestingly seemed to correlate with a positive match outcome in our results. Perhaps, a discussion regarding their interim year pursuits and how they are a stronger candidate this year reflects strong work ethic and persistence, which is admired in any medical field. Although our study highlights the outcomes of unmatched applicants, it would be beneficial to discuss measures to prevent an applicant from not matching in the first place, including mentoring and parallel planning. The 2018 NRMP Charting Outcomes presentation states "If you are applying to a competitive specialty (or are less competitive for your chosen specialty) and you want to have a residency position in the event you are unsuccessful in matching to a program in your preferred specialty, also rank your most preferred programs in an alternate specialty"^[@R8]^. For an applicant applying for a competitive subspecialty, the following advice is provided: rank all programs in which you would accept a position, include a mix of highly competitive and less competitive programs, and if you want a residency position in the event you are unsuccessful in matching to your preferred specialty, then also rank your most preferred programs in an alternate specialty. Programs cannot see the other specialties applied to, so it may be important to have parallel planning. However, if the applicant is considering this, they may be less likely to match according to the NRMP. In 2018, unmatched applicants applied to 1.3 distinct specialties, whereas matched applicants applied to 1.1 specialities^[@R2]^. This may imply that applicants who applied to another specialty while applying into orthopaedics were less likely to match into orthopaedics, and this may be because of weaker applications. If that is the case, the applicant should give long thought to their application process and discuss their application with a trusted mentor. Before applying for orthopaedic surgery, the applicant should build relationships and connections with people in the field. A mentor who can provide advice throughout the application process can be invaluable and perhaps can even advocate on behalf of the applicant. Equally important is connecting with current senior medical students or junior residents who successfully navigated the application process and can provide valuable and contemporary insight into the application process and residency. There are several limitations to this study, and our findings should be viewed in light of these. Our results were based on applicant responses to our survey that was distributed online. Therefore, there is some selection bias because every applicant did not respond to the questionnaire. Over a 4-year period, we had only 81 responses from unmatched applicants. The questionnaire was sent online immediately after the Match Day to obtain accurate information; however, this is an emotional time for many of these applicants and may have affected the response rate. However, because we acquired our data through applicant responses and in a timely manner, our data are more likely to be accurate. We attempted to mitigate the small sample size by sending the survey over a 4-year period to acquire as much data as possible. Our data are also based on contemporary outcomes because we included match days from 2016 to 2019. However, because of our small sample size, some of our data may not have been sufficiently powered. For instance, there was a higher proportion of applicants who matched after pursuing a surgical year compared with those who pursued a research year. Although there was no statistical difference, we may not have been powered to detect a difference. Last, resident selection encompasses factors that are nonquantifiable, and we could not accurately study factors such as letters of recommendation, interview skills, interpersonal relationships, and other similar factors that go into ranking an applicant. In conclusion, our findings demonstrate that most orthopaedic applicants matched during their subsequent attempt, which involved applying to more programs and increasing their number of publications. Women and those with AOA status had a significantly higher match rate than their counterparts. There was no difference in the outcomes between those who pursued a research year or surgical internship, Step 1 or 2 CK scores, and the number of publications. Applicants are unlikely to be interviewed by the same programs as the first cycle. Further study is needed to properly analyze the risk factors for not matching on a subsequent attempt. Appendix {#app1} ======== Supporting material provided by the authors is posted with the online version of this article as a data supplement at [jbjs.org](http://jbjs.org) (<http://links.lww.com/JBJSOA/A178>). This content was not copy-edited or verified by JBJS. Investigation performed at the Rothman Institute at Thomas Jefferson University, Philadelphia, Pennsylvania **Disclosure:** The **Disclosure of Potential Conflicts of Interest** forms are provided with the online version of the article (<http://links.lww.com/JBJSOA/A177>).

Mitochondrial function ====================== Mitochondria, originating from bacterial precursor cells that were able to generate energy, provide a platform for the generation of ATP, the energy currency of the cell. As ATP is essential for many cellular processes, mitochondrial function (and mitochondrial dysfunction) plays an important role in metabolic health and cellular fate. Mitochondrial function can be defined in a number of different ways, but for the purpose of this review we have largely focussed on the role of mitochondria in metabolic processes including oxidative phosphorylation and substrate oxidation (summarised in [Fig. 1](#fig1){ref-type="fig"}). The regulation of mitochondrial function is complex and still not fully understood. It involves rapid adaptations to changing metabolic conditions, such as fusion and fission, mitophagy as well as mitochondrial biogenesis. All of these will be discussed in the following sections, mainly in relation to changes in the obese state and under conditions of impaired insulin action (i.e. insulin resistance (IR)). Mitochondrial dysfunction ========================= The diverse roles of mitochondria in different cellular processes and the multitude of methods used to examine mitochondrial function have led to variations in the definition of 'mitochondrial dysfunction'. For example, mitochondrial function has been assessed by changes in mRNA levels of mitochondrial markers (either by targeted PCR or in more global microarray approaches) [@bib1], alterations in protein level (by immunoblotting) [@bib1] or in enzymatic activity of key components of mitochondria-driven oxidation [@bib1], as well as changes in mitochondrial size and shape (by electron microscopy) [@bib5] and substrate oxidation [@bib6]. Accordingly, some groups referring to mitochondrial dysfunction as diminished mitochondrial content, others as a decrease in mitochondrial activity and oxidative phosphorylation, while others focus on different aspects such as reactive oxygen species (ROS) generation. In the context of this review, we refer to the term 'mitochondrial dysfunction' as a decrease in the mitochondrial oxidation of substrates, including lipid and carbohydrate, resulting from a general decrease in oxidative phosphorylation. As well as defining mitochondrial dysfunction, it is important to give an overview about the possible mechanisms by which impairments in mitochondrial oxidative metabolism (i.e. mitochondrial dysfunction) could affect insulin sensitivity. Mitochondrial dysfunction can result from a decrease in mitochondrial biogenesis, reduced mitochondrial content and/or a decrease in the protein content and activity of oxidative proteins 'per unit of mitochondria' (such as a decrease in the complexes of the electron transport chain (ETC)) ([Fig. 2](#fig2){ref-type="fig"}). All such changes would presumably lead to a decrease in substrate oxidation ([Fig. 2](#fig2){ref-type="fig"}A). The reduced oxidation of fuels, particularly fatty acids, results in lipid accumulation, including deposition of metabolically active lipid mediators such as diacylglycerols (DAG) and ceramides (CER). Both DAG and CER have been shown to inhibit insulin signalling: DAG through protein kinase C activation translocates to the plasma membrane and inhibition of the insulin receptor [@bib11], and CER through inhibition of the protein kinase AKT ([Fig. 2](#fig2){ref-type="fig"}C) [@bib12]. DAG and CER accumulation is therefore a plausible link between mitochondrial dysfunction and IR. One potential caveat to this model is that muscle has an enormous spare respiratory capacity (i.e. oxygen consumption can increase ten to 20-fold above resting), and it has been proposed that a deficit in mitochondrial function of the magnitude observed in many obese and insulin-resistant individuals might not be expected to have major effects on substrate oxidation under resting conditions [@bib14]. However, it should be noted that in situations where muscle does dramatically increase respiration (e.g. exercise), there are typically large increases in muscle blood flow, marked changes in ATP demand, activation of multiple signalling pathways and large alterations in metabolite concentrations; in the absence of these changes (i.e. resting conditions), it is plausible that even relatively small decreases in substrate oxidation over time may partially lead to ectopic lipid accumulation and subsequently IR. An additional potential mechanism linking mitochondrial dysfunction to IR is that decreases in substrate oxidation affect electron flow through the ETC, causing electron leakage towards oxygen and the formation of superoxide. Superoxide and other ROS damage various mitochondrial and cellular components (including oxidative damage to mitochondrial DNA, protein aggregations and lipid peroxidation), and potentially results in either mitophagy (removal of damaged mitochondria and prevention of cell death) or, under high stress levels, apoptosis ([Fig. 2](#fig2){ref-type="fig"}B). Removal of mitochondria through mitophagy could reduce mitochondrial number, resulting in decreased substrate oxidation, further aggravating lipid accumulation. The debate about mitochondrial dysfunction and IR ================================================= Mitochondrial dysfunction was first described in the context of glucose intolerance ∼40 years ago [@bib15], and the majority of studies in this area since that time have focussed on changes in skeletal muscle, which will be the main organ discussed in this review. Several studies in humans (from the late 1990s) suggested the existence of mitochondrial dysfunction in obese and insulin-resistant patients, with these individuals exhibiting lower oxidative enzyme activities and decreased lipid metabolism in muscle compared with lean control subjects [@bib6]. In addition, Kelley *et al*. [@bib7] published in 2002 that skeletal muscle of obese subjects with type 2 diabetes (T2D) exhibited lower NADH:O~2~ oxidoreductase activity and reduced mitochondrial size when compared with lean control subjects. One year later, two major microarray studies carried out in muscle showed mitochondrial biogenesis and oxidative phosphorylation pathways to be downregulated in T2D patients and non-diabetic individuals with a family history (FH+) of T2D when compared with healthy controls [@bib2]. These two studies were of particular interest to metabolic researchers as i) they showed a decrease in peroxisome proliferator coactivator 1a (PGC1α), the master regulator of mitochondrial metabolism ([Fig. 1](#fig1){ref-type="fig"}), and therefore for the first time suggested a mechanism for the decrease in mitochondrial function, and ii) they provided evidence for genetic predisposition to mitochondrial defects and its occurrence in the 'pre-diabetic' state. Following these initial observations, several studies in humans showed similar downregulation of metabolic and mitochondrial pathways in obesity and IR (reviewed in [@bib16]). Defects in the expression of mitochondrial genes were found at the mRNA level [@bib1] as well as at the protein level [@bib1]; this was accompanied by a decrease in oxidative enzyme activity [@bib1] and mitochondrial size and density [@bib5]. To some extent, it still remains unclear whether the observed defects could be primarily due to a decrease in the number of mitochondria 'per unit of muscle tissue' or due to actual metabolic changes within the mitochondria [@bib19]. Disparate results have also been reported with regards to the intramuscular populations of mitochondria that are affected (subsarcolemmal vs intermyofibrillar) [@bib5] and between different muscles across the body [@bib24]. Although several human (as above) as well as rodent studies [@bib25] have described associations between diminished mitochondrial function and obesity/IR, various independent publications have failed to show such a correlation. For example, several studies have shown that muscle mitochondrial function was not impaired in obese and T2D human subjects when compared with controls [@bib19]. In addition, non-obese sedentary humans that were overfed for 28 days exhibited peripheral IR (determined as a decrease in glucose infusion rate during hyperinsulinemic--euglycaemic clamps) without changes in several markers of mitochondrial content in muscle [@bib28]. Similarly, rats fed a high-fat diet exhibited unchanged mRNA levels of various energy and glucose metabolism markers in muscle [@bib29], as well as similar hepatic mitochondrial and peroxisomal fatty acid oxidation capacity when compared with low-fat diet controls [@bib30]. Besides evidence for scenario 1 (a decrease in mitochondrial function with IR) and scenario 2 (unchanged mitochondrial function despite IR), several research groups, including ours, have shown a compensatory increase in mitochondrial oxidative capacity with increased lipid supply [@bib31]. Mice and rats fed high-fat diets exhibited impairments in glucose tolerance and insulin sensitivity, but simultaneously an increase in fatty acid oxidative capacity, as well as protein content and activity of mitochondrial oxidative proteins in muscle [@bib31]. Both the increase in mitochondrial content and oxidative capacity [@bib35], as well as the development of IR [@bib36], occur at around 3--4 weeks of high-fat feeding. Furthermore, in a recent comparison of mouse strain, our group showed that this mitochondrial adaptation to high-fat feeding was present in several different mouse strains (C57BL/6, 129X1, DBA/2 and FVB/N) that were prone to fat-induced obesity and glucose intolerance [@bib37]. The implications from these studies are that while there is a compensatory increase in mitochondrial oxidative capacity in rodents in response to dietary lipid oversupply, the timing and magnitude of these changes are not sufficient to cope with the dramatically enhanced lipid availability, and thus there is still ectopic lipid accumulation and IR. In support of this, dietary or genetic manipulations that enhance oxidative capacity in muscle above the normal adaptive response do ameliorate IR [@bib32]. Collectively, the three possible scenarios described above (i.e. decreased, unchanged or a compensatory increase in mitochondrial function) suggest that mitochondrial dysfunction is not a requisite feature of IR in all circumstances and the presence of mitochondrial dysfunction is dependent on its definition, the population studied, the model system examined (e.g. human vs rodent models) and the methodological approach (e.g. association vs intervention studies). Alterations of mitochondrial function: effects on insulin sensitivity ===================================================================== As association studies of IR and mitochondrial function, as commonly reported for humans, are unable to delineate whether changes in mitochondrial function are a cause or consequence of IR, various groups have relied on genetic manipulations and interventional approaches to define the cause-or-consequence question of whether changes in mitochondrial function have effects on tissue and whole-body insulin sensitivity. Various transgenic approaches have been taken to try to answer this question. As it is not possible to cover all of these studies, we will give examples of alterations in gene expression of three key metabolic regulators, *TFAM*, *PGC1* and acetyl-CoA carboxylase 2 (*ACC2*), and discuss their effects on mitochondrial function and insulin sensitivity. TFAM ---- Mitochondrial biogenesis is a complex process, involving a coordinated regulation of mitochondrial and nuclear genomes. An important protein controlling the transcription of mitochondrial proteins is the nuclear-encoded transcription factor *TFAM*, whose expression is regulated by nuclear respirator factor 1 (Nrf1) [@bib40]. Manipulating the gene expression of this key regulator of mitochondrial transcription is therefore an excellent approach to alter mitochondrial function. Muscle-specific *Tfam*-knockout mice exhibit abnormally appearing mitochondria in their muscles as well as progressively deteriorating respiratory chain function [@bib41]. Interestingly, ATP levels are almost unchanged due to a substantial increase in mitochondrial mass [@bib41]. In a subsequent study, the same researchers investigated glucose homeostasis in muscle-specific *Tfam*-knockout mice and their results suggested that mitochondrial dysfunction in skeletal muscle is not a primary etiological event in the development of IR, as knockout mice exhibited improved glucose clearance during a glucose tolerance test and increased glucose uptake into muscle (likely due to increased expression of the glucose transporters GLUT1 and GLUT4 and increased AMP-activated protein kinase (AMPK) activity) [@bib42]. An independent group has recently created adipose tissue-specific *Tfam*-knockout mice, and their results are in line with the previous findings of Wredenberg *et al*. [@bib43]. As expected, *Tfam*-knockout mice exhibited decreased mtDNA copy number and diminished protein content of mitochondrially encoded proteins in brown and white adipose tissue, but displayed greater oxygen consumption due to higher uncoupling. Increased mitochondrial oxygen consumption resulted in elevated whole-body energy expenditure, which is a possible mechanism for these mice being protected against diet-induced obesity, glucose intolerance and lipid accumulation in their livers [@bib43]. Taken together, tissue-specific knockouts of *TFAM* indicate that experimentally inducing abnormalities in mitochondrial function does not necessarily lead to deterioration of glucose homeostasis and insulin sensitivity, highlighting a dissociation between IR and mitochondrial dysfunction. Peroxisome proliferator coactivator 1 ------------------------------------- Although the knockout of *TFAM* demonstrated a clear dissociation between mitochondrial function and insulin sensitivity, alterations in gene expression of members of the *PGC1* network have shown that metabolic downstream effects are variable, with complex compensatory interactions making interpretation of data difficult. *PGC1* *α* is a master-regulator of mitochondrial biogenesis, interacting with a large complement of transcription factors and nuclear hormone receptors that are associated with mitochondrial function (including *Nrf1*, *ERR* *α*, *YY1*, *PPAR* *α*, *PPAR* *γ*) [@bib44]. Global *Pgc1* *α*-knockout mice are viable; however they exhibit an abnormal energy metabolic phenotype. With age, *Pgc1* *α*-KO mice show an increase in whole-body as well as hepatic fat deposition, which is accompanied by a decrease in liver and muscle mitochondrial oxidative capacity. Surprisingly, KO mice were less susceptible to diet-induced IR than WT controls [@bib45]. Muscle-specific knockout of *PGC1* *α* also leads to increased fat mass; however in contrast, glucose tolerance was impaired in old mice (24 months), whereas it was unaltered in young (3 months) KO mice [@bib46]. This discrepancy between age groups may be explained by a compensatory increase in PGC1β in young, but not in aged mice [@bib46]. PGC1α and PGC1β regulate a large number of overlapping genes, therefore metabolic compensation is not surprising. In addition to *PGC1* *α* knockout, other studies have shown that also muscle-specific PGC1α overexpression causes IR and glucose intolerance, potentially due to increased fatty acid delivery into muscle and decreased *GLUT4* gene expression [@bib47]. With these previous alterations of PGC1 protein levels, it should be noted that detrimental metabolic effects might be due to complicated adaptations due to life-long overexpression or knockout. In contrast, acute overexpression of *PGC1* *α* and *PGC1* *β* has shown that elevated PGC protein levels improve diet-induced IR in muscle [@bib38]. Therefore, acute and targeted activation of PGC1-dependent pathways is likely to have therapeutic potential for the treatment of IR [@bib50]. Acetyl-CoA carboxylase 2 ------------------------ As lipid oversupply is a major factor involved in the development of IR, a prevailing view was that increasing mitochondrial fatty acid oxidation would improve insulin sensitivity. Mitochondrial fatty acid oxidation is, in part, regulated by AMPK, which phosphorylates and inactivates the mitochondrial enzyme ACC2, leading to reduced malonyl-CoA levels, elevated CPT1 activity and increased entry of long-chain fatty acids into mitochondria for oxidation [@bib51]. In a recent report by Hoehn *et al*. [@bib51] activation of AMPK by the AMP-mimetic aminoimidazole carboxamide ribonucleotide (AICAR), as well as global *Acc2*-knockout in mice resulted in increased whole-body fatty acid oxidation. Interestingly, despite a significant increase in lipid oxidation, *Acc2*-knockout mice were not protected from high-fat diet-induced weight gain, glucose intolerance and deterioration of skeletal muscle glucose disposal, indicating that simply accelerating mitochondrial fatty acid oxidation alone does not prevent from a deterioration of insulin sensitivity [@bib51]. Similar observations were made by Olson *et al*. [@bib52], who reported that selective deletion of *Acc2* in mouse skeletal muscle, as well as inactivation in the germline had no effects on body weight, food intake, body composition or glucose homeostasis as compared with controls on chow or high-fat diet. These recent studies of unchanged skeletal muscle and whole-body insulin sensitivity in *Acc2*-knockout mice contrast with earlier findings by Abu-Elheiga *et al*. [@bib53], who reported that *ACC2* deletion leads to increased fat oxidation, reduced body weight and protection against diet-induced obesity and IR [@bib54]. Phenotypic differences observed in these different lines of *Acc2*-knockout mice could potentially be due to multiple experimental differences (e.g. genetic background, diet composition and cloning strategy), but highlight that seemingly identical genetic manipulations can give contrasting results, complicating advances in our understanding on the importance of mitochondrial function in the development of IR. In the section above, we have noted a number of examples emphasising that due to variable adaptations in different mouse lines, more direct genetic manipulation of mitochondrial function has still been unable to clearly define the impact of altering mitochondrial capacity on tissue and whole-body insulin sensitivity. However, since many insulin-resistant individuals do show mitochondrial defects, it is possible that the mitochondrial defects could be a consequence of reduced insulin action, which is discussed below. Regulation of mitochondrial function by insulin =============================================== Insulin is an anabolic hormone that is known to play a major role in the regulation of metabolic pathways and protein synthesis in many tissues. A number of groups have examined the effect of insulin on mitochondrial function (largely via insulin infusions) and have also attempted to selectively alter glucose tolerance and insulin sensitivity in a previously 'metabolically healthy' environment to determine whether these changes affect mitochondrial function. The methodological approach taken for the latter has been to manipulate the components of the insulin signalling cascade to determine whether alterations directly affect insulin sensitivity (primary effect) and if changes in insulin sensitivity could potentially lead to alterations in mitochondrial capacity and function (secondary effect). Insulin infusions ----------------- Insulin is known to affect various metabolic pathways in lipid and glucose metabolism, by either affecting gene expression or the phosphorylation of metabolic proteins [@bib57]. In humans, several studies have also demonstrated direct effects of insulin to modify mitochondrial function, with insulin infusions leading to increased expression of mitochondrial proteins and higher oxidative enzyme activity and elevated ATP synthesis in muscle [@bib60]. Further evidence of an effect of insulin on mitochondrial function came from a study by Karakelides *et al*. [@bib62], who temporarily deprived type 1 diabetic patients of their insulin treatment and showed that insulin deficiency decreased muscle mitochondrial ATP production and expression of oxidative phosphorylation genes. This direct effect of insulin on mitochondrial function is diminished in subjects with IR [@bib60] and in rats after high-fat feeding [@bib63], suggesting that insulin is able to activate mitochondrial biogenesis and oxidative capacity, and that IR, particularly if it is present over a prolonged period, could in part contribute to mitochondrial dysfunction. Insulin receptor substrate gene manipulation -------------------------------------------- With regards to direct genetic manipulation of insulin signalling components, the best characterised models have manipulated insulin receptor substrate (IRS) proteins. In skeletal muscle, selective disruption of IRS1 or IRS2 had only minor effects on insulin sensitivity, whereas simultaneous deletion of both members of the insulin signalling cascade had dramatic effects on insulin signalling and glucose tolerance [@bib64]. Isolated muscles exhibited complete resistance to insulin and diminished insulin-stimulated glucose uptake. In addition, the lack of insulin signalling in double-KO muscle impaired mitochondrial oxidative phosphorylation and ATP production [@bib64]. In addition to the findings in skeletal muscle, similar mitochondrial impairments were also observed in hepatocytes lacking both *IRS1* and *IRS2* [@bib65]. Cheng *et al*. [@bib65] also found larger deformed and 50% fewer mitochondria in livers of these double-knockout (DKO) mice, with a simultaneous increase in markers of mitochondrial fission and fusion. Furthermore, many hepatic genes were deregulated in DKO livers, including genes known to control mitochondrial function, biogenesis and dynamics [@bib65]. Also, similar to the metabolic changes in skeletal muscle, selective silencing of *IRS1* or *IRS2* in liver had only minor effects on insulin signalling, whereas disruption of both isoforms led to impaired glucose tolerance and insulin sensitivity [@bib66]. These results in muscle and liver suggest that direct genetic induction of IR has major effects on mitochondrial capacity, and that IR itself is able to lead to the development of mitochondrial dysfunction. Mitochondrial effects of *IRS1/2* deletion are likely mediated through the IRS-PI3K-Foxo pathway, as additional deletion of *Foxo1* (triple knockout) was able to rescue the metabolic phenotype [@bib65]. These studies of genetic manipulation of components of the insulin-signaling cascade, as well as direct insulin infusion or insulin deprivation, raise the possibility that mitochondrial defects observed in some insulin-resistant subjects could be a consequence of the IR itself. Ectopic lipid accumulation: effects on other mitochondrial parameters ===================================================================== Ectopic lipid accumulation is commonly observed in obesity and T2D and is one of the earliest changes observed during the development of IR [@bib36]. Mitochondria respond to cellular stress (including excess lipid availability) in various ways, for example through increase in reactive species generation, complementation by fusion and fission and activation of cell death pathways. Interestingly, cells have developed a mechanism to sequester and degrade non-functional mitochondria, a process termed mitophagy, before cell death can occur. In this section, we will discuss scientific findings with regards to excess lipid availability and IR and the role of mitochondrial oxidative stress, mitophagy and stress mitigation through mitochondrial fusion and fission pathways. Oxidative stress ---------------- Mitochondria are an important source of superoxide generation in the cell, with complexes I and III of the ETC having the greatest capacity for superoxide production [@bib67]. Most ROS within cells originate from superoxide and hydrogen peroxide, a product resulting from degradation of superoxide by the superoxide dismutase enzymes, or from biochemical reactions with these primary ROS [@bib68]. Under physiologically relevant ADP-stimulated conditions, mitochondrial superoxide represents only around 0.018% of total oxygen consumption [@bib69], being therefore 'concentration-wise' a minor but 'implication-wise' a major player in mitochondrial function. Several studies from the 1990s proposed a link between oxidative stress and the development of IR. Lipid infusions in humans increased plasma thiobarbituric acid reactive substance (TBARS) levels and simultaneously inhibited insulin-stimulated whole-body glucose disposal [@bib70]. In contrast, infusions of reduced glutathione exhibited the opposite effect on both oxidative damage and insulin sensitivity [@bib70]. More direct evidence connecting IR and mitochondrial ROS generation came from a recent study by Anderson *et al*. [@bib72]. In both rodents and humans, a high-fat diet increased the H~2~O~2~-emitting potential of mitochondria without any changes in oxidative capacity. Attenuating mitochondrial H~2~O~2~ emission, by treating rats with a mitochondrial-targeted antioxidant or via overexpression of catalase in mouse muscle mitochondria, completely preserved glucose tolerance and insulin sensitivity [@bib72]. Similar beneficial effects for insulin action were observed when mitochondrial superoxide was reduced by genetic or pharmacological means [@bib73]. These and various other *in vitro* studies [@bib74] and genetic manipulations of antioxidant expression (e.g. targeted overexpression of catalase to mitochondria [@bib76] or overexpression of mitochondrial peroxiredoxin 3 [@bib77] highlight mitochondrial superoxide and H~2~O~2~ as a primary mechanism in the development of IR. The studies noted above support a role for oxidative stress in the development of IR, and there have been a number of antioxidant supplementation trials which have reported that decreasing oxidative stress improves insulin sensitivity and glucose tolerance in various human populations [@bib78] and animal models [@bib83]. However, it should be noted that there is still substantial controversy in this area, as various independent studies have failed to show such association. For example, antioxidant supplementation had minimal impact on insulin sensitivity in various human populations [@bib87]. Similarly, directed reduction in mitochondrial ROS generation using a mitochondria-targeted antioxidant did reduce oxidative stress levels in C2C12 myotubes as well as in mice, but did not affect glucose tolerance and insulin sensitivity [@bib91]. These disparate findings suggest that depending on the experimental conditions, oxidative stress may be one of a number of factors contributing to IR. However, in circumstances where elevated mitochondrial ROS production is present during the development of IR, it is likely tightly coupled with autophagic removal of damaged mitochondria, a process termed mitophagy. Mitophagy --------- Upon increased cellular and mitochondrial stress, mitochondria are able to activate both cell death pathways and mitophagy, with the two opposing forces in the cell, demonstrating a tight balance between life and death [@bib92]. The targeted removal of damaged mitochondria requires two steps: the initiation of autophagy and selective priming of mitochondria for removal [@bib93]. Priming is carried out by either the Pink1--Parkin pathway or through the activation of Nix and Bnip3 [@bib94]. Under metabolically healthy conditions, Pink1 is rapidly cleaved by mitochondrial proteases and degraded by the proteasome. However, upon loss of mitochondrial membrane potential Pink1 accumulates on the outer mitochondrial membrane and communicates with the E3 ubiquitin ligase Parkin. Parkin rapidly translocates to mitochondria and signals the initiation of mitophagy by ubiquinating mitochondrial proteins (VDAC1, mitofusin 1, mitofusin 2 and MIRO) [@bib93]. Furthermore, Parkin recruitment is suggested to be dependent on translocation of HSP72 to depolarised mitochondria, with HSP72-knockout mice exhibiting impaired Parkin action, enlarged and dysmorphic mitochondria, reduced muscle oxidative capacity, and muscle IR [@bib95]. This is one of few studies potentially suggesting mitophagy to be a beneficial metabolic event, important for the maintenance of mitochondrial quality and insulin sensitivity. Mitochondrial fusion and fission -------------------------------- To maintain a metabolically healthy environment, removal of damaged mitochondria through mitophagy requires cells to be able to adapt quickly through changes in mitochondrial fusion and fission pathways. Mitochondria are dynamic organelles able to constantly undergo fusion and fission events to maintain their function [@bib96]. Fusion of mitochondria enables them to mix their contents, including mitochondrial DNA and metabolic intermediates, and to recover the activity of damaged or depolarised membranes [@bib97]. Mitochondrial fusion is predominantly controlled by three GTPases, mitofusin 1 and 2*(Mfn1*/2), both located on the outer mitochondrial membrane, and optic atrophy1 (*Opa1*), localised on the inner membrane of mitochondria [@bib98]. Fission on the other hand increases the number of mitochondria and prepares the cell for cell division and meiosis [@bib100]. It is regulated through the activity of *Fis1*, located throughout the outer membrane, and the dynamin-related protein 1 (DRP1). DRP1, lacking a mitochondrial targeting sequence, is usually located in the cytosol, but gets recruited to the mitochondrial outer membrane by *Fis1* during the initiation of the fission process [@bib101]. Changes in mitochondrial dynamics could contribute to mitochondrial dysfunction observed in certain obese and insulin-resistant human and rodent populations. Skeletal muscle of obese and T2D humans and rodents shows reduced *MFN2* expression (reviewed in [@bib102]). Silencing of *MFN2* in myotubes leads to decreased oxygen consumption and glucose oxidation [@bib103], with a simultaneous decrease in glucose incorporation into glycogen, as well as reductions in pyruvate and palmitate oxidation in myotubes [@bib104]. Changes in oxygen consumption and substrate oxidation with reduced *MFN2* are likely due to a decrease in the expression of subunits of the electron transport complexes, which leads to reduced activity [@bib102]. These metabolic effects of *MFN2* are independent of its role in mitochondrial fusion, as a mutant form of mitofusin 2, lacking the ability to induce fusion, was still able to stimulate energy metabolism [@bib104]. Decreased oxygen consumption and mitochondrial dysfunction were also observed *in vivo* in mice with simultaneous deletion of *Mfn1* and *Mfn2*, which was suggested to be related with mtDNA depletion in early stages of life before physiological abnormalities developed [@bib105]. Interestingly, separate deletion of *Mfn1* or *Mfn2* did not result in any metabolic abnormalities (e.g. normal cytochrome c oxidase/succinate dehydrogenase (COX/SDH) histological staining pattern and no indication of respiratory deficiency), suggesting possible compensatory interactions between both isoforms [@bib105]. Due to the observed role of mitofusins in metabolism and their reported decrease in obesity and IR, these major regulators of mitochondrial dynamics are potentially important mediators of mitochondrial dysfunction. This is also supported by the fact that *Mfn2* levels show a positive correlation with insulin sensitivity in morbidly obese subjects and after bariatric surgery [@bib106]. Targeting mitochondria to treat obesity and IR ============================================== As described in the preceding sections, there is substantial controversy on the exact role of mitochondria in the generation of IR. However, because of their role as the key site for substrate oxidation and ATP generation, targeting mitochondrial energy metabolism may has some benefit for metabolic disorders such as obesity and T2D. Below we discuss the evidence for various approaches (lifestyle interventions that affect mitochondrial oxidative metabolism and insulin action and more direct alterations of mitochondrial oxidative metabolism) aimed at improving aspects of mitochondrial function for potential therapeutic purposes. We also highlight the intriguing work showing that under certain circumstances, mild inhibition of mitochondrial function can also result in beneficial metabolic effects. Lifestyle interventions ----------------------- Exercise is known to have major impact on both mitochondrial function and insulin sensitivity in skeletal muscle. A 12-week exercise intervention programme in T2D patients significantly increased muscle mitochondrial respiration and mitochondrial content [@bib108]. Similarly, an 8-week cycling exercise regime increased muscle fatty acid oxidative capacity and in parallel improved insulin-mediated glucose disposal [@bib109]. An improvement in insulin sensitivity and mitochondrial function (mitochondrial density and oxidative enzyme activity) was also reported by Toledo *et al*. [@bib110] in T2D patients after 4 months of exercise training, by Nielson *et al*. [@bib111] in male T2D subjects after 10 weeks of exercise, and by Meex *et al*. [@bib112] in T2D patients after a 12-week exercise bout. In addition to exercise, caloric restriction is an effective treatment improving obesity and IR. In obese humans, caloric restriction and weight loss were shown to improve insulin action [@bib113], reaching a 40% improvement in insulin sensitivity after 6 months of caloric restriction [@bib116]. Calorie restriction has also been reported to enhance mitochondrial function in humans [@bib117]. The NAD^+^-dependent deacetylase sirtuin 1 (SIRT1) is suggested to be the principal modulator of the downstream pathways responsible for the beneficial metabolic effects of calorie restriction [@bib118]. In line with this, the SIRT1 activator resveratrol increases mitochondrial content, ameliorates IR and prolongs survival [@bib120]. Recently, novel compounds have been characterised, which are 1000-fold more potent than resveratrol [@bib122], with these compounds inducing a robust increase in mitochondrial content and respiration, which is associated with improved insulin sensitivity in mice [@bib123]. Mitochondrial uncoupling ------------------------ The coupling of mitochondria describes the molar ratio of the yield of ATP (per oxygen consumed), also referred to as the P/O ratio, while mitochondrial uncoupling is defined as futile cycling of protons across the mitochondrial inner membrane, without coupling to ATP generation [@bib125]. The inefficiency induced by mitochondrial uncoupling leads to increased energy expenditure and mitochondrial substrate oxidation, as well as improved insulin action [@bib126]. Pharmacological agents, such as 2,4-dintirophenol (DNP), can induce mitochondrial uncoupling by transporting protons across the mitochondrial inner membrane into the matrix. DNP was successfully used as an anti-obesity agent in the 1930s, suggesting that increased energy expenditure through mitochondrial uncoupling could have therapeutic potential [@bib129]. However, due to its narrow therapeutic window, overdoses led to serious imbalances of energy metabolism (and even to death), and DNP had to be taken off the market in 1938. More recently described uncoupling agents may potentially have a safer profile for use in humans [@bib130]. For example, derivatives of rhodamine 19 were shown to reduce membrane potential and increase oxygen consumption. However, In contrast to DNP, the activity of those derivatives was highest at high membrane potentials and decreased with a loss of membrane potential, therefore exhibiting self-limitation and potentially being a safer option than DNP [@bib130]. In addition, Lou *et al*. [@bib131] identified various mitochondrial uncouplers, that appear to act through the adenine nucleotide translocase and have a much greater therapeutic window than previously observed with DNP (dynamic range of 10(6)). Natural compounds ----------------- Asian countries have a long history of using natural compounds for the treatment of metabolic disease. One of the compounds is Berberine, a natural plant alkaloid that was shown to improve insulin sensitivity in rodents and humans [@bib133]. Interestingly, the effects of Berberine are not through enhancement of mitochondrial function (as attempted with many other therapies), but appear to be through inhibition of complex I of the ETC, subsequent AMPK activation and the consequent beneficial metabolic effects [@bib135]. Interestingly, this counter-intuitive pattern of mild inhibition of mitochondrial oxidative metabolism has been reported in other insulin-sensitising medicinal plants [@bib136] and is also a characteristic of metformin and thiazolidinediones, which are frontline anti-diabetic therapies [@bib135]. While it is not completely resolved how mild, transient inhibition of mitochondrial function can lead to beneficial effects, it likely involves a number of mechanisms including reductions in deleterious lipid metabolites (due to decreased lipid synthesis and increased fatty acid oxidation following AMPK activation) and potentially decreases in ROS production [@bib139]. Increased fatty acid oxidation, despite a mild inhibition of complex I, is likely to occur due to AMPK-mediated lowering of malonyl-CoA levels and increased CPT1-mediated fatty acid entry into mitochondria. Increased flux of fatty acids into β oxidation generates both NADH and FADH~2~, with the entry of electrons into the ETC via complex II allowing substrate metabolism to continue despite a transient block at complex I. In addition, a decrease in ROS generation could be potentially mediated via reduced electron backflow and superoxide production from the ubiquinone reduction site of complex I with FADH~2~ (generated during each β oxidation cycle) as the electron donor. Concluding remarks ================== During the last 20 years, many studies have reported changes in mitochondrial function in various models of metabolic disease. Interestingly, these changes have been of opposing character, with some studies reporting a decrease in mitochondrial function (i.e. mitochondrial dysfunction), while others have reported either no change or an increase in mitochondrial oxidative metabolism in obese and insulin resistant humans and rodents. Similarly, studies using genetically manipulated mice to alter mitochondrial function have also produced confounding results. Collectively, these studies highlight that the relationship between mitochondria and insulin action is highly complex and there is still much to learn in this area. Some potential approaches for closing these gaps in knowledge in future studies include developing methods (e.g. with stable isotope tracers) to assess dynamic changes in mitochondrial function *in vivo*, not just in the typical fasting state, but also under a variety of other free-living conditions (e.g. in response to meals and during normal daily activities). Furthermore, increasing evidence of multiple post-translational modifications in mitochondria highlights that future studies in isolated mitochondria or tissue preparations must be performed in the presence of appropriate inhibitors to maintain the mitochondria in a similar state that observed *in vivo*. Studies such as these may help to more clearly define the extent to which mitochondrial dysfunction contributes to the development of IR, and also determine how effective targeting mitochondria may be for the treatment of IR. Declaration of interest ======================= The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the review. Funding ======= Work in the laboratory of the authors is supported by the National Health and Medical Research Council of Australia (NHMRC). M K Montgomery is supported by a NHMRC Early Career Fellowship (APP1071143) and N Turner by an Australian Research Council Future Fellowship (FT120100371). {#fig1} {#fig2}

Objective ========= To compare central venous pressure (CVP) measurements obtained in two different locations (jugular or subclavian veins and femoral veins). Setting ======= A 16-bed medical--surgical ICU. Materials and methods ===================== The patients enrolled had central venous catheters (CVC) in two different locations, one placed in the internal jugular or subclavian veins and a second in a femoral vein. Simultaneous measurements of CVP were undertaken by two different operators, with a pressure transducer zero-referenced at the mid-chest. Standard CVC with similar features (20 cm length) were used. Patients with intra-abdominal pressure (IAP) \>15 mmHg were excluded. The IAP was previously evaluated in all patients using the method described by Sugrue and Hillman. A linear correlation analysis was performed, considering significant *P*\> 0.05 and a correlation coefficient \>0.85. Results ======= Twenty four patients were studied and three patients were excluded. The mean age was 61.2 ± 9.3 years, the ICU stay was 9.8 ± 4.1 days, the APACHE II score was 24.8 ± 5.7, and SAPS II was 52.7 ± 10.4. The mean CVP measured with jugular or subclavian access was 12.1 ± 4.1 mmHg and 12.9 ± 4.2 mmHg at the femoral access. A good correlation between measurements was found with a correlation coefficient and *P*\> 0.001. Conclusion ========== CVP can be accurately measured in femoral accesses, using standard CVC in patients with normal intra-abdominal pressure.

INTRODUCTION {#SEC1} ============ In DNA nanotechnology, achieving controllable motion is a key step to creating the next generation of dynamic DNA nanodevices ([@B1]). One strategy for actuating DNA structures involves binding of external biomolecules to reconfigure DNA devices ([@B8]). A popular method is toehold-mediated strand displacement, where one of the strands in an existing DNA duplex is displaced by an externally provided strand that can form a more stable duplex ([@B11]). In this manner, hybridized connections between device components may be released, or reestablished to enable reversible actuation. This method offers the benefits of sequence specificity of displacement strands and ease of introducing displacement sites within devices. However, due to the need for external strands, release of waste strands, and slow kinetics of the displacement reaction, the method can be invasive and slow. Another actuation strategy involves integrating stimuli-responsive molecular entities into the devices, which makes the actuation less invasive and more responsive to environmental stimuli. These entities include base-stacking motifs, DNA triplexes, azobenzene moieties and I-motifs that respond to environmental cues like light, temperature, pH and ions to establish or disrupt interactions between device components ([@B12]). A third strategy involves using external forces to reconfigure devices. These forces may be applied via electrical or magnetic fields ([@B17],[@B18]), via depletants ([@B19]), or via optical traps and atomic force microscope tips ([@B20],[@B21]). Since the forces are applied directly to the structures and can be modulated fairly rapidly with existing capabilities, these methods can achieve rapid actuation response times, albeit with advanced fabrication and instrumentation requirements. Recently, we proposed a promising actuation method that combines the noninvasiveness of environmental triggers with the specificity of DNA hybridization to enable rapid reconfiguration of devices with response times comparable to force-based methods ([@B22]). The method involves introducing multiple, weakly complementary pairs of single-stranded DNA overhangs to the device components to be actuated, and using changes in solution cation concentration to trigger rapid hybridization or dehybridization of the overhangs to drive conformational transitions in the device. Because this process involves collective hybridization of many short strands, and also does not involve any diffusion of strands or displacement of one hybridized strand with another, this method exhibits much faster response times compared to the strand-displacement method. As demonstration, the method was applied to a DNA origami hinge that nominally exhibits open conformations with its arms subtending large angles about the vertex (Figure [1](#F1){ref-type="fig"}). By introducing short, complementary overhangs to the inner surfaces of the two hinge arms, the modified hinges could be efficiently actuated between conformations with open and closed arms using a variety of cations, including mono-, di- and trivalent ions. The actuation responses---fraction of hinges exhibiting closed arms versus cation concentration---were characterized as a function of the number and length of overhangs and the bending stiffness of the hinge joint. The results revealed strong, intriguing variations with respect to these design variables, suggesting high tunability of the actuation responses. {#F1} Based on our current understanding, the actuation mechanism may be explained as a competition between (i) the intrinsic predisposition of the 'bare' device (minus overhangs) to exhibit open conformations and (ii) the hybridization of overhangs that favors closed conformations. The former effect stems from the underlying structural design of the bare device which dictates intra- and intermolecular interactions between components, and hence their preferred conformations. In DNA hinges, the open conformation appears to arise from the mechanics of the DNA joint connecting the hinge arms, and not from electrostatic repulsion between them, given that the bare hinges exhibited similar hinge-angle distributions across a wide range of cation concentrations. The latter effect provides the driving force necessary for the device to overcome its natural propensity for open conformations and transition to a closed conformation. Importantly, the strength of this driving force depends on cation concentration. At low ion concentrations, the overhangs prefer to remain unhybridized, and the device retains the open conformation it exhibits without overhangs. As ion concentration is raised, the hybridized state of the overhangs becomes increasingly stabilized, causing an increasingly larger fraction of structures to exhibit closed conformations. In the original study ([@B22]), we expressed both these effects in thermodynamic terms, the former in terms of a free energy difference Δ*G*~bare~ between the open and closed states, and the latter in terms of a hybridization free energy Δ*G*~hyb~(*c*) that depends on ion concentration *c*. Assuming that all overhang pairs are indistinguishable, we formulated a simple, two-state thermodynamic model for the probability of observing closed hinges as a function of ion concentration and the two free energies. Since the hybridization free energy of the tethered, confined overhangs must be distinct from that of free strands in solution, we cast Δ*G*~hyb~(*c*) as an additive correction Δ*G*~corr~ to the *solution*-state hybridization free energy obtained from secondary-structure prediction algorithms. Using the unknowns Δ*G*~bare~ and Δ*G*~corr~ as fitting parameters, we were able to obtain good agreement with the experimentally measured actuation responses. While this description was useful for establishing the importance of bare-device mechanics and overhang hybridization in actuation and for fitting experimental data, our knowledge of the actuation mechanism remains very qualitative. For instance, the free energies contributed by the device and the overhangs, and how they vary with the 'reaction coordinate' and ion concentration to 'tilt' the overall free energy towards the open or closed conformations remain unknown. How these free energies change with the various design parameters and the associated molecular mechanisms also are not known. Elucidating these energetic and molecular mechanisms would be crucial to facilitate future design and optimization of actuation devices and responses. Here, we provide such a quantitative, mechanistic basis of this actuation method through calculation of free energy landscapes via molecular simulations, using the DNA hinge as our model system. Ideally, all-atom simulations should provide the most accurate description of the hinges. However, due to the large system size and slow timescales of hybridization and dissociation, such simulations would require prohibitive computational costs. Hence, we use the coarse-grained model, *oxDNA* ([@B23],[@B24]), to represent our hinges. oxDNA has been applied to a variety of DNA nanostructures, ([@B23],[@B25]) and shown to yield accurate descriptions of their mechanics, conformational dynamics, and ion-dependent hybridization thermodynamics, suggesting that this model should provide reasonable predictions for the ion-actuated DNA hinges studied here. To further alleviate computational costs, we devised a strategy that involves splitting the system into the bare hinge (large structure with short relaxation time) and the different pairs of overhangs (small systems with long relaxation times), and computing their free energies separately using efficient simulation approaches. Consequently, we were able to obtain not only the overall free energies of the overhang-modified DNA hinges as a function of its conformation (hinge angle), but also the free energy contributions arising from the hinge joint and each of the overhang pairs. By computing such energy landscapes for a range of ionic conditions and overhang design parameters, analyzing the conformations of the overhangs, and relating features of the landscape to actuation responses and rates, we were able to provide a more complete, molecular-level understanding of the actuation mechanism that could be used for designing and optimizing actuation responses. The results obtained here would be important not only for hinge actuation, but many other systems involving DNA hybridization in confined media, including DNA-tethered nanoparticles ([@B33]), DNA microarrays ([@B34]) and assembly of DNA origami tiles ([@B35]). MATERIALS AND METHODS {#SEC2} ===================== We studied here the same DNA origami hinge used earlier to experimentally demonstrate the ion-mediated actuation approach ([@B22]). Briefly, the hinge is composed of two stiff arms, each assembled from a 3 × 6 bundle of interconnected double-stranded DNA helices organized in a square lattice. The arms are connected by a linear array of 2 bases- and 16 bases-long single-stranded scaffold connections that form the hinge rotation axis (Figure [1A](#F1){ref-type="fig"}). Without overhangs, the hinge exhibited mostly open conformations with its arms subtending a broad distribution of angles about a mean angle of ∼80°. To actuate the hinge between such open conformations and a closed conformation, the hinge arms were modified to include short, complementary single-stranded DNA overhangs distributed evenly across the inner face of each arm at reciprocal locations (Figure [1B](#F1){ref-type="fig"}). Because of the helical structure of DNA and the square-lattice arrangement of DNA helices in the hinge arms, the overhangs could only be introduced at specific locations on the helices lining the inner surface of the arms, which restricted the separation distance between adjacent overhangs on a helix to multiples of 10.6 nm, i.e. the length of 32 bp, or 3 turns of DNA. The experiments explored actuation using 10, 20 and 30 such overhangs per arm (Figure [1B](#F1){ref-type="fig"}). The overhang pairs were each 7 bases long of sequence \|\|-TTTCGAC-5′ and 3′-GTCGTTT-\|\|, where '\|\|' denotes hinge arms. Thus, only the terminal four bases contributed to hybridization across the arms, and the inner three bases played the role of flexible linkers. The caDNAno and geometrical designs of the hinges along with a close-up of the hinge joint showing connections between the two arms are provided in [Supplementary Figures S1 and S2](#sup1){ref-type="supplementary-material"}. Here, we seek to compute the free energy *G*~hinge~(*θ*) of these overhang-modified hinges as a function of the angle *θ* subtended by the two arms about the hinge joint (vertex) representing the coordinate most descriptive of the conformational transition between the open and closed state. While all-atom molecular dynamics (MD) simulations are the ideal resolution for computing such free energy landscapes, such calculations would entail prohibitive computational costs given the large dimensions of the hinges (∼50 nm), slow timescales of the rotation of hinge arms via Brownian motion (tens of microseconds based on simulations ([@B31]) and theory ([@B36],[@B37])), and even longer timescales for the hybridization-dissociation of the overhangs (milliseconds ([@B38])). To this end, we used a coarse-grained model to treat the hinges, namely, the oxDNA model ([@B23],[@B24]) in which each nucleotide is modeled as a rigid body with three interaction sites that capture Watson-Crick base-pairing, base stacking, excluded volume, and backbone connectivity. We have shown ([@B31]) that this model can accurately capture the conformational dynamics of bare hinges, and others have shown that the model accurately describes the molecular mechanism and thermodynamics of DNA hybridization ([@B23],[@B27],[@B30]). In spite of the coarser representation, the computation of *G*~hinge~(*θ*) is still highly challenging, as it would require simulations of the entire hinge over timescales longer than their slowest relaxation time, associated with hybridization of overhangs. To tackle this problem, we considered splitting the overall free energy into two contributions, as given by *G*~hinge~(*θ*) = *G*~bh~(*θ*) + *G*~oh~(*θ*) (Figure [2A](#F2){ref-type="fig"}). The first term is the contribution arising from the bare hinge (without overhangs) that accounts for joint mechanics and intermolecular interactions between the arms. *G*~bh~(*θ*) was calculated via the umbrella sampling approach that involved performing a set of harmonically-restrained MD simulations of the bare hinge (Figure [2B](#F2){ref-type="fig"}) and analyzing the resulting hinge angle distributions using the weighted histogram analysis method (WHAM). The second term is the contribution from all overhangs, including their interactions amongst themselves and with the hinge arms. Based on several assumptions, we were able to divide *G*~oh~ into independent contributions *G*~oh,\ *i*~ from each of the *n* pairs of overhangs attached at different locations on the arms (Figure [2A](#F2){ref-type="fig"}), i.e. $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$G_\text{oh}(\theta ) = \sum _{i=1}^n G_{\text{oh},i}(\theta )$\end{document}$, and also replace the molecularly-detailed hinge arms with simple 'repulsion' planes representing the inner surface of the arms that prevent the phosphate and nucleoside groups of the overhangs from crossing the planes (Figure [2C](#F2){ref-type="fig"}). This allowed us to use highly efficient virtual-move Monte Carlo (VMMC) simulations for carrying our umbrella sampling of overhang conformations, which together with a modified WHAM approach provided us *G*~oh,*i*~(*θ*) as well as free energy contribution *G*~oh,*i*~(*θ*, *ξ*) from the different hybridization states *ξ* of each overhang pair. See [Supplementary Material and](#sup1){ref-type="supplementary-material"} associated [Supplementary Figures S3--S6](#sup1){ref-type="supplementary-material"} for methodological details of these calculations, a more detailed discussion of the underlying assumptions, and the checks performed to ensure adequate sampling of the hinge arms and the overhangs. {#F2} The end result of these simplifications is that the prohibitive calculation of *G*~hinge~(*θ*) is now reduced to less demanding calculations of *G*~bh~(*θ*) and *G*~oh,\ *i*~(*θ*): the calculation of *G*~bh~(*θ*) is no longer limited by the long simulation timescales required for sampling the hybridization states of the overhangs, while the *G*~oh,*i*~(*θ*) calculations are efficiently able to sample all their hybridization states because of the much smaller system size. Using such an approach, we computed the free energy landscape of the experimental overhang-modified hinges, focusing on contributions from the bare hinge and from each of the overhangs. Unless otherwise stated, we used the overhang sequences specified earlier attached at the 10 different locations on the hinge arms depicted in Figure [1B](#F1){ref-type="fig"}. To investigate how these overhangs 'close' the hinge arms with increasing ionic strength, we performed simulations at different ion concentrations to elucidate how the energy landscapes are modulated by ionic conditions. Since the oxDNA model has only been parameterized for Na^+^ ions, the simulations were performed with 0.2, 0.4 and 1.2 M Na^+^ concentrations at 300 K. Lastly, we also investigated the effects of the lengths of the linker and binding portions of the overhangs, two design parameters expected to affect the hybridization energy of overhangs under confinement. RESULTS {#SEC3} ======= Free energy landscape of the overhangs {#SEC3-1} -------------------------------------- In general, the free energy landscape *G*~oh,*i*~(*θ*) of the overhang pairs computed at various cation concentrations and tethered at different locations on the hinge arms exhibit a plateau at large hinge angles, a sharp minimum at small angles, and an energy barrier separating the two. Figure [3A](#F3){ref-type="fig"} presents one such landscape computed at \[Na^+^\] = 0.2 M for overhangs tethered at the closest connection point from the hinge vertex (labeled C1 in Figure [1B](#F1){ref-type="fig"}) that exhibits a free energy gain of ∼−4*k*~B~*T* at the minimum and a large barrier of ∼12*k*~B~*T*, relative to the plateau. ![Free energy landscape and conformations of an overhang pair. (**A**) Free energy *G*~oh,*i*~(*θ*) for overhangs with four complementary bases and 3-base linkers attached at location C1 and computed at \[Na^+^\] = 0.2 M and 300 K. The steric-repulsion (*Sz*), hybridization (*Hz*), dissociation (*Dz*) and non-interaction (*NIz*) zones are marked by distinct color backgrounds. Inset shows contributions *G*~hyb,*i*~(*θ*) (blue) and *G*~dis,\ *i*~(*θ*) (red) to *G*~oh,*i*~(*θ*) (circles) from partly- to fully-hybridized states and dissociated states, respectively. (**B**) Free energy contributions *G*~oh,*i*~(*θ*, *ξ*) from the different hybridization states ξ of the overhangs. Empty region in the plot indicates states whose free energies could not be reliably computed due to the extremely low probabilities of occurrence of those states. (**C**) Representative conformations of the overhangs corresponding to angles marked by arrow heads on the energy landscape. Nucleotides are colored as per their base-pairing probability, where cyan means fully dissociated and magenta mostly hybridized states.](gkz1137fig3){#F3} To investigate the molecular basis for this characteristic shape of the landscape, we analyzed the free energy contributions *G*~oh,*i*~(*θ*, *ξ*) arising from all possible hybridization states *ξ* = 0, 1, ⋅⋅⋅, 4 of the overhangs, spanning fully dissociated (*ξ* = 0) to fully associated overhangs (*ξ* = 4) (Figure [3B](#F3){ref-type="fig"}). The five states represent mutually exclusive 'subensembles' of the overall ensemble of overhang conformations, and hence their free energies combine exponentially to yield the overall free energy, that is, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$e^{ -G_{\text{oh},i}(\theta )/k_\text{B} T} = \sum _\xi e^{-G_{\text{oh},i}(\theta ,\xi )/k_\text{B} T}$\end{document}$. The magnitudes of *G*~oh,*i*~(*θ*, *ξ*) allow us to assess the relative and absolute importance of each hybridization state at various points (hinge angles) along the energy landscape. Also provided are representative conformations of the overhangs captured from simulations at key points along the landscape (Figure [3C](#F3){ref-type="fig"}). The following molecular picture of the overhangs emerges from these analyses: At large angles, the overhangs are too far from each other to interact, and the free energy remains flat. The *G*~oh,*i*~(*θ*, *ξ*) map confirms that the overhangs exhibit only the dissociated state (*ξ* = 0) at these large angles (Figure [3B](#F3){ref-type="fig"}). At *θ* ≈ 40°, the strands begin to interact (configuration 4 in Figure [3C](#F3){ref-type="fig"}). As the angle decreases further, the strands begin to form unstable base pairs, as noted from the relatively large free energies of the hybridized states (*ξ* = 1--4), which arises from the stretching of the overhangs to form base pairs (configuration 3). Concurrently, the free energy of the dissociated state (*ξ* = 0) begins to rise, evidently from the increasing confinement imposed by the hinge arms, which leads to increasing entropy loss and electrostatic repulsion between the overhangs. It is this increase in the dissociated-state free energy that is apparently responsible for the observed increase in the overall free energy with decreasing angle. At the peak of the energy barrier, the hybridized and dissociated overhangs exhibit similar free energies and are therefore more or less equally prevalent. At smaller angles, the hybridized states start to become increasingly dominant, with the fully-hybridized state (*ξ* = 4) becoming highly favored at the free energy minimum (configuration 2). Thereafter, the angle becomes severely constricted, and the free energy of all overhang states, both hybridized and dissociated, shoot up due to strong compression of the overhangs by the two arms (configuration 1). The *G*~oh,*i*~(*θ*, *ξ*) map in Figure [3B](#F3){ref-type="fig"} shows a visible shift in the population of overhang conformations from dissociated to hybridized states with decreasing angle. This transition can be better captured and related to the shape of the energy landscape by examining the *combined* free energy of all partly- to fully-hybridized states $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$G_{\text{hyb},i}(\theta ) \equiv \sum _{\xi =1}^{4} G_{\text{oh},i}(\theta ,\xi )$\end{document}$ along with that of the fully dissociated state *G*~dis,*i*~(*θ*) ≡ *G*~oh,*i*~(*θ*, *ξ* = 0), where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$e^{-G_{\mathrm{oh},i}/k_{\mathrm{B}}T} = e^{ -G_{\text{hyb},i}/k_{\mathrm{B}}T} + e^{-G_{\text{dis},i}/k_{\mathrm{B}}T}$\end{document}$. The *G*~hyb,\ *i*~(*θ*) and *G*~dis,\ *i*~(*θ*) profiles plotted in Figure [3A](#F3){ref-type="fig"} inset exhibit opposite trends with respect to *θ*, namely, *G*~hyb,*i*~(*θ*) becomes more favorable with decreasing angle due to increased stabilization of base-pairing interactions while *G*~dis,*i*~(*θ*) becomes less favorable due to entropy loss, and both profiles rise sharply at small angles due to strand compression. Apart from echoing these results gleaned from the *G*~oh,*i*~(*θ*, *ξ*) map, the *G*~hyb,*i*~(*θ*) and *G*~dis,*i*~(*θ*) profiles importantly also illustrate the sharp nature of the transition from dissociated to hybridized states as the hinge angle is decreased and its role in producing the energy barrier in the landscape. In particular, the two profiles intersect at an angle (say *θ*~p~) that coincides with the location of the *peak* of the barrier, with hybridized states dominating the landscape at smaller angles and dissociated states dominating at larger angles. This is noted from *G*~hyb,*i*~(*θ*) and *G*~dis,*i*~(*θ*) profiles virtually tracing out the *G*~oh,*i*~(*θ*) landscape in the *θ* \< *θ*~p~ and *θ* \> *θ*~p~ regions. The narrowness of the angle space about the barrier peak where *both* the hybridized and dissociated states contribute substantially to the overhang free energy landscape illustrates the sharp nature of this transition with respect to the hinge angle typical of a first-order transition. Based on these results, the free energy landscape may be divided into the four 'zones' as depicted in Figure [3A](#F3){ref-type="fig"}: (i) *Steric repulsion* zone, where the overhangs are severely compressed between the hinge arms, resulting in large steric and electrostatic repulsion that prevents duplex formation but allows intermittent base-pairing interactions. (ii) *Hybridization* zone, where the separation distance between the overhang attachment points and the free volume between the hinge arms is in the range for the overhangs to form a stable duplex. (iii) *Dissociation* zone, where the separation distance goes beyond this ideal range for strand hybridization, causing the overhangs to prefer the dissociated state even though they continue to collide within the confined space and feel some steric and electrostatic repulsion from each other. (iv) *Non-interaction* zone, where the separation distance is so large that the strands do not interact with each other and exhibit dynamics independent of each other. Effects of overhang position and cation concentration {#SEC3-2} ----------------------------------------------------- The free energy landscape *G*~oh,*i*~(*θ*) of overhang pairs corresponding to 10 distinct connection points (C1--C10) at 3 ion concentrations (\[Na^+^\] = 0.2, 0.4 and 1.2 M) are collectively shown in Figures [4A](#F4){ref-type="fig"}--[C](#F4){ref-type="fig"}; the free energy contributions *G*~oh,*i*~(*θ*, *ξ*) from the various overhang hybridization states for each of these free energy landscapes are provided in [Supplementary Figure S7](#sup1){ref-type="supplementary-material"}. Most of the landscapes exhibit the characteristic features of a plateau, a barrier and a sharp minimum at large, intermediate, and small hinge angles discussed earlier. However, some of the landscapes, namely those computed at low \[Na^+^\] for overhangs attached far from the hinge vertex (C6--C10 at 0.2 M, and C9 and C10 at 0.4 M), do not display the minimum or the complete barrier. For these systems, the minimum is either missing or appears at very small angles where overhang conformations were challenging to sample in simulations. ![Effects of attachment location and salt concentration on the overhang free energy landscape. (A--C) Free energies *G*~oh,*i*~(*θ*) plotted as a function of hinge angle for a pair of overhangs with 4 complementary bases attached at the various possible attachment sites (C1 through C10) computed at three different cation concentrations: (**A**) \[Na^+^\] = 0.2 M, (**B**) \[Na^+^\] = 0.4 M and (**C**) \[Na^+^\] = 1.2 M. (**D--F**) Same free energies plotted instead as a function of the separation distance between the two attachment sites of the overhangs. The free energy curves are vertically shifted based on their minimum value for clarity.](gkz1137fig4){#F4} More importantly, the landscapes display considerable differences between overhangs attached at the different connection points. In particular, the heights of the energy barriers at all three ionic conditions were found to decrease with increasing separation distance between the overhangs and the hinge vertex, that is, going from C1 to C10. Furthermore, the onset of the energy barrier and its width in angular terms both decrease with increasing distance of the overhangs from the vertex. This effect is largely geometric in nature, as overhangs distant from the vertex require smaller angles to come into close proximity to interact compared to those attached near the vertex. Hence, a more informative coordinate for revealing differences in interactions between overhang pairs connected at different distances from the vertex is the Euclidean distance *d* between the pair of connection points on the arms, which we term 'end-to-end distance' and calculate as *d* = 2*l*  sin (*θ*/2), where *l* is the distance of the connection points from the vertex. Indeed, when the landscapes of C1 through C10 are plotted in terms of their end-to-end distances (Figures [4D](#F4){ref-type="fig"}--[F](#F4){ref-type="fig"}), the barrier onset and width become more similar across all overhangs. However, these 'normalized' landscapes now exhibit the opposite trend whereby the barrier onset and width *increases* with increasing distance of the overhang attachment point from the vertex, suggesting the intriguing proposition that overhang pairs attached far from the vertex exhibit longer-ranged interactions compared to those close to the vertex. The free energy landscapes also exhibit different sensitivities to changes in ion concentration depending on the attachment position of the overhangs, which are better visualized in Figure [4D](#F4){ref-type="fig"}--[F](#F4){ref-type="fig"}. While the energy landscapes for overhangs attached close to the hinge vertex C1 to C3 are negligibly affected by changes in ion concentration, those for the more distant overhangs C4 through C10 show an increase in free energy gain from strand hybridization at small hinge angle with increasing ion concentration. To investigate the origin of these attachment position-dependent differences in the free energy landscapes of the overhangs, we picked for further analysis two extreme locations of the overhangs, namely positions C1 and C10 closest and farthest from the vertex. The C1 overhang exhibits the narrowest and largest energy barrier and low sensitivity to ionic conditions, whereas the C10 overhang exhibits the broadest and lowest barrier and highest ion sensitivity. Using the procedure described earlier, we decomposed the overall free energy of the two overhangs obtained at two ionic concentrations (0.2 and 1.2 M) into contributions *G*~hyb,*i*~ arising from the subensemble of partly- to fully-hybridized states and *G*~dis,*i*~ arising from dissociated overhangs (Figure [5A](#F5){ref-type="fig"} and [B](#F5){ref-type="fig"}). We also carried out simulations of a single overhang attached to a repulsion plane (without the opposite overhang and repulsion plane), and analyzed the resulting conformational ensemble of the overhang. The results are plotted as a probability density map of the positions of the overhang bases projected onto a plane normal to the repulsion plane (bottom panel of Figure [5D](#F5){ref-type="fig"}). ![Effects of ion concentration and hinge confinement on the free energy landscape of the overhangs. (**A**) Comparison of free energy contributions from hybridized (dashed lines) and dissociated states (dotted lines) at \[Na^+^\] = 0.2 M (blue) and 1.2 M (red) for C1 overhangs. (**B**) Same comparison carried out for the C10 overhangs. (**C**) Free energy landscape of *free* overhangs at \[Na^+^\] = 1.2 M (solid green lines) along with contributions from hybridized and dissociated states overhangs (dashed and dotted green lines). The free energy landscape for confined C1 overhangs at the same ion concentration is shown for reference (solid red lines). (**D**) Probability distribution in the position of nucleotide bases for free overhangs (top) and overhangs confined by repulsion plane (bottom) along with cartoon representations of the probability distributions used in (A)--(C) to illustrate differences in the frequency of interactions between overhangs and or their ability to avoid each other (see text). Increasing brightness in the distribution maps and increasing color intensity in cartoons indicate increasing probability.](gkz1137fig5){#F5} The significantly larger energy barrier to hybridization displayed by the C1 overhang pairs compared to the C10 overhangs obviously stems from the much sharper rise in *G*~dis,*i*~(*d*) of C1 overhangs with decreasing distance *d* between repulsion planes as compared to C10 overhangs (*cf*. red dotted curves in Figure [5A](#F5){ref-type="fig"} and [B](#F5){ref-type="fig"}). As discussed earlier, this rise in *G*~dis,*i*~ is related to the entropic penalty of squeezing the overhangs in their fully dissociated state. This implies that the C1 overhang pair that interact within the confinement imposed by tilted repulsion planes incur much larger entropic penalty than the C10 overhangs, which interact within near-parallel planes. To explain this difference in entropy, we turn to the conformational behavior of a surface-tethered overhang strand provided in the bottom panel of Figure [5D](#F5){ref-type="fig"}. We observe that the overhang bases reside mostly within a narrow and short conical region about the attachment point, and the base probability density decays sharply to zero outside this region. Consider now the overlap between two such conical distributions representing interactions between a pair of surface-tethered overhangs over an 'intermediate' distance (e.g. *d* ∼ 3 nm) where strands interact with each other but cannot form stable base pairs. When the repulsion planes are tilted relative to each other, corresponding to the C1 overhangs, their conical distributions are both pushed towards one side, implying that the two overhangs are highly likely to 'bump' into each other, explaining the large loss in their conformational entropy (Figure [5A](#F5){ref-type="fig"}, left cartoon). In contrast, parallel planes corresponding to the C10 overhangs give the strands the best chance to 'avoid' each other, and hence this configuration leads to small entropic losses (Figure [5B](#F5){ref-type="fig"}, left cartoon). The earlier onset of the barrier of the C10 overhangs (4.5--6 nm for the three ionic conditions) compared to C1 overhangs (3.5--4 nm), which also leads to differences in their barrier widths, may also be explained using their conformational behavior. As discussed above, the two conical density distributions point head-on towards each other in the parallel-plane geometry exhibited by the C10 overhangs (Figure [5B](#F5){ref-type="fig"}, right cartoon), whereas the distributions approach each other at an angle in the tilted-plane geometry of the C1 overhangs (Figure [5A](#F5){ref-type="fig"}, right cartoon). At the large separation distances relevant here (e.g. *d* ∼ 5 nm), both sets of conical distributions appear to exhibit similar overlaps. However, the C10 distributions overlap with their higher probability-density regions, which results in some entropy loss, whereas the C1 distributions overlap with their low probability-density regions, which sacrifices negligible entropy. Thus, the *G*~dis,*i*~(*d*) profiles for the C10 overhangs begin to rise at large distance as compared to C1 overhangs. The free energy contributions shown in Figures [5A](#F5){ref-type="fig"}, B also reveal that the primary source of the difference in the sensitivity of the C1 and C10 overhangs to ionic conditions is the free energy of partly- to fully-hybridized states. Specifically, the *G*~hyb,*i*~ curve for the C10 overhangs shifts downwards by ∼10*k*~B~*T* when \[Na^+^\] is increased from 0.2 to 1.2 M, consistent with the increased stabilization of the hybridized states of the overhangs due to increased electrostatic screening of their charged phosphate backbones. However, the corresponding *G*~hyb,*i*~ curve for the C1 overhangs exhibits a much smaller shift, indicating that the tilted orientation of the repulsion planes somehow makes strand hybridization less sensitive to salt concentration. While the origin of this effect is not fully understood, it is likely related to the stronger confinement imposed by the tilted planes that limits the conformational space available to the overhangs, as explained above. In this regime dominated by steric interactions, the effects of salt concentration become less important, leading to smaller variations in *G*~hyb,*i*~ with changing \[Na^+^\]. As expected, the dissociated-state free energy curves *G*~dis,*i*~ of *both* C1 and C10 overhangs remain largely unaffected by changes in ionic concentration, given that dissociated overhangs by definition remain physically separated from each other and therefore exhibit weak electrostatic interactions. Lastly, we examined how the free energy landscapes of the *confined* overhangs shown in Figure [4](#F4){ref-type="fig"} compare against those of *free* overhangs in solution (without repulsion planes). Figure [5C](#F5){ref-type="fig"} presents such a comparison for a pair of free overhangs at 1.2 M Na^+^ against the confined C1 overhangs at the same salt concentration. Since a hinge angle cannot be defined for free strands, all landscapes are plotted with respect to the end-to-end distance. Interestingly, the free overhangs exhibit a larger energy barrier than the confined overhangs, even the most confined ones (C1), and also do not exhibit a globally stable hybridized state unlike the confined strands. This is evidently due to the larger entropy loss incurred by the free strands when they hybridize compared to confined strands whose conformational freedom is already quite constrained as a result of the repulsion planes. Thus, for the free strands, the free energy gained by strand hybridization is insufficient to overcome the large entropy loss, resulting in a metastable hybridized state at 0.5 nm end-to-end distance. In addition, the free strands exhibit a broad energy barrier comparable to that of the C10 overhangs, but wider than that of the C1 overhangs. This observation may be explained using the same effect used earlier to explain the broader barrier displayed by the C10 strands *versus* the C1 strands. In other words, the spherically symmetric density distribution of the opposing free strands (Figure [5D](#F5){ref-type="fig"}, top panel) point head-on towards each other similar to the conical distributions of the C10 strands (Figure [5C](#F5){ref-type="fig"}, left cartoon), which implies that the two free strands 'feel' each other earlier than the C1 overhangs which approach each other at an angle (Figure [5C](#F5){ref-type="fig"}, right cartoon). Effects of overhang length and sequence {#SEC3-3} --------------------------------------- Apart from overhang attachment position (relative to hinge vertex) and salt concentration, the sequence design of the overhangs is also expected to affect their free energy landscape. We have so far examined 7-base long overhangs with 4-base 'sticky' ends (GTCG and complement CGAC) and 3-base linkers (TTT); we denote this design by '4b3T'. To explore the effects of overhang sequence and gain deeper understanding of strand hybridization under confinement, we examined three *additional* designs, namely, overhangs with (i) longer, 6-base sticky ends of complementary sequences GTCGGC and GCCGAC, but unchanged linkers, which we denote by '6b3T'; (ii) longer, 5-base linkers (of sequence TTTTT) but unchanged sticky ends denoted by '4b5T'; and (iii) longer sticky ends *and* linkers of the above sequences denoted by '6b5T'. Figures [6A](#F6){ref-type="fig"}--[C](#F6){ref-type="fig"} compares the free energy landscapes of the four overhang designs for three different attachment positions (C2, C5 and C10). Our results show that changing the lengths of the linkers and the sticky ends both have strong effects on the landscape. Increasing the linker length from 3 to 5 bases, while keeping the sticky end unchanged, leads to higher and broader energy barriers and a shallower energy minimum. Both effects are nicely captured in Figure [6D](#F6){ref-type="fig"}, which shows a zoomed view of the landscapes of the 4b3T and 4b5T overhangs (attached at position C2) along with free energy contributions *G*~hyb,*i*~ and *G*~dis,*i*~ arising from their hybridized and dissociated states. The broader and higher barrier exhibited by the 4b5T overhangs is directly related to their longer *overall* length as compared to the 4b3T overhangs. Thus, the 4b5T overhangs begin to interact with each other at larger hinge angles, explaining the earlier onset of their barrier, and they also incur larger entropic penalty than the 4b3T overhangs at similar levels of confinement (hinge angles), explaining the higher barrier and lower stability of the hybridized state. {#F6} Increasing the length of the sticky end from 4 to 6 bases, keeping linker length unchanged, leads to significantly deeper energy minima, and generally slightly higher and wider barriers (see Figure [6E](#F6){ref-type="fig"} comparing the landscapes of 4b3T and 6b3T overhangs at position C2). The deeper minimum displayed by the 6b3T overhangs obviously arises from their longer sticky ends which exhibit stronger hybridization; their higher and wider barrier is again related to their longer overall length compared to 4bT3 overhangs, as discussed above. Lastly, increasing the lengths of *both* the linker and the sticky end, i.e. in going from 4bT3 to 6bT5, leads to a combination of the two sets of effects discussed above, which we again demonstrate using the example of the C2-attached overhangs (Figure [6F](#F6){ref-type="fig"}). First, we observe that the depth of the energy minimum for the 6bT5 overhangs is intermediate to that of the 4bT3 and 6bT3 overhangs discussed earlier. While the sticky ends in the 6bT5 and 6bT3 overhangs are identical, the former are overall longer, which leads to stronger entropic repulsion and thereby less favorable hybridization. Second, based on their longer overall length, the 6bT5 overhangs exhibit higher and wider energy barriers than both 4bT5 and 6bT3 overhangs. The longer length also implies that the location of the energy minimum for the 6bT5 overhangs is the most shifted to wider hinge angles. Interestingly, the free energy landscapes for the 4bT5 and 6bT3 overhangs with different linkers and stick ends *but* identical overall lengths exhibit similar locations of their minima. The above results thus demonstrate that the lengths of the linker and sticky portions of the overhangs both affect the stability of the hybridized state of the overhangs and the height and width of the energy barrier. In general, long sticky ends with short linkers will prefer the fully hybridized state (e.g. 6b3T overhangs) and short sticky ends with long linkers will prefer to stay dissociated (e.g. 4b5T overhangs). Bare hinge and overall free energy landscapes {#SEC3-4} --------------------------------------------- Having thoroughly studied the free energies *G*~oh,*i*~(*θ*) of individual overhangs, we next investigated the free energy landscape *G*~bh~(*θ*) of the bare hinge and the free energy landscape *G*~hinge~(*θ*) of the full hinge complete with *all* the overhangs. The computed free energy landscape of the bare hinge is shown in Figure [7A](#F7){ref-type="fig"}. The landscape exhibits a largely parabolic shape with a minimum at 80°, which matches very well with the equilibrium hinge angle measured experimentally ([@B22]). However, the energy landscape rises much more steeply compared to that inferred from the experimental hinge-angle distribution (Figure [7A](#F7){ref-type="fig"} and [Supplementary Figure S8](#sup1){ref-type="supplementary-material"}). This difference could arise due to various factors not accounted in our model, including hinge deformations, structural defects, and ionic and solvation effects, that might relieve the stresses associated with closing the hinge arms. Hence, we chose to use the bare-hinge landscape derived from experiments to keep all subsequent analyses as realistic as possible. ![Overall free energy landscape of the hinge and contributions from the bare hinge and the overhangs. (**A**) Computed free energy landscape of the bare hinge (brown symbols), and the landscape inferred from experiments (violet symbols) along with its harmonic fit (violet line). (**B**) Cumulative free energy landscape at \[Na^+^\] = 0.2 M (cyan lines) and 1.2 M (blue lines) from one overhang attached at each of the 10 positions C1 through C10 (dashed lines) and from nine overhangs attached at each of those positions except C1 (solid lines). (C, D) Overall hinge free energy landscapes obtained at \[Na^+^\] = 0.2 M (**C**) and 1.2 M (**D**) for hinges with one (orange), two (red) and three overhangs (brown) attached at each of the nine positions C2 through C10.](gkz1137fig7){#F7} The experiments studied hinges with 10, 20 and 30 overhang connections corresponding to one, two, and three pairs of overhangs located at each of the positions C1 through C10 on the hinge arms (see Figure [1A](#F1){ref-type="fig"}). Consistent with these experiments, we attempted to compute the cumulative free energy *G*~oh~(*θ*) ≡ ∑~*i*~*G*~oh,*i*~(*θ*) contributed by 10, 20 and 30 pairs of overhangs. However, we found that the overhang pair attached at C1 closest to the vertex contributed excessively large steric repulsion for angles smaller than 10° (Figure [7B](#F7){ref-type="fig"}, dashed lines), essentially preventing the hinge from closing its arms even at the highest salt concentration considered here. This is not consistent with experiments which clearly show hinges in the closed state exhibiting near-zero bending angles. We therefore speculate that the experimental hinges did not properly incorporate these overhangs close to the vertex or that the hinge joint expanded slightly to mitigate the steric repulsion arising from the strongly squeezed C1 overhangs, an effect that is not accounted for by our planar and impenetrable repulsion planes. In addition, the interhelix spacing tends to open up in between crossover junctions, which can provide some room for the overhangs to occupy and reduce steric repulsion. Thus, for all remaining analyses, we considered only the overhangs attached at locations C2 through C10, that is, examining hinges with 9, 18 and 27 overhang connections. Figure [7C](#F7){ref-type="fig"} and [D](#F7){ref-type="fig"} presents the overall free energy landscape of the hinges with 9, 18, and 27 connections at low and high salt concentrations (0.2 and 1.2 M), obtained by summing up contributions from the bare hinge and each overhang pair. Our results reveal that all three hinges strongly prefer to remain open at \[Na^+^\] = 0.2 M, and their closed conformation appears only as a metastable state. In addition, the relative stability of the open state (with respect to the closed state) increases from ∼15 to 36 *k*~B~*T* as the number of connections increases from 9 to 27. In contrast, the hinges strongly prefer the closed state at high salt concentration of 1.2 M. The relative stability of the closed state increases from roughly 13 to 45 *k*~B~*T* with increasing number of connections. Interestingly, the open and closed states are separated by a fairly broad energy barrier (spanning \>20°) whose height also rises with increasing number of connections (from ∼18 to 47 *k*~B~*T*). The generally broad and flat shape of this barrier largely arises from the superposition of barriers contributed by individual overhangs, which are much narrower and offset from each other. These results thus demonstrate how changes in salt concentration could be used to drive the hinges from the open to the closed conformation, and *vice versa*, how changing the number of the connections can be used to sharpen the relative stabilities of the open and closed states and modulate the height of the barrier separating the two states. The hinges explored above considered a fixed distribution of overhang positions, namely, equal number of overhangs at each of the attachment sites C1--C10. However, this distribution could also be used as a variable to tune the actuation response of the hinges, given the sensitivity of overhang free energy landscapes to attachment positions (Figure [4](#F4){ref-type="fig"}). For instance, overhangs attached *very close* to the hinge vertex (e.g. at position C1) facilitate early closing of the hinge, i.e. at large angles, but due to strong steric repulsion, such overhangs cannot fully close the hinge arms. Furthermore, these overhangs introduce a large barrier to hinge closing, and a free energy of hybridization that is favorable at both weak and strong ionic conditions. This suggests that hinges containing only these overhangs will stay partially open, irrespective of ionic conditions, and will thus be unable to undergo salt-mediated actuation. In contrast, overhangs attached at *intermediate* distances (e.g. at positions C2 to C5) should allow the hinge arms to close fully. With increasing distance from the vertex, these overhangs present increasingly smaller barriers and smaller angles for initiating the hinge-closing transition. While these overhangs also display a propensity to stably hybridize under weak and strong ionic conditions, they do so with lower stability at low ionic concentrations. This implies that with a sufficiently large barrier to hinge closing contributed by the bare-hinge landscape, these overhangs should be able to support reversible actuation of hinges via changes in salt conditions. Lastly, overhangs attached *far* from the vertex (e.g. at positions C6 to C10) not only present small energy barriers but also the hybridization thermodynamics necessary for reversible salt-mediated actuation, that is, stable hybridized state at high ion concentration and stable dissociated state at low concentration. However, these overhangs on their own would introduce a narrow energy barrier that does not provide sufficient angular distinction between the open and closed states; in other words, the open hinge state would still allow very small angles to be sampled. Thus, a wide distribution of overhangs spanning distances both proximal and distal from the vertex (but not too proximal like the C1 overhangs) would help produce a broad energy barrier for stronger separation of the open and closed hinge states. Such distribution of overhangs will importantly also prevent accumulation of large energy barriers that might result from colocalization of individual energy barriers contributed by overhangs attached at similar distances from the vertex. Interestingly, our experiments inadvertently used such a distribution strategy to achieve rapid actuation of hinges with millisecond time scales ([@B22]). DISCUSSION {#SEC4} ========== We used molecular simulations to investigate the free energy landscape of reconfigurable DNA origami hinges, specifically to elucidate how collective hybridization and dehybridization of short, single-stranded DNA connections between hinge arms triggered by changes in salt concentration could be used to actuate the hinges between open and closed conformations. To tackle this computationally challenging problem, we devised a strategy where the overall free energy of the hinge was decomposed into contributions arising from the bare hinge and from the individual DNA overhangs attached at different locations on the arms. The two contributions were then separately computed using efficient methods tailored for sampling global hinge motions and local base-pairing interactions, enabling the free energy landscape of the hinges to be determined within reasonable computational costs. Such breakdown of free energies also helped us gain crucial insights into the roles of individual hinge components in dictating the overall actuation response of the hinges. From a broader perspective, this strategy could enable challenging free-energy calculations on other large DNA structures and biomolecular complexes such as proteins involving large, rigid components with fast relaxation times and small, associating elements with slow relaxation times. Our calculations revealed the free energy landscape of individual overhang pairs attached on the hinge arms, providing one of the first detailed picture of DNA hybridization under angular confinement. The landscapes were found to exhibit a sharp minimum at small angles and a broad barrier at intermediate angles that are respectively flanked by a steep repulsive wall at smaller angles and a plateau at larger angles. We analyzed the molecular origins of these features, revealing that: the energy barrier arises from the combined effect of the unhybridized configurations of the overhangs incurring confinement-induced entropy loss and electrostatic repulsion, and the partly and fully-hybridized configurations undergoing distortion to form base pairs; the energy minimum arises from the increased stabilization of the hybridized state brought about by confinement that more than compensates the accompanying entropy loss and electrostatic repulsion; and the repulsive wall arises from steric compression of the overhangs. Interestingly, the tilted confinement imposed by the hinge arms was found to stabilize the hybridized state of the overhangs, which preferred to stay dissociated in the absence of confinement. We showed how free strands incur much larger entropic penalties to hybridization as compared to confined ones whose conformational freedom is already quite restricted due to the hinge arms at large hinge angles. Our results thus reveal a fundamental distinction between DNA hybridization under angular confinement *versus* that in solution. These findings on confined hybridization of end-tethered DNA strands extends beyond the case of salt-mediated actuation; for instance, such a scenario appears during DNA-mediated assembly of faceted nanoparticles into crystals where the surfaces of particles are brought into close proximity at parallel or tilted angles through hybridization of surface-tethered DNA strands ([@B33]). We also studied in detail how the overhang landscapes were impacted by their length and attachment position, and salt concentration. As expected, longer sticky domains in the overhangs led to deeper minima, or stronger hybridization. Overall longer overhangs, irrespective of whether the length is added to the sticky or linker portions, led to higher energy barriers, which was explained in terms of the increased confinement entropy loss incurred by the longer strands. Placing the overhangs farther from the vertex resulted in lower energy barriers, which are also wider when the free energy was plotted as a function of the distance between the complementary overhang attachment sites but narrower when plotted in terms of hinge angle. The first two effects are not so obvious. These were rationalized based on how surface-tethered overhangs exhibit a cone-shaped conformational distribution and how differently two such distributions interact with each other for overhangs attached at different distances from the hinge vertex. The last effect is obviously geometric given that a distant overhang requires smaller changes in hinge angle to traverse the same distance as an overhang attached proximally to the vertex. Lastly, salt concentration seemed to only predominantly affect the landscapes of distal overhangs, causing increased stabilization of the hybridized states with increasing concentration as would be expected from electrostatic screening effects. The landscapes of proximal overhangs were negligibly affected, which we attributed to the dominance of steric effects in such overhangs. Our results thus suggest that: the hybridization strength of the overhangs is best tuned by the length of their sticky ends; the barrier to hybridization is best tuned by the attachment position or length of the linker regions of overhangs, and salt-mediated actuation is best carried out by overhangs distal from the vertex. Finally, we analyzed the overall free energy landscape of the full hinge obtained by combining the landscapes computed for all overhangs and the bare hinge. Such a landscape holds crucial information about the actuation behavior of the hinges: First, the salt dependence of the free energy difference Δ*G*~c-o~(\[Na^+^\]) between the closed (c) and open (o) states dictates the actuation response *P*~c~(\[Na^+^\]) of the hinges via *P*~c~ ≃ exp (−Δ*G*~c-o~/*k*~B~*T*)/\[1 + exp (−Δ*G*~c-o~/*k*~B~*T*)\], where *P*~c~(\[Na^+^\]) is the equilibrium probability of observing the closed state. Second, the shape of the energy landscape governs the kinetics of actuation; for instance, based on Kramers theory ([@B39],[@B40]), the rate constant *k* of the hinge-closing transition would be given by *k* ≃ *k*~0~exp (−Δ*G*~b-o~(\[Na^+^\])/*k*~B~*T*), where Δ*G*~b-o~ is the height of the energy barrier (b) relative to the open state and *k*~0~ is related to the solvent friction coefficient and the curvatures of the free energy minimum and barrier. We started by examining the landscapes of three experimentally-studied hinge designs containing different numbers of overhang connections distributed evenly across the hinge arms. The computed landscapes exhibited an energy minimum at the closed state and a broad energy barrier separating the closed and open states. The landscapes nicely captured the increasing propensity of the experimental hinges to remain open (at low salt) or closed (at high salt) with increasing number of connections. The landscapes further revealed proportional increase in the height of the barrier with the number of connections, suggesting that while simple scale-up of the number of connections helps sharpen the actuation response of the hinge, it could also lead to slower actuation kinetics. Next, we demonstrated how the actuation response and kinetics, related to features of the landscape, could be tuned by manipulating the positional distribution of the overhangs, enabling combinations of sharp or dull actuation responses with fast or slow kinetics to be achieved. This feature along with the ability to tune and predict individual energy landscapes contributed by each overhang pair should enable design of hinges with the desired responses and kinetics. While our computations provided many useful insights and predictions on confined hybridization and salt-mediated actuation, several approximations in the approach may limit its accuracy and applicability. First, the oxDNA model used for modeling the hinge in our approach uses a Debye-Hückel approximation to treat electrostatic screening by ions. Hence, our approach is limited to studying actuation by monovalent ions such as Na^+^, and not multivalent ions such as Mg^2+^ and Spd^3+^ that have more complex effects. Second, our previous work showed that DNA hinge joints allow for some secondary motions like sliding and twisting, in addition to its primary bending motion. These additional modes, which could provide opportunities for the hinge arms to relieve strong steric repulsion from the overhangs at small hinge angles, are currently ignored in our free energy calculations, which assume a single reaction coordinate, the hinge angle. Third, the repulsion planes we used for enabling the otherwise computationally challenging overhang free energy calculations treat the hinge arms as rigid planes. In reality, the hinge arms display a corrugated surface, with gaps between DNA helices that become wider between crossover junctions, and the arms can also potentially undergo deformation during severe compression of the overhangs. As discussed earlier, both these effects could also help mitigate steric interactions between overhangs at small hinge angles. Fourth, the overall free energy landscapes presented in Figure [7C](#F7){ref-type="fig"} and [D](#F7){ref-type="fig"} for hinges with 18 and 27 overhang pairs assumed that all copies of overhang pairs at positions C2--C10 exhibit identical free energies. However, this may not be true as part of these copies of overhangs may be attached at the outermost helices, where they will experience lesser confinement effects. Further work is required to address each of these effects and to improve upon these approximations to provide finer, more accurate descriptions of salt-mediated actuation. CONCLUSION {#SEC5} ========== We recently proposed a new approach for actuating DNA nanodevices that involves integrating short, complementary ssDNA overhangs to their structural elements, which can then be latched together or unlatched via collective hybridization or dehybridization of the overhangs triggered by changes in solution ionic conditions. In this study, we used molecular simulations and free energy calculations to establish the molecular-thermodynamic basis of this actuation approach, using DNA origami hinges as our model system. To enable calculation of free energies at reasonable costs, we decomposed the overall free energy of the hinge into contributions from the bare hinge and from each pair of overhang attached to the hinge arms, allowing each contribution to be computed separately in a tractable manner. The resulting free energy landscapes revealed the stable, metastable and transition states exhibited by the hinges, and also provided predictions on the relative stability of the open *versus* closed states of the hinges and of the height of energy barrier separating the two states. Our free energy decomposition strategy allowed us to dissect these features of the landscape in terms of contributions from the bare hinge and from each hybridizing pair of overhangs. By elucidating the dependence of the overhang free energy contributions on salt concentration, we were able to uncover the molecular origins of our salt-based actuation method. Furthermore, by elucidating the effects of the length, sequence, and attachment position of the overhangs, we were able to provide simple guidelines for designing and tuning the actuation behavior of the hinges. Lastly, we discovered a stabilizing role of confinement on the hybridization between tethered DNA strands, an effect that has applications beyond this actuation method. Together, these results provide a significantly deeper fundamental understanding of the mechanism behind a new, promising approach for reconfiguring nanodevices. Supplementary Material ====================== ###### Click here for additional data file. We thank Dr Carlos Castro and Dr Thomas Ouldridge for useful discussions. SUPPLEMENTARY DATA {#SEC6} ================== [Supplementary Data](https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkz1137#supplementary-data) are available at NAR Online. FUNDING {#SEC7} ======= National Science Foundation (NSF) \[CMMI-1921955 to G.A.\]; Computational resources were provided by the NSF XSEDE program \[ACI-1053575\]. Funding for open access charge: NSF \[CMMI-1921955\]. *Conflict of interest statement*. None declared.

Introduction ============ Fatigue is a common non-specific symptom experienced by many people and is associated with many health conditions. Often defined as an overwhelming sense of tiredness, lack of energy and feeling of exhaustion, fatigue relates to a difficulty in performing voluntary tasks.^[@bib1]^ Fatigue accumulation, if not resolved, leads to overwork, chronic fatigue syndrome (CFS), overtraining syndrome, and even endocrine disorders, immunity dysfunction, organic diseases and a threat to human health. There are many different fatigue classification methods. According to its duration, fatigue can be classified into acute fatigue and chronic fatigue. Acute fatigue can be quickly relieved by rest or life-style changes, whereas chronic fatigue is a condition defined as a persistent tiredness lasting \>months that is not ameliorated by rest.^[@bib2],\ [@bib3],\ [@bib4]^ Fatigue can also be classified as mental fatigue, which refers to the cognitive or perceptual aspects of fatigue, and physical fatigue, which refers to the performance of the motor system.^[@bib1]^ Muscle fatigue is defined as a decrease in maximal force or power production in response to contractile activity.^[@bib5]^ It can originate at different levels of the motor pathway and is usually divided into central and peripheral components. Peripheral fatigue is produced by changes at or distal to the neuromuscular junction. Central fatigue originates at the central nervous system (CNS), which decreases the neural drive to the muscle.^[@bib5],\ [@bib6]^ Muscle fatigue is a commonly experienced phenomenon that limits athletic performance and other strenuous or prolonged activity. It is also increases and restricts daily life under various pathological conditions, including neurological, muscular and cardiovascular disorders, as well as aging and frailty. This review primarily focuses on muscle fatigue, particularly during intense exercise, to provide a basic understanding and potential therapies for muscle fatigue. Factors that affect muscle contraction and fatigue ================================================== The production of skeletal muscle force depends on contractile mechanisms, and failure at any of the sites upstream of the cross-bridges can contribute to the development of muscle fatigue, including nervous, ion, vascular and energy systems.^[@bib7]^ Specifically, metabolic factors and fatigue reactants during the process of contraction, such as hydrogen (H^+^) ions, lactate, inorganic phosphate (Pi), reactive oxygen species (ROS), heat shock protein (HSP) and orosomucoid (ORM), also affect muscle fatigue. Neural contributions -------------------- Central neurotransmitters, especially 5-HT, DA and NA, play important role during whole-body exercise and fatigue. 5-HT produces a negative effect, whereas methylphenidate, a DA-releasing enhancer and reuptake inhibitor, produces a positive effect in exercise performance.^[@bib8]^ The so-called central fatigue hypothesis states that exercise induces changes in the concentrations of these neurotransmitters, and fatigue arises from changes within the CNS (or proximal to the neuromuscular junction). However, recent data have shown that drugs influencing the neurotransmitter systems scarcely perturb performance under normal ambient temperatures but significantly improve endurance under high ambient temperatures. For example, the NA reuptake inhibitor reboxetine and a dual DA/NA reuptake inhibitor, bupropion, have a negative effect^[@bib9],\ [@bib10],\ [@bib11]^ on exercise performance under normal temperature. However, under heat, reboxetine decreases, whereas bupropion increases performance, thus suggesting that the thermoregulatory system may have an important influence on exercise performance. The CNS, via a central neurotransmitter, produces various excitatory and inhibitory inputs on the spinal motoneurons, thus ultimately activating motor units (MUs) to achieve the force output. The strength and timing of contraction are controlled by the firing of the motoneurons. When first recruited in a healthy system, MUs usually fire at 5--8 Hz. During brief nonfatiguing voluntary contractions in humans, the mean MU firing rates are 50--60 Hz.^[@bib12]^ MUs are recruited or derecruited in an orderly fashion on the basis of the motoneuron size, and they essentially control the amount of muscle tissue being activated.^[@bib13]^ Slowing or cessation of MU firing contributes to the loss of force that marks fatigue. Motoneuron firing is influenced by intrinsic changes in the motoneuron properties, descending drive and afferent feedback. During fatiguing maximal contractions, motoneuron firing rates decrease because of the following factors: (1) Repetitive activation (repeated firing) of motoneurons leads to a decrease in their excitability to excitatory synaptic input;^[@bib14]^ (2) the excitatory drive from the motor cortex or other supraspinal area to the motoneurons is lower;^[@bib14]^ (3) the firing of group III/IV muscle afferents is increased,^[@bib15],\ [@bib16]^ thus decreasing motoneuron firing; (4) the firing of muscle spindles (sensory receptors) is decreased, thus decreasing firing of group Ia muscle afferents, increasing presynaptic inhibition, and finally decreasing motoneuron firing;^[@bib17],\ [@bib18]^ (5) specifically, group III/IV muscle afferents also exhibit feedback interaction with cardiovascular and respiratory processes via the autonomic nervous system, thereby improving muscle blood flow and oxygenation and consequently slowing the development of fatigue of the muscle itself.^[@bib14]^ Ca^2+^ ------ Neural activation results in signal transmission from the brain to the muscle's transverse tubules, inducing calcium release from the sarcoplasmic reticulum (SR) into the cytosol and initiation of cross-bridge cycling. This excitation-contraction coupling process involves the following events: the action potential (AP) is generated at the neuromuscular junction and propagates along the surface membrane and into the transverse tubules, where it is detected by voltage-sensor molecules (the dihydropyridine receptors, VS/DHPRs), which in turn open the ryanodine receptor-Ca^2+^ release channels (RyR1 isoform in skeletal muscle) in the adjacent SR and cause release of Ca^2+^ into the sarcoplasm.^[@bib19]^ The binding of Ca^2+^ to troponin moves tropomyosin away from the myosin-binding site on actin, thus permitting cross-bridge cycling. The removal of Ca^2+^ from the cytoplasm by Ca^2+^ ATPase results in the recovery of tropomyosin to its blocked position, and relaxation occurs.^[@bib20]^ Impaired calcium release from the SR has been identified as a contributor to fatigue in isolated skeletal muscle fibers. Several possible mechanisms have been proposed: (1) AP involves Na^+^ influx, and the subsequent repolarization involves K^+^ efflux in muscle cells. High-frequency stimulation may lead to extracellular K^+^ accumulation, which may decrease voltage sensor activation and the action potential amplitude; (2) Most of the ATP in a rested fiber is Mg^2+^ bound. Fatigue can induce a decrease in intracellular ATP and an increase in free Mg^2+^, thus decreasing the effectiveness of SR Ca^2+^ channel opening; (3) Exposure to myoplasmic phosphate causes a sustained decrease in SR Ca^2+^ release in skinned fiber because inorganic phosphate can enter the SR and precipitate Ca^2+^, thus decreasing the free Ca^2+^ and amount of Ca^2+^ available for release.^[@bib21]^ Blood flow and O~2~ ------------------- Blood flow can bring oxygen necessary for aerobic ATP production and remove by-products of metabolic processes in working muscles, thus playing an important role in the maintenance of force output. Muscle voluntary contractions increase the mean arterial blood pressure,^[@bib22]^ which consequently decreases the net blood flow to the working muscle and induces fatigue.^[@bib23]^ The occlusion of blood flow to a working muscle substantially decreases the time to exhaustion ^[@bib24],\ [@bib25],\ [@bib26]^ and increases the magnitude of the decline in force,^[@bib27],\ [@bib28]^ thus indicating the potential importance of blood flow in fatigue prevention. However, despite changes in blood flow accompanying the development of muscle fatigue, decreased blood flow does not seem to be a key factor in the development of fatigue. Wigmore *et al.*^[@bib29]^ have used venous occlusion plethysmography to decrease blood flow of the ankle dorsiflexor muscles, and have found that the decline in MVC force precedes significant changes in blood flow to the muscle. One of the important roles of blood flow is to provide O~2~ to the working muscles. It has been well documented that decreased oxygen availability to exercising muscle has profound consequences on muscle fatigue. Breathing hypoxic air can significantly increase muscle fatigue *in vivo*,^[@bib30],\ [@bib31]^ and enhanced O~2~ delivery to the exercising muscles^[@bib32]^ directly attenuates muscle fatigue and increases muscle efficiency. However, O~2~ availability affects the fatigue process at moderate work intensities. Generally, oxygen uptake and ATP utilization are increased until the VO~2max~ is reached. During exercise at a very high intensity (usually the VO~2max~ is already reached), the demand for more ATP cannot be met by increases in oxygen delivery, thus resulting in an imbalance of metabolic homeostasis and leading to fatigue.^[@bib33]^ Energy ------ Muscular work must be supported by a ready supply of ATP energy. There are three major ATPases that require ATP for muscle activity: Na^+^/K^+^-ATPase, myosin ATPase and Ca^2+^ ATPase. The Na^+^/K^+^-ATPase pumps Na^+^ back out and K^+^ back into the fiber after an action potential. The myosin ATPase uses ATP to generate force and do work, and the Ca^2+^ ATPase pumps Ca^2+^ back into the SR, thus allowing for muscle relaxation. The activities of these enzymes account for 10%, 60% and 30% of total ATP use, respectively.^[@bib34]^ Glycogen is the carbohydrate energy store for ATP production. There are three distinct subcellular localizations of glycogen: (1) intermyofibrillar glycogen, located between the myofibrils and close to SR and mitochondria; (2) intramyofibrillar glycogen, located within the myofibrils and most often in the I-band of the sarcomere; and (3) subsarcolemmal glycogen, located beneath the sarcolemma and primarily next to mitochondria, lipids and nuclei. Approximately 75% of the total glycogen store in the cells is intermyofibrillar glycogen.^[@bib35],\ [@bib36]^ It is a fundamental concept in exercise physiology that glycogen is an important fuel during exercise.^[@bib37]^ As early as the 1960s, a strong correlation between muscle glycogen content and exercise endurance was found.^[@bib38]^ When glycogen stores are limited, exercise cannot continue.^[@bib39]^ Glycogen oxidation is a major source for ATP regeneration during prolonged exercise (\>1 h) and high-intensity intermittent exercise.^[@bib40]^ Furthermore, glycogen may be important because it produces tricarboxylic acid cycle intermediates, thus contributing to the maintenance of oxidative metabolism.^[@bib41]^ Excitation-contraction coupling and relaxation have been reported to be affected by glycogen levels.^[@bib37],\ [@bib42],\ [@bib43]^ Low-muscle glycogen and/or glycolytic-derived energy are associated with impaired SR Ca^2+^ release, reuptake, and Na^+^/K^+^-pump function.^[@bib43],\ [@bib44]^ However, how glycogen depletion affects the series of events and ultimately results in fatigue are not fully understood. Metabolic factors ----------------- Muscle contractions activate ATPases and promote glycolysis, thus leading to an increase in intracellular metabolites, such as H^+^, lactate, Pi and ROS, which contribute to the changes in cross-bridge activity. Historically, H^+^ has been thought to have a role in the development of muscle fatigue. Glycolysis leads to the production of pyruvate, which feeds into the TCA cycle for oxidation. If pyruvate production exceeds its oxidation, excess pyruvate is converted into lactic acid, which dissociates into lactate and H^+^. The accumulation of H^+^ lowers the pH, thus potentially interfering with SR Ca^2+^ release, troponin C sensitivity to Ca^2+^ and cross-bridge cycling and resulting in impaired muscle force.^[@bib45]^ However, the role of decreased pH as an important cause of fatigue is now being challenged.^[@bib46]^ Several recent studies have shown that decreased pH may have little effect on contraction in mammalian muscle at physiological temperatures. Furthermore, there is a lack of association between changes in pH and MVC throughout fatiguing exercise and in recovery in humans.^[@bib47]^ In addition to acidosis, anaerobic metabolism in skeletal muscle also involves hydrolysis of creatine phosphate (CrP) to creatine and Pi. The concentration of Pi can increase rapidly from approximately 5--30 m[M]{.smallcaps} during intense fatigue. Creatine has little effect on contractile function, whereas Pi, rather than acidosis, appears to be the most important cause of fatigue during high-intensity exercise.^[@bib48]^ Increased Pi substantially impairs myofibrillar performance, decreases SR Ca^2+^ release and therefore contributes to the decreased activation.^[@bib49]^ Mitochondrial respiration produces ATP and consumes O~2~, a process that generates ROS. The most important ROS include superoxide (O~2~•−), hydrogen peroxide (H~2~O~2~), and hydroxyl radicals (OH•). As the work intensity increases, ROS production increases. The most convincing evidence that ROS contribute to fatigue comes from experiments showing that pretreatment of intact muscle with a ROS scavenger significantly attenuates the development of fatigue. ROS affect muscle fatigue mainly through the oxidation of cell proteins such as the Na^+^--K^+^ pump, myofilaments, DHPR and RyR1,^[@bib50]^ thus leading to the inhibition of SR Ca^2+^ release and myofibrillar Ca^2+^ sensitivity. In addition, ROS activate the group IV muscle afferents^[@bib51]^ and directly inhibit motoneurons. Other metabolites with probable roles in fatigue include ATP, ADP, PCr and Mg.^[@bib52]^ For example, muscle ADP increases with intense contractile activity. In skinned fibers, ADP decreases fiber velocity but increases force, presumably because of more cross-bridges in the high force states. However, the more important role of ADP in eliciting fatigue appears to be related to the inhibition of the SR Ca^2+^ pump and the resulting disturbances in ECC rather than direct effects on the cross-bridge.^[@bib53]^ Fatigue reactants ----------------- Organisms have different levels of adaptive responses to fatigue stress, including the CNS nervous system, sympathetic nervous system, endocrine system (hypothalamus-pituitary-adrenal axis, HPA axis), and innate immune system (that is, non-specific cytokines, complement system and natural killer cells). Many fatigue reactants, such as cortisol, catecholamine, IL-6 and HSPs, may have roles in muscle function.^[@bib54]^ HSPs are involved in the adaptation to fatigue stress. Within the family of HSPs, HSP25 protein is abundantly expressed in skeletal muscle and increases with muscle contractile activity.^[@bib55]^ Interestingly, Jammes *et al.* have reported that a widespread HSP25 response to fatigue in a single hindlimb muscle is responsible for a global adaptive response to acute localized stress and have demonstrated that group III and IV muscle afferents play an important role in the fatigue-induced p-HSP25 response; moreover, the sympathetic nerve supply to the muscles and kidney comprises the efferent arm of the p-HSP25 activation.^[@bib56]^ Skeletal muscle HSP25 has been reported to stabilize muscle structure and repair damaged muscle proteins,^[@bib57]^ as well as to decreases apoptosis in cultured muscle C2C12 cells by inhibiting the intrinsic and extrinsic apoptotic cell death pathway.^[@bib58]^ Orosomucoid (ORM) is an acute-phase protein, with a very low pI of 2.8--3.8 and a very high carbohydrate content of 45%. It is predominantly synthesized in the liver, and many extra-hepatic tissues have also been reported to produce ORM under physiological and pathological stress.^[@bib59]^ Our studies have found that the expression of ORM is markedly increased in the serum, liver and skeletal muscle in response to various forms of fatigue, including sleep deprivation, forced swimming and treadmill running. Interestingly, exogenous ORM increases muscle glycogen and enhances muscle endurance, whereas ORM deficiency results in decreased muscle endurance, thus indicating that ORM is an endogenous anti-fatigue protein. Further studies have demonstrated that ORM binds to C--C chemokine receptor type 5 (CCR5) on muscle cells and activates AMPK, thus promoting glycogen storage and enhancing muscle endurance, and representing a positive feedback mechanism for resisting fatigue and maintaining homeostasis.^[@bib60],\ [@bib61]^ Modulation of the level of ORM and CCR5 signaling may be a novel strategy for the management of muscle fatigue. Non-invasive techniques for the ASSESSMENT OF sites of muscle fatigue ===================================================================== Muscle fatigue is manifested most naturally in the intact organism. Non-invasive techniques of site-specific stimulation can now be used to evaluate the potential sites of the entire system for force production in human studies. All evoked muscle responses are recorded via electromyography (EMG) electrodes placed on the muscle. Transcranial magnetic stimulation --------------------------------- Transcranial magnetic stimulation involves applying magnetic stimulation to the motor cortex and is optimized to activate the muscle of interest.^[@bib1]^ The stimulation-induced muscular response recorded by EMG is known as the motor-evoked potential (MEP). MEP is influenced not only by cortical excitability but also by spinal cord motor neuron excitability and muscle factors. MEP depression can occur in the relaxed muscle after a fatiguing exercise, possibly as a result of afferent input from the fatigued muscle. MEP is increased in the upper- and lower-limb muscles during sustained submaximal isometric contractions and is regarded as an augmentation of the central drive to the lower motoneuron pool that allows a constant level of force to be maintained despite the development of peripheral fatigue. During sustained MVC, MEP has been reported to increase during the first seconds and then to level off, increase linearly or remain stable, depending on the protocol used (that is, continuous vs intermittent) and the muscle investigated.^[@bib1]^ Cervicomedullary region electrical stimulation ---------------------------------------------- Electrical stimulation in the cervicomedullary region aims to activate the corticospinal tract at a subcortical level, thereby eliminating cortical contributions to the evoked muscle response. The muscular response recorded by EMG is known as the cervicomedullary motor-evoked potential (CMEP). Comparison of MEP and CMEP is helpful for the localization of excitability at the cortical or subcortical level. During a sustained 30% MVC of the plantar flexors, a large increase in MEP and only a slight increase in CMEP have been reported, thus suggesting a small contribution of spinal factors to the increase in corticospinal excitability during submaximal fatiguing contractions. In contrast, during 50% MVC of the elbow flexors to task failure, similar MEP and CMEP kinetics has been found, thus indicating that central changes occur almost entirely at the spinal level.^[@bib62],\ [@bib63],\ [@bib64]^ Peripheral nerve low-intensity electrical stimulation ----------------------------------------------------- Low-intensity electrical stimulation of the peripheral nerve preferentially activates the Ia sensory fibers, which synapse with the α-motoneuron in the spinal cord. The signal is then carried along the motor neurons to the muscle, generating a response in the muscle known as the Hoffmann reflex (H-reflex). The H-reflex is used to assess spinal excitability and inhibition. Although there are several of an increase^[@bib65]^ or no change,^[@bib66]^ the general consensus is that there is an overall decline in the amplitude of the H-reflex with the development of muscle fatigue, thus indicating a decrease in spinal excitability.^[@bib67],\ [@bib68]^ The rate and degree of decrease in H-reflex amplitude appear to be dependent on the type of fatiguing task. Peripheral nerve high-intensity electrical stimulation ------------------------------------------------------ High-intensity stimulation of the peripheral nerve directly activates the α-motoneuron, evoking a motor response (m-wave) from the muscle. The m-wave is a compound action potential recorded with surface EMG and is used to assess peripheral excitability of the muscle membrane and transmission at the neuromuscular junction. A change in the twitch force without a change in the m-wave indicates a failure of excitation-contraction coupling. Short-duration fatiguing contractions (\~20 s) induce an enhancement in the amplitude and area of the m-wave.^[@bib69]^ A longer (4-min) sustained maximal contraction does not induce changes in the amplitude of the m-wave^[@bib70]^ but results in a significant decline in the central activation, thus suggesting that central factors contributing to fatigue can occur in the absence of a peripheral change in membrane excitability. However, more longer-duration contractions that induce fatigue (\~17 min) can also induce a decline in the muscle membrane excitability and m-wave size.^[@bib69]^ Biomarker for the diagnosis of muscle fatigue ============================================= At present, there are still no specific factors that have been consistently associated with a particular type of fatigue. Exercise types (for example, aerobic/anaerobic, short or long term), contraction type (for example, incremental/constant, isometric/non-isometric, concentric/eccentric), and fatigue degree and duration all affect the biomarker profile. According to the mechanism and metabolic changes during muscle fatigue, three categories of biomarkers have been determined: (1) ATP metabolism biomarkers, such as lactate, ammonia and hypoxanthine; (2) Oxidative stress biomarkers (ROS), such as lipid peroxidation, protein peroxidation, and antioxidative capacity; and (3) Inflammatory biomarkers, such as TNF-α, leukocytes, and interleukins.^[@bib71]^ ATP metabolism biomarkers ------------------------- Under normal circumstances, the total adenine nucleotide pool (ATP+ADP+AMP) remains constant. When the ATP supply fails to meet the consumption of ATP during exercise, fatigue occurs. To maintain the ATP/ADP ratio, two molecules of ADP may be converted to one molecule of ATP and one molecule of AMP. AMP is subsequently degraded by AMP-deaminase to IMP and ammonia.^[@bib72]^ IMP is degraded to inosine and hypoxanthine, and ammonia is further converted to urea nitrogen (BUN), thus increasing blood BUN. In the case of inadequate oxygen supply, oxidative phosphorylation of ADP to generate ATP fails to meet the energy requirement, and the ATP production shifts from aerobic processes (the processing of glucose/glycogen, lipids or amino acids) to anaerobic glycolysis or glycogenolysis,^[@bib73]^ thereby resulting in lactate accumulation. The best-known biomarkers of muscle fatigue from ATP metabolism include lactate, ammonia, and hypoxanthine.^[@bib74],\ [@bib75]^ Lactate and ammonia are usually determined in the serum. Hypoxanthine is usually analyzed in the serum or urine. Serum lactate increases with exercise intensity in healthy and diseased subjects.^[@bib76]^ However, serum lactate does not appear to be related to age, sex, and physical fitness. Under the conditions of workload standardization, serum lactate appears to be a promising biomarker of muscle fatigue.^[@bib73]^ Serum ammonia closely follows the lactate response during exercise ^[@bib73]^ and increases during exercise. Serum ammonia is not associated with age^[@bib77]^ and remains low in physical fitness, but is higher in men than in women.^[@bib78]^ Serum hypoxanthine significantly increases immediately after exercise.^[@bib79]^ There exists a sex difference ^[@bib80]^ but a lack of reliable data on age- or physical fitness-dependency on serum hypoxanthine. Oxidative stress biomarkers --------------------------- Reactive oxygen species (ROS) remain at a low level in resting skeletal muscle but increase in response to contractile activity. ROS products lead to protein, lipid or nucleic acid oxidation accompanied by a marked decrease in the antioxidant capacity,^[@bib81]^ thus ultimately inducing fatigue. Promising biomarkers to assess oxidative damage in muscle fatigue include lipid peroxidation biomarkers (that is, thiobarbituric acid-reactive substances (TBARS) and isoprostanes), and protein oxidation biomarkers (that is, protein carbonyls (PCs). Biomarkers to evaluate the antioxidant capacity include glutathione (GSH), glutathione peroxidase (GPX), catalase, and the total antioxidant capacity (TAC).^[@bib71]^ TBARS are indicators of lipid peroxidation and oxidative stress, which form during the decomposition of lipid peroxidation products that react with thiobarbituric acid and form a fluorescent red adduct. Isoprostanes are prostaglandin-like compounds derived from the peroxidation of essential fatty acids catalyzed by ROS. PCs are mainly derived from the oxidation of albumin or other serum proteins and are regarded as markers of oxidative protein injury. GSH is a pseudotripeptide that is present in nearly all cells and plays an important role in ROS scavenging. GPX and catalase are both enzymes that scavenge hydrogen peroxide into water and oxygen. TAC is defined as the sum of the antioxidant activities of the nonspecific pool of antioxidants. TBARS, PC, catalase and TAC are usually determined in the serum, but TBARS are also detectable in the saliva. Isoprostanes are usually measured in the serum, urine, or other body fluids and blood cells. GSH and GPX are present in cells and are detectable in serum and saliva.^[@bib82]^ With increasing exercise intensity, the levels of TBARS, isoprostanes, PC, catalase, TAC and GPX all increase, and that of GSH decreases.^[@bib76],\ [@bib82],\ [@bib83],\ [@bib84]^ With age, the levels of TBARS, isoprostanes and TAC increase,^[@bib85],\ [@bib86],\ [@bib87]^ those of GSH, GPX and catalase decrease,^[@bib88],\ [@bib89],\ [@bib90]^ and changes in PC remain controversial.^[@bib91],\ [@bib92]^ With physical fitness, the levels of TBARS, PC, GSH and GPX increase,^[@bib93]^ whereas the changes in catalase, PC and TAC still lack definite data.^[@bib94]^ The levels of TBARS, isoprostanes, PC, catalase and TAC have been reported to be lower in females than in males,^[@bib90],\ [@bib95],\ [@bib96],\ [@bib97]^ whereas GSH and GPX levels show an opposite trend.^[@bib89],\ [@bib98]^ Inflammatory biomarkers ----------------------- In addition to the depletion of ATP and ROS production, exercise and fatigue also induce local or systemic inflammatory reaction. Promising biomarkers to evaluate inflammation in muscle fatigue include leukocytes, IL-6 and TNF-α.^[@bib71]^ T-lymphocytes, especially CD4+ and CD8+ lymphocytes, are mobilized from peripheral lymphoid compartments into the blood after exercise.^[@bib99]^ In addition, neutrophils show a significant increase immediately after exercise. These changes represent a nonspecific immune response induced by ischemia in a stressed tissue, while there is a lack of a real injury.^[@bib100]^ IL-6, acting as an important pro-inflammatory (monocytes and macrophages) cytokine, is also now regarded as one of myokines released from muscle in response to contractions.^[@bib101]^ The levels of TNF-α, predominantly produced by macrophages, also increase as a result of muscle fatigue. Generally, IL-6 and TNF-α levels are determined in the serum. IL-6 levels can also be determined in the saliva. With age, the change in T-cells expressing CD8 remains controversial,^[@bib102],\ [@bib103]^ whereas the change in IL-6 is age independent. Sex differences in T-cell immune responses are particularly evident in graft-versus-host disease, with a stronger effect in females,^[@bib104]^ and IL-6 levels are also markedly lower in females.^[@bib102]^ TNF-α levels appear to be independent of age, sex and physical condition. There are still many potential immunological biomarkers, including C-reactive protein (CRP), IL-8, IL-10, IL-15, HSP27, HSP70, plasma DNA and orosomucoid (ORM).^[@bib72],\ [@bib101],\ [@bib105]^ For example, IL-15 has been found to accumulate within the muscle after regular training.^[@bib106]^ ORM, an acute-phase protein with immune-modulating activity, significantly increases in serum, muscle and liver tissues in response to various forms of muscle fatigue in rodents.^[@bib60]^ Of course, there are still several biomarkers that are unsuitable for the diagnosis of muscle fatigue, such as elastase, IL-1β and complement C4a, because their concentrations do not change substantially after exercise.^[@bib107]^ Potential treatment for muscle fatigue ====================================== Improper exercise, long time combat, military training and some related diseases (for example, cancer and stroke) can cause muscle fatigue, which negatively affects athletic achievement, military combat ability and patient recovery. At present, there are still no official or semi-official recommendations for the treatment of muscle fatigue. However, some nonspecific treatments, such as synthetic products (for example, amphetamine and caffeine), natural products (for example, American ginseng and rhodiola rosea) and nutritional supplements (for example, vitamins and minerals and creatine), have been used clinically or experimentally, and have shown some effects in various studies. Synthetic products ------------------ Amphetamine, ephedrine, caffeine, for example, are all synthetic products that excite the central nervous system or sympathetic nervous system and promote resistance to muscle fatigue. Almost half of the stimulant abuse in sport involves amphetamines and ephedrine, as reported by WADA (World Anti-Doping Agency) in 2005. The use of amphetamines, amphetamine derivatives, propanolamine and ephedrine remains illegal in competition. However, caffeine and pseudoephedrine have been accepted at any level since 2004. ### Amphetamine Amphetamine is a phenethylamine-type stimulant and antidepressant that is highly addictive and produces euphoria and an elevated mood. Amphetamine at low to moderate doses enhances the physical performance of humans and animals.^[@bib108],\ [@bib109],\ [@bib110]^ However, the underlying mechanism remains largely unknown. High body temperature is one of the strongest exhaustion signals. Recently, Morozova E has reported that amphetamine may mask or delay fatigue in rats by slowing down the exercise-induced elevation in core body temperature. Although amphetamine usage is prohibited during competitions, it may be used in some situations, such as in combat, to improve performance by delaying exhaustion.^[@bib111]^ ### Caffeine The use of caffeine as a sports-related enhancement drug is well documented. High caffeine dose consumption enhances performance during extended periods of exercise.^[@bib112],\ [@bib113]^ Indeed, the performance-enhancing effects of caffeine have been described for both prolonged aerobic exercises and prolonged activities involving resistance.^[@bib114]^ The effects of caffeine on short periods of intense aerobic activity (5--30 min) have been reported to be significantly beneficial, but its effects on very short-term anaerobic exercise, for example, sprinting, are inconclusive.^[@bib115]^ Mechanistically, caffeine has been reported to increase the epinephrine and norepinephrine response associated with exercise.^[@bib116]^ In addition, caffeine potentiates muscle contractility via the induction of SR calcium release, inhibition of phosphodiesterase isoenzymes, inhibition of glycogen phosphorylase enzymes and stimulation of the sodium/potassium pump.^[@bib115]^ ### Others Other sympathomimetic stimulants, such as ephedrine, pseudoephedrine and phenylpropanolamine, are several times less potent than amphetamines in improving performance.^[@bib116],\ [@bib117],\ [@bib118]^ Bell *et al.*^[@bib119]^ have provided clear evidence that ephedrine at a high dose improves endurance exercise in subjects running 10 km. In addition, taltirelin, a synthetic thyrotropin-releasing hormone (TRH) analog, effectively improves sports activity.^[@bib120]^ Cocaine, which causes a rapid sympathetic response, significantly increases endurance during high-intensity exercise.^[@bib121]^ Modafinil, a new drug type that acts on the central nervous system and keeps subjects awake,^[@bib122]^ markedly prolongs exercise time to exhaustion ^[@bib123]^ and has been widely used in the war to allow people to resist fatigue. Benzamide derivatives, such as 1-(1, 3-benzodioxol-5-ylcarbonyl) piperidine (1-BCP), significantly prolong the time of forced swimming in mice, through an unclear mechanism.^[@bib124]^ Natural products ---------------- More than half of the drugs introduced worldwide are derived from or are inspired by natural products. In the past few decades, health scholars and athletic physiologists have been searching for natural products that can improve athletic ability and resist or eliminate fatigue in human beings. Now, more and more natural products and their extracts have been revealed as potentially anti-fatigue agents. ### Araliaceae ginseng species American ginseng, panax ginseng C. A. Meyer and radix notoginseng all belong to the araliaceae ginseng species. American ginseng is the root of panax quinquefolium, which is currently grown in Canada and eastern USA. Panax ginseng C.A. Meyer. (ginseng) is an edible and medicinal Chinese herb that is often used in Asian countries. Panax notoginseng (Burk.) F.H. Chen is cultivated throughout Southwest China, Burma, and Nepal. The root, a commonly used part of this plant, is called radix notoginseng or Sanchi. All of them contain multiple active components, such as saponins, polysaccharides, flavonoids, vitamins and microelements, which are responsible for the effects in the improvement of physical fatigue in humans and animals. For example, saponins or protein extracted from American ginseng significantly lengthens the swimming time in mice via increasing the levels of liver glycogen and muscle glycogen.^[@bib125]^ Polysaccharides, Ginsenoside Rb1, Ginsenoside Rg3 or small molecule oligopeptides, derived from Panax ginseng C. A. Meyer, have all been reported to have marked anti-fatigue activity in mice swimming or grasping test.^[@bib126],\ [@bib127],\ [@bib128]^ One particular type of ginseng, red ginseng, has been found to have a positive effect on sports performance in 11 volunteers undertaking repetitive anaerobic exercise.^[@bib129]^ Multiple mechanisms are involved in the anti-fatigue effects of panax ginseng C. A. Meyer, including enhancing lactate dehydrogenase (LDH) activity, increasing hepatic glycogen levels, retarding the accumulation of serum urea nitrogen (SUN) and blood lactic acid (BLA), inhibiting oxidative stress and improving mitochondrial function in skeletal muscles. Regarding panax notoginseng, a single dose has been reported to enhance aerobic capacity, endurance and mean blood pressure (MAP) in young adults.^[@bib130]^ Total saponins extracted from panax notoginseng, the principal active ingredients, have been found to extend the exhaustive swimming time of mice, delay the increase in lactate in the blood, and increase the tissue glycogen content.^[@bib131]^ ### Rhodiola rosea Rhodiola rosea (R. rosea), belonging to the family Crassulaceae and genus Rhodiola, is a commonly used plant in folk medicine in Eastern Europe and China. It is also an important resource against fatigue. The ingredients of rhodiola rosea include salidroside and rosavin. Rosavin is the only constituent unique to R. rosea from the Rhodiola genus, and salidroside is common to most other Rhodiola species. The natural ratio of rosavins to salidrosides in R. rosea is approximately 3:1. Salidroside has been identified as the main anti-fatigue ingredient in Rhodiola rosea. Acute intake of Rhodiola rosea containing 3% rosavin+1% salidroside plus 500 mg starch has been found to improve endurance exercise capacity in young healthy volunteers.^[@bib132]^ Fermented R. rosea extract has also been found to significantly increase swimming time, hepatic superoxide dismutase content, and serum lactate dehydrogenase in mice.^[@bib133]^ ### Garlic Garlic (*Allium sativum*) is an herb that is used mainly as a food in many countries. Garlic was given to soldiers and athletes as a tonic in ancient Rome. Recently, the anti-fatigue effect of garlic has been reported by many researchers. Garlic-processing methods affect the anti-fatigue effects.^[@bib134]^ The main methods for processing raw garlic can be classified as (1) production of garlic powder, obtained after the drying of raw garlic; (2) production of garlic oil, distilled by steaming raw garlic; (3) production of oil macerate, extracted from raw garlic with vegetable oil; and 4) production of aged garlic extract (AGE).^[@bib135]^ Ushijima *et al.* have examined the effect of raw garlic juice, heated garlic juice, dehydrated garlic powder and AGE on physical strength and recovery from fatigue. They have found that raw garlic and AGE prolongs the treadmill running time of mice and enhances the speed of recovery of rectal temperature after immersion in cool water. These effects are related to the improvement of peripheral circulation, an action of anti-stress, and improvement of nutrition.^[@bib136]^ Recently, clinical studies have revealed many intriguing findings.^[@bib137]^ Verma *et al.* have investigated the effects of garlic oil on cardiac performance and exercise tolerance in 30 patients with coronary artery disease. After an initial treadmill stress test, the subjects were administered garlic oil for 6 weeks, and treadmill stress tests were repeated. In comparison with the initial test, garlic significantly decreased the heart rate at peak exercise and work load on the heart, thus leading to the better exercise tolerance. ### Others Enhancing the energy metabolism effectively helps to improve exercise capacity. Chinese yam and fructus aurantii have been reported to improve muscle glycogen, liver glycogen and other indicators.^[@bib138],\ [@bib139]^ Increasing numbers of natural products and their active components have been reported to have certain curative effects against physical fatigue, such as ophiopogon root, astragalus, Chinese wolfberry, caltrop, *Acanthopanax giraldii*, *Cordyceps sinensis*, *Ganoderma lucidum*, eucommia, *Ginkgo biloba*, radix paeoniae alba, gynostemma, acanthopanax, angelica, radix rehmanniae and radix polygoni multiflori. However, most of them still require extensive studies to determine their anti-fatigue effects and mechanisms. Nutritional supplements ----------------------- Several nutritional factors that may limit exercise performance have been identified, thus leading to the widespread use of nutritional strategies. Nutritional supplementation is regarded as legal by the International Olympic Committee (IOC) and, therefore, has gained popularity as a way to achieve performance enhancement. Nutritional supplements can be grouped into dietary supplements (for example, multivitamins, fish oils and glucosamine sulfate/chondroitin), ergogenic aids (for example for example, protein powder/amino acids and creatine) and sports foods (for example, sports drinks and meal replacement).^[@bib140]^ The most commonly used products are sports drinks and vitamin/mineral supplements, followed by creatine and protein supplements.^[@bib141]^ ### Vitamins and minerals Despite their relative paucity in the diet and the body, vitamins and minerals are key regulators of health and function, including work performance. They are not direct sources of energy but facilitate energy metabolism. Water-soluble vitamins include B vitamins (thiamin, riboflavin, niacin, pyridoxine, folate, biotin, pantothenic acid, vitamin B12 and choline) and vitamin C. Fat-soluble vitamins include vitamin A, D, E, and K. Vitamin A, C and E are also antioxidants. Twelve minerals are designated essential nutrients. Magnesium, iron, zinc, copper and chromium have the potential to affect physical performance.^[@bib142]^ Researchers have shown that vitamin and mineral deficiencies may result in decreased physical performance, and their supplementation improves physical performance in persons with preexisting deficiencies. For example, severe deprivation of folate and vitamin B12 result in anemia and decrease endurance work performance. Iron supplementation improves progressive fatigue resistance in iron-depleted, nonanemic women.^[@bib143]^ However, the use of vitamin and mineral supplements does not appear to improve performance in people consuming adequate diets.^[@bib142]^ Dietary supplementation in athletes at the Australian Institute of Sport for up to 8 months, including vitamins B1, B2 (riboflavin), B6, B12, C, E, A, folate and copper, magnesium, zinc, calcium, phosphorus, as well as aluminum, has not been found to improve athletic performance.^[@bib144]^ ### Fish oil Fish oil, a dietary supplement, has been shown to have beneficial effects on performance. Fish oil is rich in omega-3 fatty acids, specifically docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which have been found to improve cardiac energy efficiency, fat metabolism and immunomodulatory responses. The consumption of fish oil (containing 1050 mg EPA+750 mg DHA) for 3 weeks in 20 healthy subjects has been found to decrease the body fat percentage and improve exercise performance and physiological recovery after exercise.^[@bib145]^ ### Creatine Creatine (Cr), a nitrogen-containing compound synthesized in the body from glycine, arginine and methionine, is also found in the diet, primarily in red meat and seafood. The creatine/phosphorylcreatine system can provide energy when the rate of ATP utilization is higher than the rate of production by mitochondrial respiration, thus maintaining ATP homeostasis.^[@bib146]^ Introduced to the general public in the early 1990s, creatine has become one of the most widely used nutritional supplements or ergogenic aids, and has been consistently shown to increase performance in high-intensity intermittent exercise.^[@bib147]^ Creatine is regarded as legal by the International Olympic Committee. Therefore, creatine supplementation is a potential ergogenic strategy to improve muscle endurance. ### Red bull Red bull contains a mixture of carbohydrates, taurine, glucuronolactone, vitamin B and caffeine, and it is a commonly used energy drink. Several small studies have reported that carbohydrate and caffeine consumption improves aerobic and anaerobic performance as well as cognitive functions such as concentration, alertness and reaction time.^[@bib148]^ It has been postulated that the effects are from the modulation of adenosinergic receptors by caffeine and taurine. ### Others Carnitine plays an essential role in fatty acid oxidation in muscle. However, there is a lack of definite evidence regarding its beneficial role in performance as a supplement. Protein supplements have been demonstrated to be ineffective except in rare cases in which dietary protein intake is inadequate. Individual amino acids, especially ornithine, arginine and glutamine, are also commonly used as supplements. However, their effects on performance are not supported by documented evidence. Acute-phase protein ORM has been reported to enhance muscle endurance after vein or intraperitoneal injection in rodents,^[@bib60]^ but it is not convenient for daily supplementation. In theory, the use of antioxidant vitamins and glutamine during periods of intensive training should be beneficial, but further evidence is still needed before they are recommended as supplements.^[@bib149]^ Conclusions =========== Muscle force production involves a sequence of events, extending from cortical excitation to motor unit activation to excitation--contraction coupling, and ultimately leading to muscle activation. Changes at any level in this pathway, including changes in the nervous, ion, vascular, and energy systems, impair force generation and contribute to the development of muscle fatigue. Metabolic factors and fatigue reactants, such as H^+^, lactate, Pi, ADP, ROS, HSP25 and ORM, also affect muscle fatigue. Site-specific stimulation via non-invasive techniques provides a method to gain systemic insight into the fatigue process under physiological conditions. Although there is a lack of consensus, a sex- and age-specific distribution in muscle fatigue has been observed, in which children, older adults and males are more resistant to fatigue than adults and females. Biomarkers of ATP metabolism, oxidative stress and inflammatory reactions are helpful for the diagnosis of muscle fatigue. Despite the lack of official or semi-official recommendations, muscle fatigue has been reported to be improved by some nonspecific treatments, including CNS-exciting drugs, natural products and nutritional supplements. More potential mechanisms, biomarkers, targets and related drugs for muscle fatigue--- for example, ORM---still need to be explored in the future. Publisher's note ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This study was supported by grants from the National Natural Science Foundation of China (81273606 and 81473259 to XL), and the National Science and Technology Major Project (2014ZX09J14103-08C to XL). The authors declare no conflict of interest. [^1]: These authors contributed equally to this work.

1. Introduction {#sec1-materials-12-04180} =============== Nanoparticles (NPs) have been explored for a wide range of applications, including biosensors and chemical sensors, bioimaging, catalysis, optics, electronics, drug delivery, and energy \[[@B1-materials-12-04180]\]. The conventional chemical or physical methods for the production of nanoscale materials depend on the utilization of toxic stabilizing and capping agents, which pose risks to the environment and human health \[[@B2-materials-12-04180]\]. Therefore, there is an increasing need to develop cost-effective, non-toxic, and environmentally green approaches that do not use toxic chemicals to produce NPs. The microbial synthesis of NPs, which takes advantage of microbial detoxification of metallic ions and precipitation of metals through reducing enzymes, intracellular or extracellular reducing reagents and metal-binding proteins, showed immense potential \[[@B3-materials-12-04180],[@B4-materials-12-04180]\]. In particular, there is an increasing interest in the exploitation of the cysteine-rich metal-binding proteins, metallothioneins (MTs), in the development of biofactories for metal NP production. MTs are found in all eukaryotes, as well as some prokaryotes, and play an important role in intracellular metal distribution, accumulation, as well as protection of cells against heavy metal toxicity and oxidative stress \[[@B5-materials-12-04180],[@B6-materials-12-04180]\]. The Cys-Xaa-Cys clusters of MTs can act as a reducing functional group for the reduction of metal ions, chelation, and accumulation of metal particles in the cells \[[@B5-materials-12-04180],[@B6-materials-12-04180],[@B7-materials-12-04180]\]. Studies showed that MTs are involved in the detoxification of several metals, including zinc, mercury, copper and cadmium in microorganisms \[[@B5-materials-12-04180],[@B6-materials-12-04180],[@B8-materials-12-04180]\]. Due to their important role in metal reduction and high metal binding affinity, studies have been seeking to integrate MTs in creating biological platforms for the production of metal NPs. Engineered bacteria expressing MT and/or phytochelatins have been shown to be able to produce diverse nanoparticles \[[@B9-materials-12-04180],[@B10-materials-12-04180],[@B11-materials-12-04180]\]. A critical aspect in developing a microbial platform for NP synthesis is choosing a proper host microorganism. It is ideal that a biofactory can make NPs with consistent properties for their applications in various field of nanotechnology. Many types of microorganism have been shown to be able to synthesize various metal NPs, but with diverse properties \[[@B12-materials-12-04180],[@B13-materials-12-04180],[@B14-materials-12-04180],[@B15-materials-12-04180],[@B16-materials-12-04180]\]. In addition, the conditions for cultivation and maintenance of microorganisms vary for different species \[[@B14-materials-12-04180],[@B15-materials-12-04180],[@B16-materials-12-04180]\]. Among the microorganisms able to serve as potential biofactories for metal NP synthesis, the readily available bacteria, *E. coli*, is of particular interest, due to the relatively easy and simple procedures to grow, maintain and manipulate them in the lab. The potential of engineered *E. coli* as a common platform for synthesis of diverse NP has been explored; however, the efficiency of NP synthesis by the engineered bacteria and the potential to upgrade to large scale NP production were not examined \[[@B9-materials-12-04180],[@B10-materials-12-04180],[@B11-materials-12-04180]\]. The goal of this study was to provide preliminary evidence for exploiting engineered *E. coil* as a platform for the large-scale production of metal NPs with controlled morphological properties. In this study, we chose *C. albicans* MT as a target protein and transferred a PCR-assembled *C. albicans* MT gene into *E. coli* DH5α cells to examine whether expression of *C. albicans* MT in *E. coli* would improve bacterial NP synthesis. Compared to *E. coli* MT, which has four cysteine out of 56 residues (7.1%), the *C. albicans* MT has a relatively high content of cysteine residues (15.8%). Importantly, the *C. albicans* has four repeats of Cys-Xaa-Cys presumably for binding metal ions, such as copper \[[@B17-materials-12-04180],[@B18-materials-12-04180]\]. To examine whether the expression of this MT protein will improve NP synthesis, we used biosynthesis of silver NPs as an example to investigate the effects of *C. albicans* MT on NP production. Our results showed that the *E. coli* cells expressing *C. albicans* MT produced silver NPs faster than *E. coli* cells transformed with pUC19 empty vectors. Particularly, the silver NPs synthesized by engineered *E. coli* are more uniform in size and shape. 2. Materials and Methods {#sec2-materials-12-04180} ======================== 2.1. Synthesis of C. Albicans MT Gene and Transformation of E. Coli DH5α Cells {#sec2dot1-materials-12-04180} ------------------------------------------------------------------------------ To obtain the *C. albicans* MT gene, first, the MT protein sequence of the *C. albicans* was obtained from the NCBI protein database. Then the *C. albicans* MT gene was codon-optimized for protein expression in *E. coli*, and eight oligonucleotides for gene synthesis were designed using DNAworks. The oligonucleotides were synthesized by Eurofins Genomics (Louisville, KY, USA). The *C. albicans* gene was synthesized in four consecutive PCR reactions, as described by D. Marsc \[[@B19-materials-12-04180]\]. Assembled gene was inserted between HindIII and BamHI sites of plasmid pUC19 through in vivo homologous recombination in *E. coli* DH5α cells. Plasmids were propagated in *E. coli* DH5α cells, and clones carrying the error-free gene are confirmed by DNA sequencing (Eurofins Genomics, Louisville, KY, USA). To obtain genetically modified *E. coli* cells for NP synthesis, a single colony of *E. coli* DH5α cells transformed with the sequence-verified plasmid was inoculated in 5 mL of Luria-Bertani (LB) medium in the presence of 100 µg/mL of carbenicillin and cultured overnight at 37 °C with a continuous shake at 250 rpm. The overnight culture was centrifuged using Sorvall Legend X1R (Fisher Scientific, Pittsburgh, PA, USA) and the cell pellet was resuspended in 15 mL of fresh LB medium containing 100 µg/mL of carbenicillin. The cells were further cultured at 37 °C with a continuous shake for 30--45 min. Then glycerol was added to the cells to reach a final concentration of 15% and cells were stored at −80 °C for future use. All chemicals and reagents were purchased from Fisher Scientific (Pittsburgh, PA, USA) unless specified. 2.2. Monitoring Cell Growth {#sec2dot2-materials-12-04180} --------------------------- To examine whether the expression of *C. albicans* MT in E. coli affects cell growth, we monitored cell growth along time of cultivation. 50 µL of the glycerol stock cells containing the *C. albicans* MT gene inserted in pUC19 plasmid (pUC-Met) was inoculated in 10 mL of LB in the presence of 100 µg/mL carbenicillin and cultured overnight at 37 °C in a shaker incubator. Cells were spun down and resuspended in 100 mL of LB with 100 µg/mL carbenicillin. Optical density at 600 nm (OD~600nm~) was monitored each hour after resuspension until OD~600nm~ reached between 0.6 and 0.8, then OD~600nm~ was measured at 24 h, 48 h and 72 h. It has been shown that MT expression was induced and upregulated when microorganisms are under heavy metal stress \[[@B5-materials-12-04180],[@B6-materials-12-04180],[@B17-materials-12-04180],[@B18-materials-12-04180]\]. To further investigate whether the expression of *C. albicans* MT in *E. coli* impact the bacterial tolerance to heavy metal, we cultivated the bacteria in the presence of 0.2 mM CuSO~4~. 0.2 mM of CuSO~4~ was added to the cells after OD~600nm~ reached between 0.6 and 0.8. Cells were further cultivated at 37 °C for 17--19 h with a continuous shake. OD~600nm~ was measured again before cells were collected by centrifugation at 4000× *g* for 10 min. *E. coli* cells transformed with pUC19 empty vector (pUC19-Vec) was used as control. The viability of the cells after they grew overnight in the presence of 0.2 mM CuSO~4~ was estimated by plating the cells from the overnight culture on agar plates. The overnight bacterial culture was diluted with LB medium at a ratio of 1:1000, 1:10,000, 1:100,000 and 1:1,000,000, and 100 µL of each dilution was plated on agar plates in the absence of CuSO~4~. Cells were allowed to grow overnight at 37 °C for 17 h to obtain colonies. Individual colonies were counted to estimate the viable cells after CuSO~4~ challenge. 2.3. Determination of MT Protein Expression {#sec2dot3-materials-12-04180} ------------------------------------------- *C. albicans* MT was identified as a copper binding protein, and its level increases under metal ion stresses \[[@B5-materials-12-04180],[@B6-materials-12-04180],[@B17-materials-12-04180],[@B18-materials-12-04180]\]. To analyze the expression level of *C. albicans* MT in *E. coli*, the preparation of overnight culture for examining protein expression was carried out similarly as described in "Monitoring Cell Growth". The overnight cell culture was centrifuged, and cells were resuspended in 100 mL of LB containing carbenicillin and grown at 37 °C. When OD~600nm~ reached between 0.6 and 0.8, bacteria were grown for another 8 h for MT protein expression analysis. Cell pellets were resuspended in lysis buffer containing 50 mM Tris-HCl, pH 8, 50 mM NaCl at a ratio of about 0.05 g wet cells/mL of buffer and sonicated to break apart the cells. The whole lysate was centrifuge again at 15,000× *g* for 30 min to separate the soluble proteins from the cell membrane debris. Soluble proteins were passed through a GE Healthcare Vivaspin™ concentrator with a 30-kDa cutoff to remove the proteins larger than 30 kDa. Subsequently, the flow through was further concentrated with GE Healthcare Vivaspin™ concentrator with a 3-kDa cutoff. 10, 20, 40 µg of the concentrated proteins smaller than 30 kDa were run on 16% T, 4% C Tricine SDS-PAGE gel to separate the proteins. Proteins were stained in buffer containing 30% methanol, 10% glacial acetic acid, 0.1% Coomassie blue and 60% water for at least 2 h. Then the gel was destained in the same buffer without Coomassie blue, for overnight with continuous shake. Gel picture was taken using Gel Doc EZ Gel Documentation System (Biorad, Hercules, CA, USA). 2.4. Biosynthesis of Silver NPs by Bacteria {#sec2dot4-materials-12-04180} ------------------------------------------- The preparation of overnight culture for silver NP synthesis was carried out similarly as described in "Monitoring Cell Growth". The overnight cell culture was centrifuged, and cells were resuspended in 100 mL of LB in the presence of carbenicillin and grown at 37 °C. AgNO~3~ was added to the culture when OD~600nm~ reached between 0.6--0.8. Cells were further cultured at 37 °C with continuous shaking for 72 h. Samples were taken to measure OD~600nm~ at 24 h, 48 h and 72 h after addition of AgNO~3~. To estimate the yield of silver NPs in a larger scale, we carried out three experiments in which bacteria were grown in 1 L LB medium in the presence of 1 mM AgNO~3~ under the same conditions as for small scales. The wet weight of pellet collected after 72 h of cultivation was measured. After each OD~600nm~ measurement, cultures sat for 30 min to allow particles to settle down on the bottom of the flask and take pictures of the precipitates. pUC19-Vec cells were used as a control in this experiment. 2.5. Collection of NPs {#sec2dot5-materials-12-04180} ---------------------- After 72 h of cultivation, cell culture was centrifuged at 4000× *g* for 10 min. The cell pellets were resuspended in 1.5 mL of 4 M urea, sonicated for three cycles of 45 s at an output of 20%, with an interval of 1 min between each cycle (Sonic Dismembrator, Model 50; Fisher Scientific, Pittsburgh, PA, USA). The lysate was then transferred to a 2-mL microcentrifuge tube and further spun at 10,000× *g* for 10 min (accuSpin™ Microcentrifuge, Fisher Scientific, Pittsburgh, PA, USA). The supernatant was discarded, and the pellet was resuspended in 1 mL of 4 M urea by 3--4 pulses of sonication, followed by centrifugation to remove the supernatant. The pellet was further washed twice with 1 mL of sterile deionized water. The final pellet was resuspended in sterile deionized water and stored in the dark at 4 °C for next step characterizations. 2.6. Fourier Transform Infrared (FTIR) Spectral Analysis {#sec2dot6-materials-12-04180} -------------------------------------------------------- FTIR was used to reveal the functional groups that may be responsible for reducing and stabilizing the bioreduced silver NPs. The isolated NPs were diluted in deionized water, and The FTIR spectra were collected at a resolution of 2 cm^−1^ in transmission mode range between 4000 and 500 cm^−1^ using NICOLET IS10 FTIR (ThermoFisher, Waltham, MA, USA). Sterile deionized water was used as blank for this experiment. 2.7. Scanning Electron Microscopic and Scanning Transmission Electron Microscopic Characterization {#sec2dot7-materials-12-04180} -------------------------------------------------------------------------------------------------- We used scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM) to determine the morphological properties of the biologically synthesized silver NPs. The samples for SEM and TEM were prepared similarly. NPs washed with urea and deionized water were resuspended in sterile deionized water. An FEI Tecnai F30 super-twin field-emission-gun transmission electron microscope (TEM) (manufactured by the FEI Company in Hillsboro, OR, USA) operating at 300 kV was used to acquire the TEM images, electron diffraction patterns and annular dark-field (ADF) scanning transmission electron microscopy (STEM) images. Chemical composition was explored using an X-ray energy dispersive spectrometer (EDS) attached to the TEM. A dual-beam focus ion beam (FIB) (FEI Helios 600 dual-beam FIB) was used to obtain the SEM images of the biosynthesized silver NPs. The size of NPs was analyzed using Gatan Digital Micrograph Software. 2.8. Statistical Analysis {#sec2dot8-materials-12-04180} ------------------------- All data are expressed as mean ±SD. Comparison between groups was evaluated with the Student t-test. Probability values of \<0.05 were considered significant. 3. Results {#sec3-materials-12-04180} ========== 3.1. MT Gene Assembly in vitro and Expression of MT in E. coli DH5α Cells {#sec3dot1-materials-12-04180} ------------------------------------------------------------------------- We synthesize the *C. albicans* MT gene instead of cloning it from the *C. albicans'* genome for two reasons. First, we could obtain the gene without the need of *C. albicans'* genomic DNA; second, it allowed us to optimize codons for improving the expression of a yeast protein in *E. coli*. Eight oligonucleotides covering the full length of *C. albicans* MT gene were designed to synthesize the gene by PCR followed the procedure described in reference 19. The full length of *C. albicans* MT gene was assembled in four consecutive PCR reactions ([Figure 1](#materials-12-04180-f001){ref-type="fig"}A,B). The expected length of the final PCR product is 288 base pairs the product of each PCR reaction was run on a 1% agarose gel. The incremental increase in DNA size indicated the successful assembly of the gene ([Figure 1](#materials-12-04180-f001){ref-type="fig"}B). The full-length *C. albicans* MT protein has 76 residues with a calculated molecular weight of 7.8 kDa, and has 15.8% (12/76) cysteine residues ([Figure 1](#materials-12-04180-f001){ref-type="fig"}C). 16% T, 4% C Tricine SDS-PAGE and Coomassie blue staining were used to analyze the expression of *C. albicans* MT protein in *E. coli*. [Figure 2](#materials-12-04180-f002){ref-type="fig"}. showed the intensity of a band with a size between 5--10 kDa was enhanced in the proteins smaller than 30 kDa from pUC-Met cells (arrow pointed). The size of this band in pUC-Met cells matched the predicted molecular weight of the *C. albicans* MT protein; while the band with a similar size was much weaker in pUC19-Vec cells. It is possible that the *C. albicans* MT was co-localized with an endogenous protein. In addition, the insertion of the *C. albicans* metallothionein gene between BamHI and HindIII of pUC19 added a few more residues to the original peptide and increased its molecular weight to \~8.5 kDa. We believe that the increase in the intensity of this band was due to the expression of the recombinant *C. albicans* MT. 3.2. Cell Growth and Survival during Extended Cultivation and Copper Ion Challenge {#sec3dot2-materials-12-04180} ---------------------------------------------------------------------------------- Once we revealed that the *C. albicans* MT protein was expressed in *E. coli*, we continue to explore whether its expression can facilitate bacterial growth, as well as metal NP biosynthesis. Our results supported the notion that the expression of *C. albicans* MT protein promoted bacterial growth and survival. Under the same cultivation conditions, it appeared that the engineered recombinant pUC-Met cells grew faster and survived better than the pUC19-Vec cells during extended cultivation. We chose OD~600nm~ between 0.6 and 0.8 as the baseline for monitoring growth rate, because this OD~600nm~ range usually indicates the bacteria are at their fastest-growing stage and an inducer is added to induce protein expression if the target gene is under the control of an inducible promoter. When the starter culture was similar, at the point when OD~600nm~ of pUC19-Vec cells reached between 0.5 and 0.6 (0.59 ± 0.044, n = 7), the OD~600nm~ of pUC-Met cells was between 0.6 and 0.8 (0.68 ± 0.097, n = 7) ([Figure 3](#materials-12-04180-f003){ref-type="fig"}A,B). At 24 h after OD~600nm~ reached between 0.6 and 0.8, the OD~600nm~ measurement of engineered pUC-Met cells was significantly higher than that of pUC19-Vec cells ([Figure 3](#materials-12-04180-f003){ref-type="fig"}A. OD~600nm~: 1.33 ± 0.15 vs. 1.47 ± 0.12; *p* \< 0.05, n = 7). Similar trends were observed at 48, 72 h of cultivation, even if the measurements were not significantly different ([Figure 3](#materials-12-04180-f003){ref-type="fig"}A). Furthermore, the pUC-Met cells performed better than the pUC19-Vec cells when the cells grew 17--19 h in the presence of 0.2 mM CuSO~4~. The growth of pUC-Vec cells was significantly slowed down, compared to the pUC-Met cells ([Figure 3](#materials-12-04180-f003){ref-type="fig"}B. OD~600nm~: 1.11 ± 0.21 vs. 1.61 ± 0.008, n = 3, *p* \< 0.05). These results suggested that the expression of *C. albicans* MT helped cell grow and survive during extended cultivation and under heavy metal challenge. To determine that the higher OD~600nm~ was due to higher survival of pUC-Met cells under stress, we plated the bacteria on agar plates in a serial of dilution after they were exposed to 0.2 mM CuSO~4~ for 17--19 h. Results of 1:1000 and 1:10,000 dilution were not shown because there were too many colonies, and it was not easy to count the individual colonies. Results of 1:100,000 and 1:1,000,000 dilutions were shown in [Figure 3](#materials-12-04180-f003){ref-type="fig"}C. We found that that the pUC-Met cells after overnight cultivation in medium containing CuSO~4~, not only gave rise to more colonies, but also these colonies were larger than the ones from pUC19-Vec cells ([Figure 3](#materials-12-04180-f003){ref-type="fig"}C). The estimated viable cell number after overnight CuSO~4~ challenge was 6.4 × 10^8^ in the pUC-Met group, while it was 2.4 × 10^8^ in pUC19-Vec group. Thus, we demonstrated that the expression of *C. albicans* MT increased the tolerance of recombinant *E. coli* to copper ion and facilitated cell survival under heavy metal stress. 3.3. Yield of Silver NPs {#sec3dot3-materials-12-04180} ------------------------ After we found that expression of *C. albicans* MT helped bacteria grow and improved their resistance to metal ions, we went on to examine whether this would allow the bacteria to produce NPs with improved yield and quality. A color change in the medium after addition of AgNO~3~ to the bacteria culture is usually a sign of NP formation. We not only observed a color change from yellow to brown, but also detected a large amount of black precipitates in all groups of cells. Pictures of precipitates at the bottom of the flasks were taken after culture containers sat at room temperature for 30 min. We observed more dark precipitates formed by pUC-Met cells ([Figure 4](#materials-12-04180-f004){ref-type="fig"}A--D) at 24 and 72 h after addition of AgNO~3~. Importantly, the wet weight of pellets obtained after centrifugation of 1 L bacterial culture was greater than that of pUC19-Vec cells (0.89 ± 0.04 g/L in pUC19-Vec vs. 1.55 ± 0.084 g/L in pUC-Met. N = 3. *p* \< 0.05) ([Figure 4](#materials-12-04180-f004){ref-type="fig"}F). Thus, our data suggested that the expression of MT facilitated cell survival, as well as the yield of NP production. 3.4. Identification of the Functional Groups Involved in Reducing and Stabilizing Silver NPs {#sec3dot4-materials-12-04180} -------------------------------------------------------------------------------------------- The mechanisms of bacterial biological synthesis of metal NPs involve the presence of metal-binding protein peptides, reducing enzymes and functional groups that can donate electrons in the bacteria \[[@B4-materials-12-04180],[@B7-materials-12-04180],[@B12-materials-12-04180],[@B13-materials-12-04180],[@B20-materials-12-04180],[@B21-materials-12-04180],[@B22-materials-12-04180]\]. FTIR is able to identify the chemical composition of biological samples. We used FTIR to analyze the possible reducing functional groups in the bacteria that are involved in reducing and stabilizing silver ions. Three peaks were identified in the silver NP samples around 1640 cm^−1^, 2080 cm^−1^ and 3440 cm^−1^. The sharp, strong peak at 1640 cm^−1^ was probably the stretch of amide I bonds (C=O), from the conversion of C--OH biomolecules. The absorption peak at 1640 cm^−1^ shows that proteins are interacting with biosynthesized nanoparticles, and also their secondary structure were not affected during reaction with Ag^+^ ions or after binding with silver NPs \[[@B23-materials-12-04180],[@B24-materials-12-04180]\], while the peak at around 2090 cm^−1^ was likely the stretch of isothiocyanate group (N=C=S). The broad strong stretch of compounds, ranging from 3200 cm^−1^ to 3600 cm^−1^, were assigned to both phenols (O--H) and amines (N--H) ([Figure 5](#materials-12-04180-f005){ref-type="fig"}). These results were generally similar to what has been reported with the exception of isothiocyanate compounds \[[@B23-materials-12-04180],[@B24-materials-12-04180]\]. There were no differences in the peaks between the two groups of cells, suggesting that the bacteria probably used similar functional groups in the reduction of metal ions and the formation of NPs. 3.5. Characterization of Silver NPs Using Scanning Electron Microscopy and Scanning Transmission Electron Microscopy {#sec3dot5-materials-12-04180} -------------------------------------------------------------------------------------------------------------------- To confirm that bacteria did produce silver NPs, and to determine the compositions and morphological properties of the NPs, we examined the NPs using EDS, SEM and Scanning TEM. X-ray EDS compositional analysis identified that the major element in the particles was silver ([Figure 6](#materials-12-04180-f006){ref-type="fig"}), confirming that the bacteria indeed synthesized silver NPs. Other elements found in the NPs included chloride, sulfur and copper ([Figure 6](#materials-12-04180-f006){ref-type="fig"}). The detection of sulfur was particularly interesting, since it showed that the thiol groups of cysteine residues were in fact involved in reducing silver ions. Importantly, the analysis of the particle images also proved that all *E. coli* cells produced silver particles in the nano-size range ([Figure 7](#materials-12-04180-f007){ref-type="fig"} and [Figure 8](#materials-12-04180-f008){ref-type="fig"}). The silver NP images in [Figure 8](#materials-12-04180-f008){ref-type="fig"} were selected for size analysis using Gatan Digital Micrograph Software. [Figure 8](#materials-12-04180-f008){ref-type="fig"} showed that the size of NPs produced by pUC19-Vec cells was slightly and significantly larger ([Figure 8](#materials-12-04180-f008){ref-type="fig"}F,G. pUC19-Vec: 23.9 ± 7.3 nm. n = 40; pUC-Met: 20.0 ± 3.7 nm, n = 49. *p* \< 0.05). Our results suggested that the NPs synthesized by pUC-Met cells were more homogeneous. Firstly, the size of the NPs formed by pUC-Met cells was more homogeneous, which was demonstrated by the difference in the standard deviation (SD) of the NP size. The SD of the size of NPs (not including the large crystals shown in [Figure 8](#materials-12-04180-f008){ref-type="fig"}C) made by pUC19-Vec cells (SD = 7.3 nm) was much greater than that produced by pUC-Met cells (SD = 3.7 nm). Furthermore, the NPs produced by pUC-Met cells mostly had a round spherical shape ([Figure 7](#materials-12-04180-f007){ref-type="fig"}B and [Figure 8](#materials-12-04180-f008){ref-type="fig"}D,E); while the NPs synthesized by pUC19-Vec cells were more diverse ([Figure 7](#materials-12-04180-f007){ref-type="fig"}A and [Figure 8](#materials-12-04180-f008){ref-type="fig"}A--C). The heterogeneity of the NPs synthesized by the pUC19-Vec cells was further exemplified by the presence of some micro-size silver crystals with shapes of cube, rectangular and hexagonal prism ([Figure 8](#materials-12-04180-f008){ref-type="fig"}C). Thus, the results of SEM and STEM examination demonstrated that the engineered cells synthesized more uniform silver NPs in both size and shape. 4. Discussion {#sec4-materials-12-04180} ============= Both wild type *E. coli* and engineered *E. coli* have been shown to be able to synthesize diverse NPs \[[@B9-materials-12-04180],[@B10-materials-12-04180],[@B11-materials-12-04180]\]; however, it is not clear whether the engineered *E. coli* cells perform better than non-engineered cells, and whether these cells have the potential to upgrade to large scale NP production. In this study, we explored the potential of application of *E. coli* DH5α cells transformed with *C. albicans* MT gene in the biosynthesis of more uniform silver NPs at lager scale. We used the *E. coli* DH5α cells transformed with pUC19 empty plasmid vector as a control, since the *C. albicans* MT gene was delivered into the cells by vector pUC19. Many studies have shown that MT is protective when cells are exposed to metal ions \[[@B6-materials-12-04180],[@B17-materials-12-04180],[@B18-materials-12-04180]\]. MT proteins have high binding-affinity for heavy metal ions and serve as important reducing agents under heavy metal stress; thus, MT proteins are critical for proper accumulation, distribution and detoxification of the metal ions \[[@B5-materials-12-04180],[@B17-materials-12-04180],[@B18-materials-12-04180]\]. Similarly, our results also showed that the *E. coli* cells expressing *C. albicans* MT (pUC-Met cells) tolerated CuSO~4~ challenge better than the pUC19-Vec cells. During the prolonged cultivation in the absence of metal ion stress, the OD~600nm~ of pUC-Met cells was higher than that of pUC19-Vec cells at 24, 48 and 72 h after the OD~600nm~ reached 0.6--0.8 ([Figure 3](#materials-12-04180-f003){ref-type="fig"}A). Importantly, when the cells were subjected to copper ion stress for 17--19 h, the pUC-Met cells grew to a significantly higher OD~600nm~ than the pUC19-Vec cells ([Figure 3](#materials-12-04180-f003){ref-type="fig"}B). In particular, assessment of cell viability confirmed that pUC-Met cells had a higher survival rate than pUC19-Vec cells after bacteria were exposed to heavy metal stress ([Figure 3](#materials-12-04180-f003){ref-type="fig"}C). The better growth and higher survival of bacteria under heavy metal challenge suggested that the pUC-Met cells had increased tolerance to the metal ions, presumably due to the expression of *C. albicans* MT protein. *C. albicans* MT has a relatively high content of cysteine residues that can be used to reduce silver ions, bind and accumulates silver particles in the cells, thus, protect the cells from the toxicity of silver ions. We proposed that the higher tolerance to metal ions would subsequently promote the cells to make more NPs if they are used as biofactories for metal NP synthesis. Indeed, when the cells were grown in the presence of 1 mM AgNO~3~, the pUC-Met cells consistently produced more silver NPs at each examination time point ([Figure 4](#materials-12-04180-f004){ref-type="fig"}A--E), and the final yield of NPs from 1liter culture by pUC-Met cells was greater than that of the pUC19-Vec cells ([Figure 4](#materials-12-04180-f004){ref-type="fig"}E, 1 g/L vs. 0.74 g/L). The yield of a wet pellet of bacterial culture in the presence of 1 mM AgNO~3~ for 72 h was not compromised by increasing culture medium volume from 0.1 L to 1 L, suggesting that upgrade to large scale NP production is very promising, although more experiments are needed to confirm the conclusion. Thus, our results demonstrated that expression of *C. albicans* MT in *E. coli* promoted cell growth and survival and enhanced biosynthesis of silver NPs in these cells. Even if the mechanisms of bacterial synthesis of metallic NPs remain to be completely elucidated, it has been postulated that the presence of reducing groups that can donate electrons to metal ions, such as phenol, amide, thiols and other unsaturated bonds, of the organic matter in bacteria play an important role in reducing and binding metal ions \[[@B12-materials-12-04180],[@B25-materials-12-04180]\]. To shed light into the mechanisms of bacterial NP synthesis, we analyzed the reducing functional groups using FTIR. FTIR identified compounds that probably contain phenol, amines, amide I and isothiocyanate functional groups in all cells ([Figure 5](#materials-12-04180-f005){ref-type="fig"}). We hypothesized that the oxidization of C--OH group in organic molecules to C=O (amide I) group probably was involved in the reduction of Ag^+^ ions to Ag^0^. In addition, the amide I groups may cap and stabilize the silver NPs once the Ag^+^ ions were converted into Ag^0^. The possible amines (N--H) of biomolecules, such as proteins, may as well act as binding and stabilizing agents of silver NPs. Furthermore, it is well accepted that the O--H stretching in alcohols and phenolic compounds are used to reduce the Ag^+^ ions into Ag^0^ in the biological synthesis of metal NPs \[[@B22-materials-12-04180],[@B23-materials-12-04180],[@B24-materials-12-04180]\]. Therefore, the FTIR analysis suggested that the bacteria in our study probably used similar reducing agents to reduce Ag^+^ ions to Ag^0^ as reported \[[@B12-materials-12-04180],[@B22-materials-12-04180],[@B23-materials-12-04180],[@B24-materials-12-04180],[@B25-materials-12-04180]\]. Interestingly, a possible isothiocyanate (N=C=S) was observed in the bacteria synthesized silver NPs. Isothiocyanates are sulfur-containing antioxidants that have antimicrobial activities \[[@B26-materials-12-04180]\]. It is true that the strong reducing agent isothiocyanates can play a role in biosynthesis of silver NPs, it is yet to elucidate how and why the engineered bacteria produced these compounds that can kill themselves. We expected that the FTIR could detect thiol groups, since the cysteine residues in the MTs contain thiol groups. However, FTIR could not catch the thiol groups associated with silver. We speculate that the thiol group, which usually is assigned to a weak peak around 2600 cm^−1^, might be buried in the stretches of the strong peaks adjacent to 2600 cm^−1^. To complement this drawback of FTIR, compositions of silver NPs were further assessed by STEM and SEM. Importantly, the compositional analysis confirmed that silver atoms were among the key elements in the NPs. Further, consistent with our prediction, sulfur atoms were indeed detected associated with these silver NPs, indicating that the thiol groups of proteins were involved in reducing silver ions and accumulating silver NPs ([Figure 6](#materials-12-04180-f006){ref-type="fig"}). Therefore, our results not only confirmed that the NPs were indeed silver NPs, but also supported that thiol groups were involved in reducing and binding silver NPs. Since *C. albicans* MT has a higher level of cysteine residues that can generate a higher reducing power and metal-binding affinity in pUC-Met cells than the pUC-Met cells, we believe that the expression of *C. albicans* MT in *E*. *coli* cells allowed the cells to convert more silver ions, and faster into silver NPs, and therefore, protected the cells from silver ion stress, as well as increased the bacterial NP yield. Another finding of the SEM compositional assessment was the detection of chloride and copper associated with silver NPs. It has been shown that chloride is one of the key elements that *E. coli* use to counter silver NP toxicity \[[@B27-materials-12-04180]\]. The identification of chloride associated with silver NPs suggested that the *E. coli* not only used thiol groups, but also chloride to reduce and accumulate silver under silver ion stress. It is not surprising that copper was also detected in the NPs, since *C. albicans* MT was originally discovered as a copper-binding protein, and its expression was induced by copper ions \[[@B17-materials-12-04180],[@B18-materials-12-04180]\]. FTIR analysis, SEM and STEM examinations obtained similar results in the NPs synthesized by pUC19-Vec and pUC-Met cells, suggesting the two groups of cells probably used similar mechanisms to reduce silver ions and produce NPs. However, the expression of *C. albicans* MT protein in *E. coli* conferred the engineered recombinant cells higher tolerance and better survival, thus, greater capacity to synthesize silver NPs when exposed to silver ions. One of the problems associated with biologically synthesized NPs has been morphological heterogeneity of the NPs \[[@B12-materials-12-04180],[@B13-materials-12-04180],[@B14-materials-12-04180]\]. A primary goal of this study was to investigate whether engineered *E. coli* cells can improve the quality of NPs. Both SEM and STEM demonstrated that the two groups of *E. coli* cells successfully produced silver particles in the nano-size range. The average size of NPs synthesized by pUC-Met cells is slightly, but significantly, smaller than the size of those produced by pUC19-Vec cells ([Figure 8](#materials-12-04180-f008){ref-type="fig"}F,G). However, there was a greater variation in the sizes of the NPs synthesized by pUC19-Vec cells (SD: 3.7 nm in pUC-Met vs. 7.3 nm in pUC19-Vec). Note that calculation of this SD did not include the large crystals mentioned below). Surprisingly, some micro-size silver crystals were formed by these cells ([Figure 8](#materials-12-04180-f008){ref-type="fig"}C). Overall, our data showed that NPs produced by pUC-Met cells not only had a more uniform size, but also appeared to have a more uniform shape. As shown in [Figure 7](#materials-12-04180-f007){ref-type="fig"}B, [Figure 8](#materials-12-04180-f008){ref-type="fig"}D,E, under SEM and STEM, it appeared that the NPs made by pUC-Met cells had a round shape, while the morphology of NPs from pUC19-Vec cells was more heterogeneous, which showed round, rectangular prism and hexagonal prism shapes ([Figure 7](#materials-12-04180-f007){ref-type="fig"}A and [Figure 8](#materials-12-04180-f008){ref-type="fig"}A,C). Thus, we demonstrated that both pUC19-Vec and pUC-Met bacterial groups were able to synthesize silver NPs. Nevertheless, the bacteria expressing *C. albicans* MT had a better control in the dimension and morphology of silver NPs and a higher yield of NPs. The mechanisms of how the pUC-Met recombinant bacteria synthesized NPs with a more uniform size and shape remains to be elucidated. We speculate that more thiol groups in pUC-Met cells have a higher reducing power and a greater binding capacity for silver ions, which probably allows the cells to produce NP faster; meantime, more thiol groups can provide a larger silver binding capacity, which is more potent to stabilize the silver NPs once the silver ions are reduced. Faster reducing reactions along with a higher capacity to stabilize the NPs probably allowed the cells to synthesize NPs with a smaller size and higher uniformity. 5. Conclusions {#sec5-materials-12-04180} ============== In summary, we revealed that the expression of *C. albicans* MT in *E. coli* cells allowed these cells to biologically synthesize more silver NPs at a higher yield. An important finding is that these engineered recombinant *E. coli* cells appeared to have better control in the size and shape of the NPs. Moreover, the procedures used to grow, propagate the engineered bacteria, as well as the methods used to produce silver NPs, in our study, are quite simple and easy to set up in any microbiology laboratories. Expansion of this engineered *E. coli* as a platform for the biosynthesis of other metal NPs, optimization of bacterial growth conditions to upgrade the method for large scale production of uniform metal NPs, as well as an exploration of the potential of in vitro NP synthesis by these engineered cells will be among our goals in the future. The results of this study provided preliminary information for genetically modifying bacteria as promising biofactories for large-scale production of size- and shape-controlled NPs for their applications in nanotechnology. The research is supported by DoD/ARO under Grant No. W911NF-18-1-0444. Research carried out in part at the Center for Functional Nanomaterials, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-SC00112704. The authors greatly appreciate: Ms. Shonda Scott at the Chemistry Program at Alabama A&M University for her help in FTIR analysis; Yong Ding at George Institute of Technology for his help in performing the TEM analysis; and Fernando Camino at Brookhaven National Laboratory for his help in obtaining the SEM pictures. Q.Y. designed the experiments, synthesized genes, produced the engineered bacteria, produced NPs analyzed the data and wrote the manuscript. Z.X. characterized the NPs, analyzed data and contributed to writing the manuscript. M.B. did FTIR to analyze the functional groups in NP samples and reviewed the manuscript. This study was supported by funding from DoD/ARO (award number: W911NF1810444). The authors declared no conflict of interest. {#materials-12-04180-f001} {#materials-12-04180-f002} {#materials-12-04180-f003} {#materials-12-04180-f004} {#materials-12-04180-f005} {#materials-12-04180-f006} {#materials-12-04180-f007} {#materials-12-04180-f008}

Background ========== The pathogenesis of endotoxemic tubular dysfunction with failure in urine concentration is poorly understood. Urea plays an important role in the urinary concentrating mechanism and expression of the urea transporters UT-A1, UT-A2, UT-A3, UT-A4 and UT-B is essential for tubular urea reabsorption. The present study attempts to assess the regulation of renal urea transporters during severe inflammation *in vivo*. Materials and methods ===================== By agreement of the animal protection committee C57BL/6J, mice were injected with lipopolysaccharides (LPS, 10 mg/kg) or proinflammatory cytokines. Hemodynamic, renal parameters and the expression of renal urea transporters were investigated. To clarify the role of cytokines and renal ischemia in the regulation of renal urea transporters, experiments were performed with cytokine knockout mice, mice treated with low-dose LPS (1, 5 mg/kg) as a sepsis model without induction of hypotension, glucocorticoid-treated mice, and mice with renal artery clipping serving as a model for renal ischemia. Results and discussion ====================== LPS-injected mice (10 mg/kg) presented with reduced glomerular filtration rate, fractional urea excretion and inner medulla osmolality associated with a marked decrease in expression of UT-A1, UT-A2, UT-A3, UT-A4 and UT-B (Figure [1](#F1){ref-type="fig"}). Similar alterations were observed after application of TNFα, IL-1β, IFNγ or IL-6. LPS-induced downregulation of urea transporters was not affected in knockout mice with deficient TNFα, IL-receptor-1, IFNγ or IL-6. Glucocorticoid treatment inhibited LPS-induced increases of tissue TNFα, IL-1β, IFNγ or IL-6 concentration, diminished LPS-induced renal dysfunction and attenuated the downregulation of renal urea transporters. Injection of low-dose LPS (1, 5 mg/kg) also led to renal dysfunction paralleled by a downregulation of renal urea transporters without alterations in blood pressure. Renal ischemia induced by renal artery clipping did not influence the expression of urea transporters. {#F1} Conclusion ========== Our findings demonstrate downregulation of renal urea transporters that probably accounts for tubular dysfunction during sepsis. Furthermore, they suggest that downregulation of renal urea transporters during LPS-induced acute renal failure is mediated by proinflammatory cytokines and is independent from renal ischemia due to sepsis-induced hypotension. Acknowledgements ================ Supported by grants from the German Research Foundation (SFB 699).

Background {#Sec1} ========== Left atrium (LA) size and function are powerful biomarkers of cardiovascular outcomes in many diseases. We sought to determine if the expected age-associated increase in arterial stiffness (AS) and left ventricular (LV)-LA afterload leads to corresponding effects on LA function and this can be measured with cardiovascular magnetic resonance (CMR). Additionally, we investigated the significance of these markers in asymptomatic individuals with cardiovascular risk factors (CRF). Methods {#Sec2} ======= Female subjects from the Twins UK cohort with no overt cardiac disease were prospectively recruited for a CMR study on a 1.5 Tesla scanner (Philips, Best, Netherlands) with tissue characterization (T1 mapping and late gadolinium enhancement). Patients with atrial fibrillation, valvular disease, regional wall motion abnormalities at rest or areas of myocardial enhancement were excluded from the analysis. LA reservoir, conduit and contractile functions were quantified by both fractional volume changes and CMR feature tracking derived strain and strain rate. Additionally, CMR feature tracking derived myocardial deformation indices and pulse wave velocity (PWV) (foot-to-foot methodology), were calculated. Results {#Sec3} ======= 40 female twins were enrolled. Baseline characteristics are shown in table [1](#Tab1){ref-type="table"}. Bivariate analysis showed that LA volume, LA reservoir, conduit and booster function correlated with LV deformation parameters and with PWV, a surrogate marker of AS (p \< 0.001 to 0.044). Furthermore, LA function components assessed by fractional volume changes were significantly different in the presence of ≥1 risk factors (p \< 0.001 to 0.012). Multivariable regression analysis confirmed that only the conduit and booster components were associated with changes in LV deformation and PWV (figure [1](#Fig1){ref-type="fig"}, panel A). Subjects with CRF had lower LA conduit function and higher booster function. We hypothesize that this can be attributed to an increase in atrial afterload in response to increased LV stiffness-AS that occurs with aging but is more pronounced subjects with CRF as is demonstrated by lower LV deformation indices and higher calculated PWV. ROC analysis showed that LA volume and function parameters outperformed LV deformation and AS parameters in the evaluation of subclinical cardiac changes in the presence of CRF (figure [1](#Fig1){ref-type="fig"}, panels B and C)Table 1Baseline characteristicsCHARACTERISTICSALLCRF = 0CRF≥1pAge, years69 ± 5.6 \[57-77\]68 ± 5.669 ± 5.60.114Women, n (%)40 (100%)2020-Cardiovascular risk factors (≥1)20 (50%)020\<0.001Hypertension, n (%)9 (21%)090.0012Diabetes, n (%)000-Dyslipidemia16 (40%)016\<0.001Smoker (former), n (%)4 (10%)040.106Pulse wave velocity \[PWV (m/s)\]8.127 ± 2.776.630 ± 2.778.127 ± 3.03\<0.001LV end-diastolic volume indexed \[LVEDVi (mL/m2)\]70 ± 14.567 ± 8.471 ± 13.10.079LV end-systolic volume \[LVESVi (mL/m2)\]41 ± 9.641 ± 6.435 ± 9.60.153LV ejection fraction \[LVEF (%)\]67 ± 7.461 ± 7.470 ± 7.40.975LV mass indexed (mg/m2)54 ± 12.553 ± 12.555 ± 6.40.637Global circumferential strain (GCs)-20.5 ± 8.6-24.7 ± 10.4-16.4 ± 3.50.001Global radial strain (GRs)29.8 ± 12.030.7 ± 6.928.9 ± 15.70.652Global circumferential strain rate peak systole \[GCSr (syst)\]-1.58 ± 0.42-1.58 ± 0.41-1.57 ± 0.450.924Global radial strain rate peak systole \[GRSr (syst)\]1.77 ± 0.962.44 ± 0.501.09 ± 0.83\<0.001Global circumferential strain rate early diastole \[GCSr (diast)\]2.17 ± 1.901.85 ± 0.522.47 ± 2.60.293Global radial strain rate early diastole \[GRSr (diast)\]-1.7 ± 0.83-2.21 ± 0.61-1.2 ± 0.71\<0.001Native T1 (mid septum, ms)979 ± 37.6966 ± 30.6993 ± 39.90.022LA volume indexed \[LAVI (mL/m2)\]42 ± 14.034 ± 9.650 ± 13.4\<0.001LA ejection fraction total \[LAEF total (%)\]61 ± 11.668 ± 7.853 ± 9.9\<0.001LA conduit function (%)34 ± 12.042 ± 10.626 ± 7.7\<0.001LA booster pump function (%)33.3 ± 9.030 ± 6.837 ± 9.70.012LA total strain (εs)28.0 ± 8.030.7 ± 7.425.2 ± 7.90.029LA passive strain (εe)18.6 ± 7.223.3 ± 5.214.0 ± 5.7\<0.001LA active strain (εa)LA active strain (εa)7.5 ± 3.611.2 ± 4.60.006LA peak positive strain rate (SRs)1.2 ± 0.41.4 ± 0.41.1 ± 0.30.016LA peak early negative strain rate (SRe)-1.2 ± 0.6-1.7 ± 0.4-0.8 ± 0.3\<0.001LA peak late negative strain rate (SRa)-1.0 ± 0.4-1.0 ± 0.3-0.9 ± 0.40.341CRF, cardiovascular risk factors.Figure 1**Scatter plots (Panel A) showing the relationships between LA-LV deformation parameters and LA-PWV (abbreviations as shown in table** [1](#Tab1){ref-type="table"}**)**. GRs (syst), GRSr (diast), and PWV were identified as independent determinants of LA mechanics in individuals with risk factors on multivariate analysis. **Panels B and C.** ROC curves comparing the sensitivity and specificity of different diagnostic parameters used in the analysis in the presence of cardiovascular risk factors. Conclusions {#Sec4} =========== LA mechanics correlates with LV deformation parameters and PWV and differs significantly in elderly subjects with CRF compared to their healthy age-matched peers. LA structural remodeling is predicted by AS (as expressed by PWV/LV parameters) independently of conventional CRF, thus supporting the hypothesis of arterial-ventricular-atrial coupling (AVAC). These novel markers of LA performance can potentially uncover abnormal AVAC in patients with CRF but no overt cardiac disease and give valuable insights into ventricular dysfunction beyond standard volumetrics.

Introduction {#s1} ============ Secretory pathway Ca^2+^-ATPases (SPCA) are important in sequestering Ca^2+^ and Mn^2+^ from the cytoplasm into the Golgi and post-Golgi vesicles where they are important for post-translational modification, sorting and quality control of cargo proteins [@pone.0067348-Missiaen1]. The two isoforms, SPCA1 (*ATP2C1*) and SPCA2 (*ATP2C2*) share significant sequence similarity, but have distinct distribution and function [@pone.0067348-Vanoevelen1], [@pone.0067348-Xiang1]. SPCA1 is ubiquitously expressed in mammalian tissues where it serves an essential housekeeping function, as evidenced by embryonic lethality in the homozygous knockout mouse [@pone.0067348-Okunade1]. In contrast, expression of SPCA2 is confined to highly secretory or absorptive epithelia, including mammary, testis, salivary glands, intestinal tract and lung [@pone.0067348-Vanoevelen1]. Because it shares similar transport characteristics as SPCA1, SPCA2 appears redundant at first glance. Recently, we showed pathologically elevated expression of SPCA2 in breast cancer derived tissue and cell culture models, leading to an investigation of its role in tumorigenicity [@pone.0067348-Feng1]. We showed that SPCA2, but not SPCA1, traffics to the plasma membrane in the breast cancer derived MCF7 cell line, where it interacts with the store-operated Ca^2+^ channel Orai1 to elicit constitutive Ca^2+^ influx. This signaling function was independent of Golgi and ER Ca^2+^ stores, and did not require ATPase or transport function of SPCA2. Constitutive elevation of Ca^2+^ influx in these cells activated MAP kinase signaling pathways and promoted tumor proliferation. Knockdown of either SPCA2 or Orai1 expression was found to attenuate Ca^2+^-signaling and inhibit tumor growth in mouse xenografts. Analysis of mutants and chimeras revealed that the membrane-anchored C-terminal tail of SPCA2 was sufficient for interaction with Orai1 to elicit Ca^2+^ influx [@pone.0067348-Feng1]. Interestingly, an alternative splice variant of *ATP2C2*, encoding a ∼20 kDa membrane-anchored C-terminal domain is expressed in several secretory tissues under control of the helix-loop-helix transcription factor MIST1 [@pone.0067348-Garside1] suggesting a physiological role for Orai1-SPCA2 interaction. However, the functional significance of this interaction remains to be elucidated. Selective and prominent lactation-induced expression of SPCA2 in mammary epithelium [@pone.0067348-Faddy1], [@pone.0067348-Anantamongkol1] provides a first insight into function. Total calcium concentration in milk, including ionized Ca^2+^ and bound calcium, ranges between 40--80 mM in various mammalian species [@pone.0067348-Neville1]. Multiple Ca^2+^ transporters, regulators and binding proteins must be upregulated to drive transcytosis of calcium while maintaining submicromolar cytoplasmic Ca^2+^ concentrations to avoid Ca^2+^ mediated toxicity and cell death. Transport and sequestration of Ca^2+^ is achieved by coordinated increase in the expression of Ca^2+^ pumps, and channels [@pone.0067348-Anantamongkol2]. Current models suggest that polarized mammary secretory cells take up Ca^2+^ at the basolateral membrane via yet to be described Ca^2+^ channel(s) [@pone.0067348-Neville1]. Ca^2+^ entering the mammary secretory cells then travels one of 2 routes to secretion into milk. In the first transport route (accounting for ∼40% of calcium in milk) Ca^2+^ is likely rapidly pumped in Golgi/secretory stores via SPCA1 and/or SPCA2 where it is bound to casein to facilitate casein micelle formation, packaged in secretory vesicles and secreted into milk primarily as Ca^2+^-casein [@pone.0067348-Faddy1], [@pone.0067348-Anantamongkol1], [@pone.0067348-Reinhardt1]. The second transcellular route (accounting for ∼60% of calcium in milk) cell Ca^2+^ is pumped directly across the mammary secretory cells apical membrane into milk by PMCA2bw [@pone.0067348-Reinhardt2], [@pone.0067348-VanHouten1]. The proteins involved in sequestering cell Ca^2+^ while it is shuttled to PMCA2bw are unknown but calbindin-9k is a candidate calcium binding protein that could provide this function [@pone.0067348-Ji1]. An alternate but yet unproven role for PMCA2bw is that PMCA2bw may also pump Ca^2+^ directly into the secretory vesicles while it is trafficked to the apical membrane [@pone.0067348-Reinhardt2]. The least understood part of mammary calcium transport into milk is the mechanism by which calcium enters the basolateral membrane of the lactating mammary secretory cell. Based on our recent findings in mammary tumor cells, we evaluated a potential role for SPCA2 in eliciting Ca^2+^ entry into the lactating mammary secretory cells, by interaction with Orai1 channels [@pone.0067348-Feng1]. In this study, we examine the interaction of SPCA2 with Orai1 channels throughout lactation, both in native mouse tissue and in a three-dimensional cell culture model derived from mammary epithelium ("mammospheres"). We show that SPCA2 and Orai1 are simultaneously induced early in lactation, colocalize, and are required for Ca^2+^ influx into mammary epithelial cells Our observations point to a new role for store-independent Ca^2+^ influx (SICE) in the sequestration of Ca^2+^ from the blood for transport to milk. Results {#s2} ======= Coordinated Induction of a Calcium Transporting Module in Lactation {#s2a} ------------------------------------------------------------------- Lactation is characterized by massive transcellular flux of calcium, from the basolateral side of mammary alveolar epithelium (blood) into lumen (milk). This involves coordinated induction of a host of Ca^2+^ channels, transporters, buffering proteins and regulators, and must be tightly modulated to avoid cytoplasmic calcium toxicity. Previously, analysis of transcripts from mouse mammary gland tissue starting at Day 10 before parturition revealed induction of all three classes of Ca^2+^-ATPases, including isoforms of SERCA, PMCA and SPCA pumps [@pone.0067348-Reinhardt4]. Of note, while PMCA2b isoform showed the largest transcriptional induction of ∼100-fold, the secretory pathway Ca^2+^-ATPase was induced early and significantly prior to parturition. Subsequently, isoform-specific differences in mRNA were noted, with SPCA2 showing higher transcriptional elevation relative to SPCA1 in lactating mouse mammary gland [@pone.0067348-Faddy1], [@pone.0067348-Anantamongkol1]. Here, we validate and quantify isoform-specific differences in SPCA proteins by Western analysis of mouse lactating mammary tissue. While the housekeeping isoform SPCA1 shows a substantive increase of ∼10-fold upon parturition ([Figure 1A](#pone-0067348-g001){ref-type="fig"}), SPCA2 protein is elevated by more than 100-fold ([Figure 1B](#pone-0067348-g001){ref-type="fig"}). Both ATPases remain elevated through lactation. Next, we evaluated transcriptional induction of Orai1 [@pone.0067348-Putney1], an SPCA2-activated Ca^2+^ channel previously described to elicit Ca^2+^ influx in breast derived tumor cells [@pone.0067348-Feng1]. Semi-quantitative PCR analysis showed early induction of Orai1, matching that of both SPCA2 and PMCA2 ([Figure 1C](#pone-0067348-g001){ref-type="fig"}). Two other isoforms, Orai2 and Orai3 were also transcriptionally induced, but at a later stage concordant with SPCA1 induction. STIM1 and STIM2, the calcium sensors in the ER, showed little relative change through the lactation cycle ([Figure 1C](#pone-0067348-g001){ref-type="fig"}). Previously, we had shown that SPCA2 interacts with Orai1 by co-immunoprecipitation from breast cancer derived MCF-7 cells and in HEK293 cells expressing a variety of chimeric and tagged proteins [@pone.0067348-Feng1]. Here, we extended these observations to endogenous proteins expressed in native mammary tissue where such interactions may be physiologically relevant to lactation. Immunoprecipitation of SPCA2 from lactating (Day 5) mouse mammary tissue confirmed a physical association with Orai1 ([Figure 1D](#pone-0067348-g001){ref-type="fig"}). {#pone-0067348-g001} Distinct Localization of Two SPCA Isoforms in Lactating Mouse Mammary Gland {#s2b} --------------------------------------------------------------------------- Previously, Faddy et al. [@pone.0067348-Faddy1] showed that whereas SPCA1 could be stained in all mammary gland cell types, including stromal and myoepithelial cells, expression of SPCA2 was restricted to the luminal epithelium, with no detectable staining outside the acini. Here, we show a further distinction between the two isoforms in subcellular localization as seen in [Figure 2](#pone-0067348-g002){ref-type="fig"}. Immunostaining of SPCA1 was discretely localized to compartments apical to the nucleus ([Figure 2A](#pone-0067348-g002){ref-type="fig"}), overlapping largely with the Golgi marker GM130 ([Figure 2A](#pone-0067348-g002){ref-type="fig"}; merge). In contrast, SPCA2 displayed a broader, punctate distribution with little colocalization with GM130 ([Figure 2B](#pone-0067348-g002){ref-type="fig"}). Thus, we conclude that in lactating mammary tissue SPCA1 has a conventional Golgi distribution, but SPCA2 is largely found in extra-Golgi vesicles. Secondary antibody controls and pre-block with antigenic peptides are shown in [Figure S1](#pone.0067348.s001){ref-type="supplementary-material"}. We also examined the distribution of Orai1 and STIM1 proteins. As expected for its known ER localization, STIM1 had a diffuse reticular distribution, whereas Orai1 was restricted to basolateral domains of the plasma membrane ([Figure 2C](#pone-0067348-g002){ref-type="fig"}). Both Orai1 and STIM1 stained myoepithelial cells, seen as patches in the tissue section. We conclude that SPCA2 and Orai1 are co-expressed in luminal epithelial cells of lactating mammary glands where their interaction may be important in mediating transepithelial Ca^2+^ flux. {#pone-0067348-g002} Expression and Localization of SPCA2 and Orai1 in Mammospheres {#s2c} -------------------------------------------------------------- In order to evaluate potential functional roles in Ca^2+^ handling for SPCA2 and Orai1 in a lactation model, we turned to a three-dimensional culture that mimicked some aspects of native lactating tissue while still retaining the advantages of *in vitro* cell culture. The mouse mammary epithelial line SCp2 responds to basement membrane (Matrigel) and lactogenic hormone (prolactin) by differentiating into alveolus-like structures characterized by induction and secretion of milk protein, β-casein [@pone.0067348-Desprez1]. Formation of mammospheres with distinct lumen and tight junctions occurred over 10 days ([Figure 3A--B](#pone-0067348-g003){ref-type="fig"}). Transcriptional analysis revealed induction of β-casein in the mammospheres, confirming lactation-induced differentiation. We show increase of SPCA2, PMCA2 and Orai1 expression ([Figure 3C](#pone-0067348-g003){ref-type="fig"}), consistent with initiation of a lactogenic program for Ca^2+^ transport as seen in native tissue. Other Orai and STIM isoforms also showed varying levels of transcriptional induction ([Figure 3C](#pone-0067348-g003){ref-type="fig"}). {#pone-0067348-g003} Immunofluorescence staining and confocal microscope imaging of mammospheres revealed punctate distribution of SPCA2 throughout the cell, reminiscent of mammary gland staining, with some concentration of puncta near the cell membranes. A merge with the basolateral marker E-cadherin showed apparent colocalization, although more careful evaluation of transverse sections suggests a juxtaposition of SPCA2 puncta immediately under the cell membrane ([Figure 3D](#pone-0067348-g003){ref-type="fig"}; Movie S1). Orai1 localization was enriched at the outer basal membrane of the mammosphere ([Figure 3E](#pone-0067348-g003){ref-type="fig"}) and a top view of the mammosphere showed a close juxtaposition of SPCA2 with Orai1 ([Figure 3F](#pone-0067348-g003){ref-type="fig"}; Movies S2 and S3). Secondary antibody controls showed no specific staining ([Figure 3G](#pone-0067348-g003){ref-type="fig"}). Taken together, these observations place a portion of SPCA2 at or near the basal membranes of mammospheres where it may be in position to functionally interact with Orai1 to regulate Ca^2+^ influx. SPCA2 and Orai1 are Critical for Ca^2+^ Entry in Mammary Epithelial Cells {#s2d} ------------------------------------------------------------------------- To investigate the potential contribution of SPCA2 and Orai1 in Ca^2+^ entry, we used shRNA constructs packaged in lentiviral vectors to knockdown their expression in SCp2 cells. [Figure 4A](#pone-0067348-g004){ref-type="fig"} is a Western analysis of cultured SCp2 cells showing significant reduction in expression of both proteins following transfection and selection of shRNA viral constructs. Examination of transcripts by semi-quantitative RT-PCR confirmed knockdown of SPCA2 and all three Orai isoforms ([Figure 4B](#pone-0067348-g004){ref-type="fig"}). We also noted small, potentially significant changes in transcript levels of SERCA2b (decreased) and SPCA1 (increased) in response to the knockdowns. SCp2 cells with either Orai or SPCA2 knockdown formed normal monolayers and grew at similar rates to control (scrambled shRNA), as seen in [Figure S2A](#pone.0067348.s002){ref-type="supplementary-material"}--B. Although Orai knockdown cells were able to polarize and form tight junctions as seen by the staining with E-cadherin ([Figure S3A](#pone.0067348.s003){ref-type="supplementary-material"}), mammosphere production was nearly absent, and was also noticeably decreased in shSPCA2 treated cells, with concomitant increase in number of small clumps of cells (spheroids; [Figure 4C-D](#pone-0067348-g004){ref-type="fig"}). {#pone-0067348-g004} We examined the effect of SPCA2 and Orai1 knockdown on Ca^2+^ signaling pathways in monolayer SCp2 cells. Resting Ca^2+^ levels were significantly lowered in both SPCA2 and Orai1 knockdown cells ([Figure 4E](#pone-0067348-g004){ref-type="fig"} inset), consistent with our previous observation in HEK293 and tumor-derived MCF7 cells [@pone.0067348-Feng1]. In control SCp2 cells, addition of thapsigargin blocks the SERCA2 Ca^2+^-ATPase resulting in passive release of SERCA2-filled stores, followed by store-operated Ca^2+^ entry (SOCE) upon reintroduction of extracellular Ca^2+^([Figure 4E](#pone-0067348-g004){ref-type="fig"}). However, thapsigargin-induced Ca^2+^ release and subsequent Ca^2+^ entry were both largely diminished upon SPCA2 and Orai1 knockdown ([Figure 4E](#pone-0067348-g004){ref-type="fig"}). One interpretation was that ER stores and SOCE were both severely depleted in these knockdowns, however their normal growth and appearance was not consistent with ER stress or subsequent cell death ([Figure S2](#pone.0067348.s002){ref-type="supplementary-material"}). Indeed, total stored Ca^2+^ released by ionomycin was similar in control and knockdown cells ([Figure 4F](#pone-0067348-g004){ref-type="fig"}). Alternatively, a decrease in thapsigargin-releasable Ca^2+^ can be explained by a shift to thapsigargin-insensitive stores consistent with the transcriptional changes shown in [Figure 4B](#pone-0067348-g004){ref-type="fig"}. Previously, we showed that overexpression of the thapsigargin resistant SPCA1 pump in HEK293 cells blocked release of stored Ca^2+^ by thapsigargin and STIM1-mediated SOCE in response to thapsigargin [@pone.0067348-Feng1]. These findings provide functional evidence for a major role in Ca^2+^ handling for SPCA pumps and Orai channels in a mammary epithelium. SPCA2 is Required for Cell Surface Trafficking of Orai1 in SCp2 Cells {#s2e} --------------------------------------------------------------------- Given the interaction between SPCA2 and Orai1, we considered the effect of gene knockdowns on their biogenesis and trafficking in mammary epithelial cells. In control SCp2 cells grown as monolayer, SPCA2 had punctate, perinuclear distribution whereas Orai1 showed both intracellular and plasma membrane staining ([Figure 5A, C](#pone-0067348-g005){ref-type="fig"}). There was minor colocalization of the two at the plasma membrane, as detected by cell surface biotinylation (not shown). Upon knockdown of Orai1, SPCA2 localization appeared more restricted to the perinuclear region, with less punctae near the plasma membrane ([Figure 5B](#pone-0067348-g005){ref-type="fig"}) although tight junction formation monitored by E-cadherin labeling appeared normal ([Figure S3A](#pone.0067348.s003){ref-type="supplementary-material"}). Strikingly, distribution of Orai1 largely shifted to the same perinuclear localization upon SPCA2 knockdown ([Figure 5D](#pone-0067348-g005){ref-type="fig"}). Consecutive confocal sections through individual cells showed that Orai1 localized around, but not in the nucleus in shSPCA2 knockdown cells ([Figure S3B](#pone.0067348.s003){ref-type="supplementary-material"}--D). Given that expression of the major Golgi Ca^2+^-ATPase SPCA1 was retained ([Figure 4B](#pone-0067348-g004){ref-type="fig"}), the results suggest that specific interaction with SPCA2 may be important for cell surface trafficking of Orai1 in mammary epithelium cells. {#pone-0067348-g005} C-terminal Domain of SPCA2 Partially Rescues Orai1 Trafficking and Restores Ca^2+^ Influx {#s2f} ----------------------------------------------------------------------------------------- To confirm the role of SPCA2 in cell surface localization of Orai1 in mouse SCp2 cells, we reintroduced full-length, GST-tagged human SPCA2 into shSPCA2 treated cells using virally packaged vectors for efficient transfection. We also introduced the membrane-anchored C-terminal domain of hSPCA2 (hSPCA2C) previously shown to be necessary and sufficient to interact with Orai1 and elicit Ca^2+^ influx [@pone.0067348-Feng1]. hSPCA2C was at least partially effective in redistributing Orai1 out of the perinuclear region to a more punctate location ([Figure 6A](#pone-0067348-g006){ref-type="fig"}). Full-length hSPCA2 fully reversed the effect of the knockdown, resulting in plasma membrane trafficking of Orai1 ([Figure 6A](#pone-0067348-g006){ref-type="fig"}). {#pone-0067348-g006} shSPCA2 knockdown and re-transfected SCp2 cells were loaded with Fura-2 for Ca^2+^ imaging. Neither full-length or C-terminal domain of SPCA2 fully restored thapsigargin-sensitive stores ([Figure 6B](#pone-0067348-g006){ref-type="fig"}), again consistent with a shift to thapsigargin-insensitive stores. Strikingly, upon addition of extracellular Ca^2+^, Ca^2+^ influx was elevated and sustained with both constructs, to levels even higher than control (empty vector, EV-GST). Store-independent Calcium Entry (SICE) Requires SPCA2 and Orai1, and is Elevated in Mammospheres {#s2g} ------------------------------------------------------------------------------------------------ Recently, we obtained evidence for a novel mode of store independent Ca^2+^ entry (SICE) elicited by SPCA2 via interaction with Orai1 (5). Following brief exposure of SCp2 monolayer cells to nominally Ca^2+^ free medium, reintroduction of extracellular Ca^2+^ was accompanied by rapid and transient influx that was abolished by knockdown of either SPCA2 or Orai channels ([Figure 7A](#pone-0067348-g007){ref-type="fig"}). Addition of thapsigargin to SCp2 at 5--30 second intervals following transfer to Ca^2+^ free medium demonstrated that the stores were largely unchanged ([Figure S4A](#pone.0067348.s004){ref-type="supplementary-material"}) as seen by the rate of Ca^2+^ release and peak height. Therefore, the Ca^2+^ influx observed in [Figure 7A](#pone-0067348-g007){ref-type="fig"} is not likely to be SOCE. Furthermore, we evaluated maximal SOCE in these cells by comparing thapsigargin-elicited elevation of Ca^2+^ levels in the presence or absence of extracellular Ca^2+^ (2 mM), shown in [Figure S4B](#pone.0067348.s004){ref-type="supplementary-material"}. Maximal SOCE estimated by this method was significantly smaller than that observed in [Figure 7A](#pone-0067348-g007){ref-type="fig"}. Therefore we conclude that SPCA2 and Orai1 contribute to a store-independent mechanism of Ca^2+^ entry (SICE) that is unmasked upon brief removal of extracellular Ca^2+^. As additional demonstration of this mechanism of Ca^2+^ entry, we evaluated SICE in shSPCA2 treated cells that were transfected with full-length or C-terminal domain of hSPCA2. Both constructs were able to confer elevated and sustained Ca^2+^ entry to the SPCA2 knockdown cells, consistent with plasma membrane delivery and functional rescue of Orai1 ([Figure 7B](#pone-0067348-g007){ref-type="fig"}). These results also suggest that a portion of the Ca^2+^ entry observed after thapsigargin addition, as seen in [Figure 6B](#pone-0067348-g006){ref-type="fig"}, was due to SICE. {#pone-0067348-g007} Finally, we investigated whether this store-independent mode of Ca^2+^ entry occurred in mammospheres, where SPCA2 and Orai1 are induced upon differentiation. Monolayer and mammosphere cells ([Figure 7C](#pone-0067348-g007){ref-type="fig"}) were loaded with Fura2-AM, washed and imaged. In differentiated mammospheres, SICE occurred with slower kinetics relative to monolayer cells, and remained elevated and sustained ([Figure 7D](#pone-0067348-g007){ref-type="fig"}), slowly returning to baseline (not shown). Basal Ca^2+^ concentrations were similar in mammospheres and monolayer SCp2 cells ([Figure 7E](#pone-0067348-g007){ref-type="fig"}). It is possible that the slow rise in Ca^2+^ upon induction of SICE is due to slower recruitment of SPCA2-containing vesicles to the mammosphere basal membrane, relative to STIM1, as well as active Ca^2+^ sequestration consistent with transcriptional induction of SPCA pumps in mammospheres. Thapsigargin elicited a smaller release of Ca^2+^ in mammospheres ([Figure 7F](#pone-0067348-g007){ref-type="fig"}), again consistent with induction of thapsigargin-insensitive SPCA pumps, but Ca^2+^ entry was significantly larger. Taken together, we propose a model in which the induction and interaction of Orai1 and SPCA2 upon lactogenic differentiation results in increased store-independent Ca^2+^ influx. Discussion {#s3} ========== Orai1 Mediates Basolateral Ca^2+^ Influx in Mammary Epithelium {#s3a} -------------------------------------------------------------- Polarized localization of Orai1 in secretory epithelium has previously been investigated only in pancreatic acinar and salivary gland cells. In pancreatic acinar cells, where secretagogue-induced Ca^2+^ signaling is accompanied by robust Ca^2+^ extrusion and refilling of stores, Orai1 co-localized with STIM1 in puncta along the basolateral membrane [@pone.0067348-Lur1], consistent with a role in store-operated Ca^2+^ entry. Paradoxically, however, the bulk of Orai1 was seen in the apical membrane where it co-localized and interacted with IP3R but not STIM1. Thus, additional roles for Orai1 separate from SOCE, are likely and remain to be elucidated. An independent study by Hong et al. [@pone.0067348-Hong1] localized Orai1 to both apical and lateral membranes of pancreatic acinar cells. These studies highlight the possibility of store-independent roles for this so-called "store-operated" Ca^2+^ influx channel. The molecular identity of the calcium influx pathways in mammary epithelial cells has been a mystery. A genome-wide screen revealed several voltage-gated and TRP channels that were expressed but not increased dramatically during Day 1 of lactation [@pone.0067348-VanHouten1]. McAndrew et al. [@pone.0067348-McAndrew1] reported an increase in Orai1, but not Orai2 or Orai3 transcript in lactating mice. Here we show early induction of Orai1, concomitant with calcium pump isoforms SPCA2 and PMCA2, whereas Orai2--3 isoforms are elevated later in lactation (Day 5, post parturition), as seen for SPCA1. Thus, the Orai channels are good candidates for Ca^2+^ entry in mammary epithelial cells. This was substantiated by a distinct localization of endogenous Orai1 to the basolateral membrane of lactating mammary epithelia and in differentiated mammospheres cultured in vitro. Finally, knockdown of all three Orai channel isoforms abolished store-independent Ca^2+^ entry in SCp2 cells, implicating a prominent role for these channels in basolateral influx. SPCA2 Interacts with Orai1 to Mediate Store-independent Ca^2+^ Entry in Mammary Epithelium {#s3b} ------------------------------------------------------------------------------------------ Previously, we had demonstrated an unexpected moonlighting function of SPCA2, independent of pumping activity, in which the N- and C- termini interacted with Orai1 to elicit Ca^2+^ entry into cells. A consequence of this signaling activity was the oncogenic role of SPCA2 in breast cancer cells, where dysregulation of expression led to constitutive Ca^2+^ influx, activation of MAP kinase pathways, high rates of proliferation and tumorigenesis. Furthermore, SPCA2-mediated activation of Ca^2+^ entry appeared to be independent of both Golgi and ER stores, and of the STIM sensors [@pone.0067348-Feng1]. Thus, in non-polarized cells, this unusual property would result in a futile cycle of energy independent activation of Ca^2+^ entry, via Orai1, and energy (ATP)-requiring Ca^2+^ efflux via active sequestration of Ca^2+^ into the secretory pathway. However, SPCA2 is restricted to polarized cells of secretory or absorptive epithelia (intestinal, lung, mammary and other glands; [@pone.0067348-Vanoevelen1]). Therefore, we hypothesized that induction of SPCA2 expression in polarized cells may allow activation of Ca^2+^ influx channels at one membrane domain, and active transport of Ca^2+^ at the other membrane domain, to facilitate transepithelial transport of Ca^2+^ across the epithelium. Here, we provide evidence for a robust physiological role of SPCA2 in promoting SICE in a model of lactating mammary epithelia. Although both SPCA isoforms are upregulated during lactation, we found that SPCA2 was elevated earlier and to significantly higher levels, compared to SPCA2. Whereas SPCA1 showed discrete localization to Golgi compartments, SPCA2 was predominantly localized to a vesicular compartment, closely associated with both apical and basolateral membranes. These differences are consistent with non-redundant roles for two isoforms of the secretory pathway pumps in mammary epithelium and suggested an isoform-specific critical function for SPCA2 in lactation. We began by showing co-expression, partial colocalization and co-immunopreciptation of endogenous SPCA2 with Orai1 in lactating mouse mammary epithelium and in differentiated mammospheres. Next, we showed significant induction of SICE concomitant with induction of Orai1 and SPCA2 upon lactogenic differentiation of SCp2 cells into mammospheres. Finally, knockdown of either SPCA2 or Orai channels virtually abolished SICE in mammospheres. Taken together, these data provide evidence for a physiologically relevant function for SPCA2 in the activation of Orai1. Further investigation should reveal whether SPCA2 also interacts with other Orai isoforms in lactation. We note that a C-terminal transcript of SPCA2 is expressed under control of MIST1, a beta helix-loop-helix transcription factor, in pancreatic acinar cells [@pone.0067348-Garside1]. This ∼20 kDa fragment lacks ATP and Ca^2+^ binding sites, and therefore, has no transport function. However, we found that a minimal membrane-anchored C-terminal domain is sufficient to activate Orai1 and elicit Ca^2+^ influx. It remains to be determined whether native expression of this membrane-embedded C-terminal domain also mediates SICE in various secretory epithelia by interaction with basolateral Orai1. Interaction between SPCA2 and Orai1 is Required for Orai1 Trafficking and SOCE in Mammary Epithelium {#s3c} ---------------------------------------------------------------------------------------------------- The severe reduction in thapsigargin-sensitive but not ionomycin-sensitive stores upon knockdown of Orai channels suggested a shift in expression and/or localization of thapsigargin-insensitive SPCA pumps. We show a redistribution of SPCA2 to a more perinuclear location, continuous with the nuclear membrane, in the absence of Orai channels. More dramatically, distribution of endogenous Orai1 was limited to a similar perinuclear reticular compartment in shSPCA2 cells, suggesting an arrest in trafficking out of the endoplasmic reticulum. Distinct from conventional endoplasmic reticulum, this specialized sub-compartment remains to be identified. The biogenesis defect was effectively reversed by introducing hSPCA2 into the knockdown cells, ruling out any non-specific effects of the shRNA reagent, with concomitant functional restoration of Ca^2+^ influx. Given the expression of SPCA1 in these cells, we reasoned that Orai1 trafficking did not require secretory pathway/Golgi Ca^2+^ stores. Indeed, the ability of the C-terminal domain of SPCA2 to partially mediate Orai1 exit from this perinuclear compartment suggests that Orai1 trafficking requires a chaperone-like interaction with SPCA2 protein. Further studies using longer fragments, or chimeric constructs with both N- and C-termini implicated in Orai1 binding [@pone.0067348-Feng1], may narrow down the precise domains of SPCA2 that mediate this function. It also remains to be seen whether SPCA2 facilitates Orai1 trafficking in other secretory epithelia where it is expressed. In conclusion, we demonstrate an isoform-specific physiological role for SPCA2 in lactating mammary epithelium. This role is mediated in large part by interaction with Orai1, although additional Ca^2+^ entry channels may also be involved. These studies lay a framework for the investigation of SPCA2 function in other secretory or absorptive epithelia, where high expression levels have been documented. Prior to this study nothing was known about the mechanisms that mediate basolateral calcium entry into the mammary secretory cell to support calcium needs for milk production. The data presented here demonstrate that SPCA2 and Orai1 function together to regulate SICE, which mediates the massive basolateral Ca^2+^ influx into mammary epithelia to support the large calcium transport requirements of lactation. Methods {#s4} ======= Animals {#s4a} ------- All animal work was conducted according to relevant national and international guidelines. The National Animal Disease Center's Animal Care and Use Committee approved all animal procedures. Pregnant or lactating 129/SV mice were housed individually, in hanging basket cages on sawdust bedding. All mice were equalized to 6 pups per mouse mother on day one of lactation. Mice were killed at times indicated. Mice were anesthetized with a 50∶50 mix of CO~2~:O~2~ followed by decapitation. Mammary tissue was removed and fixed as described below or flash frozen in liquid N~2~, and stored at −70°C until processed as described. Cell Culture {#s4b} ------------ SCp2 cells (gift of Andrew Ewald, Johns Hopkins University) [@pone.0067348-Desprez1] were cultured in monolayer media-DMEM/F-12 (1∶1) with 5% HI-FBS and insulin (5 µg/ml) at 37°C in a 5% CO~2~ incubator with ample humidity. Upon 80% confluence, they were trypsinized in 0.25% Trypsin with EDTA (Invitrogen) and centrifuged at 8 rpm for 2 minutes to remove excess trypsin. Cell growth was monitored using Cell Titer 96 AQ from Promega according to manufacturer's instructions. For mammospheres, trypsinized cells were then mixed with a solution containing 50% Matrigel (Sigma) and 50% differentiation media (DMEM/F-12 (1∶1), hydrocortisone (1 µg/ml), insulin (5 µg/ml) and recombinant mouse prolactin (3 µg/ml). Cells were plated at 1×10^3^ on a 100% Matrigel matrix on 25 mm round coverslips. Matrix was again allowed to solidify prior to the addition of differentiation media to cover the layered coverslip. Mammosphere culture was allowed to differentiate for 10 days, changing the media every second day, prior to assays for differentiation. Mammosphere Culture {#s4c} ------------------- SCp2 cells were plated on 25 mm coverslips for three-dimensional culture at 10^3^ cells per coverslip. These coverslips were placed in a standard cell culture incubator and allowed to differentiate. Differentiation liquid media was changed every two days and replaced with fresh media. Cellular clusters (consisting of dense groups of \>4 cells each) were counted and categorized into one of three categories: (1) undifferentiated clusters- cells which maintained characteristic of monolayer culture but were in close proximity to one another (single cells); (2) spheroids- partially differentiated cell clusters with characteristics which showed lack of tight junction formation and lumen; (3) mature mammospheres- cells which grouped into a differentiated mass with tight junctions and a lumen. All three classifications were considered clusters (belonging to group 1) but the proportion of spheroids to mature mammospheres and the percentage of cells, which went through the entire development program, were noted. RNA Collection and RT-PCR {#s4d} ------------------------- 10^6^ cells were washed with sterile Hank's Balanced Salt Solution (HBBS; Invitrogen) prior to RNA extraction. An RNAeasy kit was used to collect the total RNA via centrifugation (Qiagen) per the manufacturer's instructions and quantified by absorption at 260 nm by spectrometry. RNA was converted to cDNA by reverse transcription using iScript (Bio-Rad Laboratories). Equal amounts of cDNA was subjected to PCR amplification for specific transcripts using the following primers: Orai1∶5′-ACCCCACGAGCGCATGCATC-3′ (*forward*) and 5′-GCTTGGTGGGGCTTGGCTGT-3′ (*reverse*); Orai2∶5′-CTGAGGTGGTCCTGCTCT-3′ (*forward*) and 5′-GGTAGAAGTGGATGGTGAAG-3′ (*reverse*); Orai3∶5′-CATCCACAATCTCAACTCTG-3′ (*forward*) and 5′-ATAGAAGCAGAGGATGGTGT-3′ (*reverse*); STIM1∶5′-AAGAGTCTACCGAAGCAGAG-3′ (*forward*) and 5′-GTGCTATGTTTCACTGTTGG-3′ (*reverse*); STIM2∶5′-GTGCGCTGGGTCGGAAGAC-3′ (*forward*) and 5′-GGGGCACCAGATCGCATCG-3′ (*reverse*); SPCA1∶5′-CCAGTGTGGCCGTGGCTGAC-3′ (*forward*) and 5′-TCAGCCTGGAGAAGGCCTGCAA-3′ (*reverse*); SPCA2∶5′-GACCTGCTGCTGCTGACGGG-3′ (*forward*) 5′-CAGGCCAGAGGCACCCAAGC-3′ (*reverse*); PMCA2∶5′-CAGGGTCTGCCACCCTCGGAG-3′ (*forward*) and 5′-CATGGTCGGGACAGCTCCCCTA-3′ (*reverse*); SERCA2B: 5′-ACTTCTTGATCCTCTACGTG-3′ (*forward*) and 5′-AGACCAGAACATATCGCTAA-3′ (*reverse*); actin: 5′-GCAGCTCCTTCGTTGCCGGT-3′ (*forward*) and 5′-TACAGCCCGGGGAGCATCGT-3′ (*reverse*). The number of amplification cycles was adjusted to ensure that generation of products was not saturated. Loading control was mActin. Membrane Preparation and Western Blotting {#s4e} ----------------------------------------- Mammary tissue microsomes were prepared as previously described [@pone.0067348-Reinhardt2]. Briefly, tissue was homogenized in 10 volumes of Buffer A containing Tris--HCl (10 mM), MgCl~2~ (2 mM), sucrose (300 mM) and a complete protease inhibitor cocktail (Boehringer Mannheim Indianapolis, IN) at pH 7.5. The homogenate was mixed with an equal volume of Buffer B (Buffer A plus 0.3 M KCl) and centrifuged at 4000 g for 10 min. The supernatant was collected, adjusted to 0.7 M KCl, and centrifuged at 100,000 g for 1 h. The supernatant was discarded and the pellets were resuspended in Buffer C (Buffer A plus 0.15 M KCl). Microsomes from mammary tissue were incubated for 15 min at room temperature in a modified Laemmli buffer containing 150 mg/ml urea and 65 mM DTT. Samples were then electrophoresed for 50 minutes at 200 volts in a 4--12% Novex NuPAGE® Bis-Tris Gel using MOPS SDS running buffer (Life Technologies, Grand Island, NY). Proteins were transferred to nitrocellulose membranes using the iBlot Dry Blotting System (Life Technologies, Grand Island, NY) at 23 volts for 7 minutes. The blots were blocked with StartingBlock T20 (Thermo Fisher Scientific Inc). SPCA1 (RS-1 \#227) or SPCA2 (orange \#2) antibodies were diluted 1/2000 in StartingBlock T20 and the blots were incubated over night at 4°C. After washing they were incubated 1 hr at RT with 1/50000 HRP goat anti-rabbit \#31460 (Thermo Fisher Scientific Inc) and washed. Blots were developed using Pierce's Supersignal (Pierce Products, Rockford IL) using the protocol provided by the manufacturer. Developed film was imaged and bands quantitated with a Gel Doc EZ imager (BioRad, Hercules, CA). 1×10^6^ cells from cell culture were washed three times with sterile HBBS (Invitrogen) then immediately lysed in buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM Na~3~EDTA, 1 mM EGTA, 5 mM Na~4~P~2~O~7~, 1 mM Na~3~VO~4~, 10 mM NaF, pH 7.4 with 1% SDS in the presence of protease inhibitor cocktail (Roche). Protein concentrations were assayed using a bicinchoninic acid assay kit (Pierce). Approximately 50 µg of protein was loaded onto a NuPAGE gel for SDS-PAGE Analysis and Western blotting. Immunoblots were made on 0.45 µm nitrocellulose membrane (Bio-Rad). Antibodies for mouse SPCA2, Orai1 and GAPDH were incubated with the membrane overnight at 1∶1000 dilution in 1×PBST (PBS plus 0.2% Tween). Membranes were washed five times in PBST then mouse or rabbit secondary antibody conjugate to HRP was added (GE Healthcare UK Limited) at 1∶2000 for one hour at room temperature on a rotator and the membranes were washed three times with PBST then four times with normal PBS to remove Tween residue. Blots were visualized using ECL kit (Pierce) on an Intelligent Dark Box Imager (Fuji Film). Immunofluorescence Microscopy {#s4f} ----------------------------- Mammary tissue was fixed in Tellyesniczky's fixative (70% ethanol, formalin, glacial acetic acid, 20∶2∶1) for 5 h at room temperature [@pone.0067348-Shillingford1] and then stored in 70% ethanol prior to paraffin embedding. Paraffin-embedded sections were cleared in xylene and rehydrated through an alcohol series. Tissue sections were immersed in antigen unmasking solution H-3300 (Vector Laboratories; Burlingame, CA) in a preheated pressure cooker. Sections remained in the pressure cooker for 20 minutes after the pressure maximized. The sections were then allowed to cool, wash 3 times with distilled, deionized water followed by PBS for 5 minutes. After permeabilization/blocking of the sections with PBS containing 0.5% Triton X-100,.01 g sodium azide and 50 mg/ml BSA, the primary antibodies were applied and incubated at 4°C overnight. Primary antibodies used were 1/50 Orai1 \# 4281 (ProSci Incorporated, Poway, CA), 1/50 Stim1 \# 610954 (BD Transduction Laboratories, San Jose, California), 1/100 GM130 \#610822 (BD Transduction Laboratories, San Jose, California). SPCA1 (1/100) and SPCA2 (1/100) antibodies were prepared and characterized as previously described [@pone.0067348-Reinhardt1], [@pone.0067348-Reinhardt3]. The slides were washed 3 times in blocking buffer and then incubated for 2 hr at 37°C using secondary antibodies Green anti-mouse at 1∶1000 Alexa Fluor A11017 488 F(ab')~2~ fragment of goat anti-mouse IgG(H+L), and Red anti-rabbit at 1∶1000 Alexa Fluor A11070 594 F(ab')~2~ fragment of goat anti-rabbit IgG (H+L) (Molecular Probes/Life Technologies, Grand Island, NY). The slides were washed 3 times with blocking buffer and mounted with VectaShield (Vector Laboratories). Slides were viewed and photographed on Zeiss Axio Scope.A1 fluorescence microscope (Carl Zeiss, Germany). Monolayer cultures were washed three times with 1×PBS prior to fixation in 4% paraformaldehyde and subsequent permeation in blocking buffer (1% BSA and 0.5% Triton-X in 1xPBS). Cells were then blocked and permeabilized in 0.1% Triton and 1% BSA in 1xPBS and washed three times in 1xPBS. Cells were incubated with antibodies to SPCA2 [@pone.0067348-Feng1], Orai1 (Sigma) and E-cadherin overnight at 4°C. The next day cells were washed again (three times in 1xPBS) and incubated with conjugated anti-rabbit and anti-mouse (AlexaFluor 288 and 388, respectively) for one hour at room temperature. Finally, cells were washed in 1xPBS three times, washed briefly in DAPI and in sterile water then mounted onto slides with DAKO Fluorescent Mounting Medium (DAKO). Mammospheres were fixed in ice-cold methanol and acetone (−20°C; 1∶1) for five minutes on ice [@pone.0067348-Faddy1]. Subsequently, the mammospheres were permeabilized and blocked in 0.1% Triton and 1% BSA in 1xPBS for 1 hour at RT. They were then washed in 1xPBS for 5 minutes three times. Primary antibodies against SPCA2, Orai1 and E-cadherin were diluted in 0.2% BSA in 1xPBS. Mammospheres were allowed to incubate in primary antibodies at 1∶250 dilutions overnight at 4°C. The next day, the mammospheres were washed gently in 1xPBS three times for 5 minutes at RT. Next, rabbit and mouse conjugated AlexaFluor secondaries (AlexaFluor 288 and 388, respectively) were incubated at a 1∶1000 dilution at RT for 1 hour in the dark, with slow agitation. Cells were washed with 1x PBS three times for 10 minutes with slow agitation in the dark for 5 minutes per wash. Cells were DAPI stained by brief submersion, washed in distilled, deionized water for 1 minute then the coverslips were permanently mounted to slides with fluorescent mounting medium (DAKO) and allowed to dry overnight in the dark. Lentiviral Production and Transfection {#s4g} -------------------------------------- pLK0.1 plasmids were obtained from Sigma (Mission shRNA constructs) for SPCA2 and Orai1. HEK293T cells were inoculated with 1 µg plasmid DNA for each accessory packaging protein for lentiviral packaging. Media was removed from the cells after 72 hours and virus was purified using Lenti-X (Sigma) per the manufacturer's instructions. Approximately 10 µg of virus was added to each well of a 6 well plate containing SCp2 cells at 50--70% confluence and allowed to incubate for 48 hours. The virus was then removed and cells were washed then inoculated with new media containing 500 µg/ml of puromycin for selection over 3 days. Once the cells were selected, they were grown in the selection media for two weeks then discarded. Each experiment was done within 5 passages of the cells to ensure complete RNA inhibition. Calcium Imaging {#s4h} --------------- Cells were cultured as a monolayers or mammospheres on 25 mm circular coverslips. Briefly, cells were washed in sterile HBBS (Invitrogen) for five minutes at a time, three times. After washing, FURA2-AM (Invitrogen) was added at a final concentration of 1 µg/ml in imaging buffer (20 mM Hepes, 126 mM NaCl, 4.5 mM KCl, 2 mM MgCl~2~, 10 mM glucose at pH 7.4) containing 2 mM CaCl~2~. After incubation at room temperature for 20 minutes, cells were washed briefly in imaging buffer without calcium and 0.15% EGTA. Cells were then washed twice in imaging buffer without calcium to wash away residual EGTA and Ca^2+^. For mammosphere imaging, protocols were kept the same, however, after FURA2 loading, two 2 mM Ca^2+^ washes were added with slow agitation to remove FURA2 from the Matrigel, prior to washing in imaging buffer without calcium. Store dependent (SOCE) and independent (SICE) Ca^2+^ entry was measured as described [@pone.0067348-Feng1]. Cells were excited at 340 nm and 380 nm, and Fura emission was monitored at 505 nm. For SOCE, cells were switched from 2 mM Ca^2+^ to nominally Ca^2+^ free recording buffer. Thapsigargin (2 µm) or ionomycin (2 µm) was added where indicated, and followed by readdition of 2 mM Ca^2+^ to measure store-dependent Ca^2+^ influx. For SICE, fluorescence was recorded from cells placed in nominally Ca^2+^ free recording buffer, followed by addition 2 mM Ca^2+^, activating store-independent Ca^2+^ influx. Supporting Information {#s5} ====================== ###### **Control experiments for immunostaining of mammary gland.** Sections of lactating mouse mammary tissue were treated with a mixture of anti-SPCA1 or anti-SPCA2 antibody either with or without preincubated with the immunogenic peptide as indicated. Secondary antibody controls used in the absence of SPCA antibodies resulted in no specific signal, as shown. Nuclei are detected by DAPI staining as described in Methods. (TIF) ###### Click here for additional data file. ###### **Morphology and Growth of SCp2 cells after transfection with shRNA constructs.** A. Growth of SCp2 cells after transfection with lentivirus carrying empty vector or shRNA against SPCA2 or Orai1 was monitored using MTT assay as described in Methods. No significant differences were observed following knockdown. B. Morphology of SCp2 cells following knockdown of SPCA2 and Orai1 is similar to that of control cells. (TIF) ###### Click here for additional data file. ###### **Confocal sections of Immunofluorescence staining in Orai1 (A) and SPCA2 (B, C) knock down SCp2 cells.** A. Consecutive optical sections, starting from the bottom, of SCp2 cells knocked down for Orai channels stained with E-cadherin (red), SPCA2 (green) and DAPI (blue). Note the formation of tight junctions indicated by E-cadherin stain and normal shape of the cells. B. Consecutive optical sections, starting from the bottom, of SCp2 cells knocked down for SPCA2 stained with Orai1 (green) and DAPI (blue). Note the separation of Orai1 stain from the nucleus at the bottom and top sections. C. Confluent SCp2 cells treated with shEV (top) or shSPCA2 (bottom) stained for Orai1 (green) and DAPI (blue). Note the change in Orai1 localization from the cell boundaries (top) to circumnuclear (bottom). (TIF) ###### Click here for additional data file. ###### **Estimation of Stored Ca^2+^ in SCp2 cells.** A. SCp2 cells were transferred to nominally Ca^2+^ free medium at time 0 and thapsigargin was added between 5--30 seconds as indicated. Ca^2+^ release was monitored by Fura2 (340/380 ratio). B. SCp2 cells were transferred to nominally Ca^2+^ free medium or not, at time 0 as indicated. Baseline Ca^2+^ before addition of thapsigargin was unchanged (*inset i*) Thapsigargin was added and Ca^2+^ release was monitored as fold-change relative to starting 340/380 ratios. The difference between the traces, indicated by gray shading, was plotted in *inset ii*. This indicates maximal SOCE resulting from thapsigargin mediated store release. Note that it is smaller than Ca^2+^ influx observed in [Figure 6B](#pone-0067348-g006){ref-type="fig"}. (TIF) ###### Click here for additional data file. ###### **Consecutive confocal sections of a mammosphere immunostained with antibody against E-cadherin (red) and SPCA2 (green), with DAPI stain of nuclei (blue).** (GIF) ###### Click here for additional data file. ###### **Consecutive confocal sections of a mammosphere immunostained with antibody against Orai1 (red) and SPCA2 (green), with DAPI stain of nuclei (blue).** (GIF) ###### Click here for additional data file. ###### **Consecutive confocal sections of a mammosphere immunostained with antibody against Orai1 (red) and SPCA2 (green), with DAPI stain of nuclei (blue).** (GIF) ###### Click here for additional data file. We thank Andrew Ewald (Johns Hopkins University) for gift of SCp2 cells and helpful advice on mammospheres culture. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: BC TR RR. Performed the experiments: BC AH. Analyzed the data: BC AH RR TR. Contributed reagents/materials/analysis tools: TR AH BC. Wrote the paper: BC TR RR.

Introduction {#Sec1} ============ Intervertebral disc (IVD) degeneration is clinically related to chronic low back pain, disc herniation, spinal canal stenosis, and spinal deformities \[[@CR1]\]. These spinal disorders are the leading causes of disability in the workforce and result in large economic and social costs \[[@CR2]\]. The etiology of IVD degeneration is complex and multifactorial, with contributions from aging, mechanical stress, smoking, infection, trauma, and heredity \[[@CR3]--[@CR5]\]. At present, the molecular mechanisms underlying IVD degeneration are largely unclear, and there are no effective therapies. IVDs, which lie between the adjacent vertebral bodies and provide flexibility and load support in the spine, are composed of two discrete components: the nucleus pulposus (NP) and the annulus fibrosus (AF). The interior structure, the NP, is an avascular cartilage-like tissue that contains extracellular matrix (ECM) proteins rich in proteoglycans. The AF, a fibrous cartilage composed of an inner and outer coaxial lamella, enwraps the NP. The NP has two different cell populations in humans: small chondrocyte-like cells and large vacuolated notochordal cells \[[@CR6]--[@CR8]\]. Because notochordal cells are rarely present after adolescence, the pathogenesis of IVD degeneration is possibly linked to loss of the cells \[[@CR6], [@CR9]\]. IVD degeneration is characterized by the reduction of water content and ECM breakdown. Biologically, this degeneration represents a loss of steady-state metabolism that probably results from an imbalance between anabolic and catabolic processes \[[@CR10], [@CR11]\]. Increased expression of proinflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor alpha (TNFα) and loss of aggrecan, a major component of proteoglycan in IVD, have been observed in degenerative discs \[[@CR12], [@CR13]\]. These changes are associated with the increased expression of matrix-degrading enzymes such as matrix metalloproteases (MMPs) and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) \[[@CR14]--[@CR17]\]. Many studies have found elevated levels of MMP-1, MMP-2, MMP-3, and MMP-13 in degenerated IVD \[[@CR14], [@CR15]\]. In addition, several lines of evidence support the presence of prostaglandins in IVD under stress and implicate the possible involvement of prostaglandins in the progression of IVD degeneration \[[@CR18], [@CR19]\]. The expression of cyclooxygenase-2 (COX-2), a key enzyme in prostaglandin biosynthesis in disc cells, has also been shown to be induced by mechanical stress, another predisposing factor that can disrupt the disc structure and initiate the degenerative cascade \[[@CR20], [@CR21]\]. These inflammatory reactions are clinically known to be the cause of lower back pain or radiculopathy in spinal disorders \[[@CR22]\]. According to the free-radical theory of aging, oxidative stress initiated by reactive oxygen species (ROS) contributes to the functional decline that is characteristic of aging \[[@CR23]\]. ROS, including the superoxide anion (O^2−^), the hydroxyl radical (OH), hydrogen peroxide (H~2~O~2~), and nitric oxide (NO), all of which can diffuse through membranes, are byproducts of cellular oxidative metabolism. Excessive ROS can overwhelm the antioxidant scavenging capacity within a cell and cause oxidative damage to DNA, lipids, and proteins as well as concomitant cellular damage. Numerous diseases are known to involve oxidative stress, including musculoskeletal diseases such as osteoarthritis and osteoporosis \[[@CR24]--[@CR26]\]. In IVD, the expression level of similar oxidative stress markers such as carboxymethyl-lysine, advanced glycation end products (AGEs), and peroxynitrite were reported to be elevated in degenerative human discs \[[@CR27]--[@CR29]\]. In cultured disc cells, the activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and Akt signaling pathways and nuclear translocation of nuclear factor (NF)-κB and Nrf2 were reported to be induced by treatment with H~2~O~2~ \[[@CR30]\]. In vivo, the mitochondria-targeted ROS scavenger rescued age-related disc degeneration in the murine model \[[@CR31]\]. However, the interaction between IVD degeneration and oxidative stress induced by excessive ROS is not yet completely understood. The primary objective of this study was to clarify the role of excessive ROS in the catabolic cascade of degenerative IVD. The secondary goal was to determine whether excessive ROS represents a therapeutic target for IVD degeneration. We clearly demonstrated that oxidative stress contributes to IVD degeneration and that antioxidant treatment rescues the phenotype of the IVD degeneration model. These results indicate that excessive ROS plays an important role in the pathogenesis of disc disease and can offer a therapeutic target to treat this debilitating and painful degenerative condition. Methods {#Sec2} ======= Human samples {#Sec3} ------------- For the experimental use of surgical samples, written informed consent was obtained from each patient according to the Keio University (Tokyo, Japan) Hospital Ethics Guideline (Keio Hospital \#15-52). A total of 10 IVD tissues were dissected from patients with degenerative disease, including eight with degenerative lumbar scoliosis, one with degenerative lumbar kyphosis, and one with adult idiopathic scoliosis. They were evaluated according to Pfirrmann's magnetic resonance classification (Table [1](#Tab1){ref-type="table"}). As a nondegenerative disc, 17-year-old male IVD dissected at autopsy was used. They were fixed in 4 % paraformaldehyde in phosphate-buffered saline and embedded in paraffin to obtain sections 4 μm thick.Table 1Patient details used for the experimentsSample numberSexAge (years)LevelGrading^a^Diagnosis1Female32L1/23Adult idiopathic scoliosis2Male55L2/33Degenerative lumber scoliosis3Male55L3/43Degenerative lumber scoliosis4Female53L3/43Degenerative lumber scoliosis5Female53L2/34Degenerative lumber scoliosis6Female75L1/24Degenerative lumber scoliosis7Female75L2/34Degenerative lumber scoliosis8Female75L3/45Degenerative lumber scoliosis9Female75L4/55Degenerative lumber scoliosis10Male77L2/35Degenerative lumber kyphosis^a^Pfirrmann disc degeneration grading Animal model of IVD degeneration {#Sec4} -------------------------------- All animals were purchased from Japan Clea (Tokyo, Japan) or born and kept under pathogen-free conditions and were cared for in accordance with the guidelines of the Keio University School of Medicine. Posterior incision above the coccyx IVD of 8-week-old female Wistar rats was made, and the soft tissues such as posterior tendons and ligaments were separated under anesthesia. Disc puncture was performed using a 23-gauge needle on the 3rd--10th coccygeal vertebrae, as in a previous study \[[@CR32], [@CR33]\]. For analysis, a total of 27 rats were sacrificed. A total of nine IVDs were used in each group. For real-time RT-PCR analysis, AF tissues were dissected through a microscope and homogenized only once 1 week after the puncture. The total RNA of the homogenized AF was extracted using TRIzol Reagent (Cosmo Bio Company, Tokyo, Japan). For western blotting, the protein of AF tissues was extracted using Tissue Protein Extraction Reagent (TPER) 1 month after puncture. Magnetic resonance imaging (MRI) was performed 2 months after the puncture. After that, AF tissues were dissected for histological analysis. The harvested discs were decalcified and embedded in paraffin, and sections 4 μm thick were cut. To assess the effect of antioxidant treatment, *N*-acetyl cysteine (NAC, 1 g/l; Sigma-Aldrich, St. Louis, MO, USA) was given orally to degenerative model rats 1 week before puncture and continued for another 2 months until MRI and histological analysis. Imaging {#Sec5} ------- Mid-sagittal T2-weighted MRI (TR/TE, 3000/86; echo train length, 12; slice thickness, 2 mm; field of view, 10 cm; matrix size, 352 × 224; number of excitations, 10 times) (GE Sigma Excite HD 1.5 T; GE Healthcare, Tokyo, Japan) of rat IVD was performed. The ratio of the high-intensity area to IVD was measured using Image Processing and Analysis in Java (Oracle Corporation, Redwood Shores, CA, USA). Histology {#Sec6} --------- Deparaffinized sections of rat IVD were stained with hematoxylin and eosin (H&E). The sections were stained with anti-nitrotyrosine antibody (diluted 100-fold; Abcam, Cambridge, UK), followed by staining with horseradish peroxidase (HRP)-conjugated goat antimouse IgG (diluted 200-fold; Sigma-Aldrich), anti-TNFα antibody (diluted 100-fold; Novus Biologicals, Littleton, CO, USA), and anti-IL-1β (diluted 100-fold; Bioworld Technology, St. Louis Park, MN, USA), followed by HRP-conjugated goat antirabbit IgG (diluted 200-fold; Sigma-Aldrich). Staining was visualized using diamino benzidine (nacalai tesque, Kyoto, Japan). Nuclei were stained with hematoxylin. Antigen retrieval was achieved by pressure-cooking in citrate buffer (pH 6.0) for 20 minutes. All specimens were viewed under a microscope (BZ-9000; Keyence Co., Osaka, Japan). The frequency of nitrotyrosine-positive cells was measured using Image Processing and Analysis in Java at a magnification of × 200. We chose three individual areas at random and calculated the average of the frequency. Isolation and culture of AF cells {#Sec7} --------------------------------- Rat AF tissues were macroscopically dissected from the lumbar and coccyx IVD of 8-week-old female Wistar rats and digested using pronase E (0.04 %) (SERVA, Heidelberg, Germany) for 1 hour at 37 °C and collagenase P (0.025 %) (Roche Diagnosis, Tokyo, Japan) for 4 hours at 37 °C. The cells were then washed with Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 5 % heat-inactivated fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS, USA) according to a previous method \[[@CR34]\]. The isolated AF cells were cultured in DMEM supplemented with 10 % FBS and 1 % penicillin--streptomycin, maintained in a humidified incubator containing 5 % CO~2~ at 37 °C, and used within the first five passages for in vitro analysis. We have confirmed there was no obvious difference in the mRNA expression level of important ECM of AF cells, type I collagen, type II collagen, and aggrecan between the second and fifth passaged cells (Additional file [1](#MOESM1){ref-type="media"}: Figure S1). Treatment of oxidative stress, TNFα, MAPK inhibitors, and antioxidants {#Sec8} ---------------------------------------------------------------------- AF cells were treated with H~2~O~2~ (0, 10, 100 μM) (Wako, Tokyo, Japan), buthionine sulfoximine (BSO; 0, 0.2, 1 mM) (Sigma-Aldrich), and TNFα (50 ng/ml) (R&D Systems, Minneapolis, MN, USA) for 24 hours. For analysis of phosphorylation, AF cells were treated with 100 μM H~2~O~2~ for 0, 5, 10, 15, 30, or 60 minutes and 50 ng/ml TNFα for 10 minutes. Cells were pretreated with MAPK signaling inhibitors for 30 minutes followed by incubation with H~2~O~2~ or BSO for 24 hours. In this assay, we utilized p38 inhibitor (SB203580, 10 μmol/l), JNK inhibitor (SP600125, 10 μmol/l), or ERK inhibitor (PD98059, 10 μmol/l). All inhibitors were purchased from Wako. To study the effect of antioxidative agents, H~2~O~2~-treated, BSO-treated, or TNFα-treated AF cells were cultured with 100 μM NAC and 20 mM α-tocopherol (Wako) solved in ethanol for 15 minutes, 30 minutes, or 24 hours. In the case of α-tocopherol experiment, ethanol was added at the same concentration to ROS-mediated or TNFα-treated AF cells. The experiment of α-tocopherol treatment was independently carried out five times, and all other experiments were independently carried out three times. Real-time RT-PCR {#Sec9} ---------------- Total RNA was isolated from AF cells using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) or TRIzol Reagent. First-strand cDNA was prepared using the Prime Script RT Reagent Kit (Takara Bio, Shiga, Japan) according to the manufacturer's instructions. Real-time RT-PCR was performed using the Thermal Cycler Dice Real-Time System and SYBR Premix Ex Taq (Takara Bio), and the results were quantified using the ddCt method. We measured the relative mRNA expression of TNFα, MMP-3, COX-2, and aggrecan normalized by the expression of β-actin or hypoxanthine phosphoribosyl transferase (HPRT). Gene-specific forward and reverse primers were as follows: *β-actin*-forward, 5′-TGAGAGGGAAATCGTGCGTGAC-3′; *β-actin*-reverse, 5′-AAGAAGGAAGGCTGGAAAAGAG-3′; *HPRT*-forward, 5′-TCCTCATGGACTGATTATGGACA-3′; *HPRT*-reverse, 5′-TAATCCAGCAGGTCAGCAAAGA-3′; *TNFA*-forward, 5′-GCAGATGGGCTGTACCTTATC-3′; *TNFA*-reverse, 5′-GGCTGACTTTCTCCTGGTATG-3′; *MMP3*-forward, 5′-GGACCAGGGATTAATGGAGATG-3′; *MMP3*-reverse, 5′-TGAGCAGCAACCAGGAATAG-3′; *COX2*-forward, 5′-TGAACACGGACTTGCTCACTTTG-3′; *COX2*-reverse, 5′-AGGCCTTTGCCACTGCTTGTA-3′; *AGC1*-forward, 5′-GGATCTATCGGTGTGAAGTGATG-3′; *AGC1*-reverse, 5′-AGTGTGTAGCGTGTGGAAATAG-3′; *Col1a1*-forward, 5′-AGCTCCTGGGCCTATCTGATGA-3′; *Col1a1*-reverse, 5′-AATGGTGCTCTGAAACCCTGATG-3′; *Col2a1*-forward, 5′-GAGGGCAACAGCAGGTTCAC-3′; and *Col2a1*-reverse, 5′-GCCCTATGTCCACACCAAATTC-3′. Intracellular ROS {#Sec10} ----------------- The dissociated cells were loaded with Mitotracker Orange CMH2TM ROS (Life Technologies, Carlsbad, CA, USA) and incubated on a shaker at 37 °C for 30 minutes. For the estimation of intracellular ROS, a FACS Calibur was used (Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA). Western blotting {#Sec11} ---------------- Total cell protein was extracted using a mammalian protein extraction reagent (Thermo Fisher Scieintific, Waltham, MA, USA). All of the wash buffers and extraction buffers included 1× protease inhibitor cocktail (Roche), NaF (1 M), and Na~3~VO~4~ (50 μM). Cell lysates mixed with loading buffer (Tris--Glycine SDS Sample Buffer (Invitrogen) with 5 % 2-mercaptoethanol) were loaded onto 15 % polyacrylamide gels and electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidenefluoride membranes (ATTO Corporation, Tokyo, Japan). The membranes were blocked using 5 % nonfat dry milk in Tris-buffered saline containing 0.1 % Triton X-100 (TBST) (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1 % Tween 20) and incubated overnight at 4 °C in 3 % nonfat dry milk in TBST with antibody against p38 (1:1000; Cell Signaling Technology, Boston, MA, USA), phosphorylated p38 (1:1000; Cell Signaling Technology), ERK (1:1000; Cell Signaling Technology), phosphorylated ERK (1:2000; Cell Signaling Technology), JNK (1:1000; Cell Signaling Technology), phosphorylated JNK (1:1000; Cell Signaling Technology), NF-κB p65 (1:1000; Cell Signaling Technology), phosphorylated NF-κB p65, and β-actin (1:1000; Cell Signaling Technology), followed by HRP-conjugated goat antirabbit IgG (1:2000; Sigma-Aldrich). Proteins were visualized using ECL Western Blotting Detection Reagent (GE Healthcare, Uppsala, Sweden). Transfections and dual luciferase assay {#Sec12} --------------------------------------- Cells were transferred to 24-well plates at a density of 5 × 10^4^ cells/well 1 day before transfection. LipofectAMINE 3000 (Invitrogen) was used as the transfection reagent. The experiment was performed following the manufacturer's recommendation. The optimized ratio of plasmids and posttransfection period were determined in previous reports \[[@CR35]\]. Cells were cotransfected with 300 ng GL4-F (OKD48) with 200 ng pRL-TK plasmid. Plasmids were premixed with the transfection reagent for each transfection. Transfected cells were harvested the next day, and the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) was used for sequential measurements of firefly and *Renilla* luciferase activities. Luciferase activities were quantified, and relative ratios were calculated using a luminometer (TD-20/20; Turner Designs, Sunnyvale, CA, USA). Three independent transfections were performed, and all analyses were carried out in triplicate. Plasmids {#Sec13} -------- P(3 × ARE)TKbasal-hNrf2(1--433)-GL4-F (OKD48) was provided by Dr Takao Iwawaki, Gunma University \[[@CR36]\]. The vector pRL-TK (Promega) harboring the *Renilla reniformis* luciferase gene was used as an internal transfection control. Statistical analysis {#Sec14} -------------------- All measurements were performed in triplicate. Data are presented as mean ± standard deviation (SD). Differences between the groups were analyzed by Student's *t* test and analysis of variance (ANOVA). *p* \<0.05 was considered to indicate statistical significance. Results {#Sec15} ======= Induction of ROS level in IVD degeneration {#Sec16} ------------------------------------------ To investigate the involvement of ROS in IVD degeneration, we first assessed one of the oxidative stress markers, nitrotyrosine, in a rat punctured model and in human degenerative IVD samples. A total of nine discs per group were used for the analysis. Mid-sagittal T2-weighted MRI findings of IVD in the rat punctured model confirmed a lower signal intensity than that in the sham group (Fig. [1a](#Fig1){ref-type="fig"}). The ratio of the high-intensity area to IVD was significantly reduced in the model (Fig. [1b](#Fig1){ref-type="fig"}). H&E staining showed a smaller NP and less-organized lamellae of AF in the punctured model (Fig. [1c](#Fig1){ref-type="fig"}). In addition, we confirmed that the mRNA expression of TNFα, MMP-3, and COX-2---catabolic molecules involved in degeneration---was significantly induced in AF of the punctured model, whereas that of aggrecan tended to be reduced, but not significantly (Fig. [1d](#Fig1){ref-type="fig"}). These results suggest that the needle punctured model is adequate for analysis of IVD degeneration. The expression of nitrotyrosine as well as TNFα and IL-1β was higher in AF of this model group compared with that of the sham group (Fig. [1e](#Fig1){ref-type="fig"}). Western blotting also showed a higher protein expression level of nitrotyrosine as well as TNFα and IL-1β in the rat degenerative model (Fig. [1f](#Fig1){ref-type="fig"}). Densitometry analysis confirmed these observations (Fig. [1f](#Fig1){ref-type="fig"}). Moreover, immunohistochemistry showed that human degenerative disc samples with each grade had a high proportion of nitrotyrosine-positive cells, accompanied by robust expression of TNFα and IL-1β, whereas human healthy disc had low expression of these markers (Fig. [1g](#Fig1){ref-type="fig"}). We assessed the frequency of nitrotyrosine-positive cells in each grade sample. Figure [1h](#Fig1){ref-type="fig"} showed that more than grade 3 degenerative discs had a significantly higher frequency of nitrotyrosine-positive cells compared with healthy disc (Fig. [1h](#Fig1){ref-type="fig"}).Fig. 1Induction of ROS level in IVD degeneration. **a** Mid-sagittal T2-weighted MRI findings of IVDs 2 months after the puncture confirm lower signal intensity than that of the sham group. **b** Compared with the sham group, the ratio of the high-intensity area to IVD areas was significantly decreased by needle puncture. **c** Hematoxylin and eosin (*H/E*) staining shows smaller NP and less-organized lamellae of AF in the punctured group. Scale bars, 300 μm. **d** Real-time RT-PCR analysis. mRNA expression of TNFα, MMP-3, and COX-2 was significantly induced in the AF of the punctured model, whereas that of aggrecan tended to be reduced. Data presented as mean ± SD of three independent experiments performed in triplicate (*n* = 3). **e** Numbers of nitrotyrosine-positive cells were increased in AF of the punctured model, correlated with elevated expression of TNFα and IL-1β. Scale bars, 100 μm. **f** Western blotting and densitometry analysis. The protein expression level of nitrotyrosine was significantly higher as well as TNFα and IL-1β in the rat degenerative model. **g** Immunohistochemistry of human degenerative disc samples with each grade including healthy disc. Scale bars, 100 μm. **h** More than grade 3 degenerative discs had significantly higher frequency of nitrotyrosine-positive cells compared with healthy disc. \**p* \<0.05. *COX* cyclooxygenase, *IL* interleukin, *MMP* matrix metalloprotease, *ns* not significant, *TNF* tumor necrosis factor Molecular phenotype of the rat AF cells treated with ROS {#Sec17} -------------------------------------------------------- To clarify the pathophysiological role of intracellular ROS, we examined the phenotype of the AF cells treated with H~2~O~2~ and BSO, which is a glutathione synthesis inhibitor that activates oxidative stress. Flow cytometry confirmed that the intracellular level of ROS was significantly increased by treatment with both H~2~O~2~ and BSO in AF cells (Fig. [2a](#Fig2){ref-type="fig"}). Next, we treated rat cultured AF cells with H~2~O~2~ and evaluated the expression of catabolic and anabolic factors of IVD degeneration by real-time RT-PCR analysis. We found that the mRNA expression of TNFα, MMP-3, and COX-2 was significantly induced, whereas that of aggrecan was reduced in a dose-dependent manner (Fig. [2b](#Fig2){ref-type="fig"}). Expectedly, real-time RT-PCR showed similar results with BSO treatment (Fig. [2c](#Fig2){ref-type="fig"}). To investigate the downstream signaling of ROS in AF cells, we evaluated the phosphorylation of MAPKs, including p38, ERK, and JNK, as well as p65 by western blotting. This analysis showed that the three signaling pathways of MAPK were maximally phosphorylated 10 minutes after treatment with H~2~O~2~ and BSO (Fig. [3a](#Fig3){ref-type="fig"}, [b](#Fig3){ref-type="fig"}). On the other hand, the phosphorylation of p65 was not activated by their treatments (Fig. [3a](#Fig3){ref-type="fig"}, [b](#Fig3){ref-type="fig"}). Next, to investigate further the involvement of MAPKs, we cultured H~2~O~2~-treated or BSO-treated AF cells with MAPK signaling inhibitors, including p38 inhibitor (SB203580), JNK inhibitor (SP600125), and ERK inhibitor (PD98059), and assessed the mRNA expression of COX-2, TNFα, and MMP-3 by real time RT-PCR analysis. Figure [3c](#Fig3){ref-type="fig"}, [d](#Fig3){ref-type="fig"} shows that all inhibitors significantly abolished H~2~O~2~-mediated or BSO-mediated induction of COX-2 mRNA expression. These results suggest that the catabolic effect of excessive ROS is mediated through the signaling pathways of p38, ERK, and JNK in AF cells. However, the results of TNFα and MMP-3 did not clearly show the effect of these inhibitors compared with COX-2 (Additional file [2](#MOESM2){ref-type="media"}: Figure S2). The gene regulatory network of TNFα and MMP3 can be more complicated than that of COX-2. The experiment concerned with the mechanisms of the difference is in progress.Fig. 2Molecular phenotype of the rat AF cells treated with ROS. **a** Flow cytometry confirmed that the intracellular level of ROS was significantly increased by treatment with H~2~O~2~ and BSO in AF cells. **b** Real-time RT-PCR analysis showed that the mRNA expression of TNFα, MMP-3, and COX-2 was significantly induced by treatment with H~2~O~2~, whereas aggrecan mRNA expression was reduced in a dose-dependent manner. **c** mRNA expression of TNFα, MMP-3, and COX-2 was significantly induced by treatment with BSO, whereas aggrecan mRNA expression was reduced in a dose-dependent manner, as determined by real-time RT-PCR. Data presented as mean ± SD of three independent experiments performed in triplicate (*n* = 3); \**p* \<0.05. *BSO* buthionine sulfoximine, *COX* cyclooxygenase, *H* ~*2*~ *O* ~*2*~ hydrogen peroxide, *MMP* matrix metalloprotease, *ns* not significant, *TNF* tumor necrosis factorFig. 3Downstream signaling of ROS in AF cells. **a**, **b** Western blot analysis showed that mitogen-activated protein kinases (*MAPKs*), including p38, ERK, and JNK, were maximally phosphorylated 10 minutes after treatment with H~2~O~2~ **a** and BSO **b**. **c**, **d** Real-time RT-PCR analysis. Treatment of AF cells with MAPK signaling inhibitors, including p38 inhibitor (*SB*), JNK inhibitor (*SP*), and ERK inhibitor (*PD*), significantly abolished H~2~O~2~-mediated **c** or BSO-mediated **d** induction of COX-2 mRNA expression. Data presented as mean ± SD of three independent experiments performed in triplicate (*n* = 3); \**p* \<0.05. *BSO* buthionine sulfoximine, *COX* cyclooxygenase, *ERK* extracellular signal-regulated kinase, *H* ~*2*~ *O* ~*2*~ hydrogen peroxide, *HPRT* hypoxanthine phosphoribosyl transferase, *JNK* c-Jun N-terminal kinase Antioxidant significantly neutralized the catabolic effect of ROS {#Sec18} ----------------------------------------------------------------- Next, to ascertain whether the treatment of antioxidant attenuates the ROS-mediated catabolic effect in AF cells, we treated AF cells with NAC, together with H~2~O~2~ or BSO. Using real-time RT-PCR analysis, we found that NAC significantly abolished the induction of TNFα, MMP-3, and COX-2 expression and reduction of aggrecan expression in H~2~O~2~-treated or BSO-treated AF cells (Fig. [4a](#Fig4){ref-type="fig"}, [b](#Fig4){ref-type="fig"}). To investigate whether NAC regulates the downstream signaling of ROS in AF cells, we assessed the phosphorylation of p38, ERK, and JNK in AF cells treated with and without NAC. Western blotting and densitometry analysis clearly showed that treatment with NAC inhibited the phosphorylation of p38 but not of JNK, ERK, and p65 (Fig. [4c](#Fig4){ref-type="fig"}, [d](#Fig4){ref-type="fig"}). We also elucidated whether the other antioxidant material α-tocopherol (vitamin E) could replicate these effects. As expected, real-time RT-PCR analysis showed that treatment with α-tocopherol also abolished the H~2~O~2~-mediated or BSO-mediated change of TNFα, COX-2, MMP-3, and aggrecan mRNA expression (Fig. [5a](#Fig5){ref-type="fig"}, [b](#Fig5){ref-type="fig"}).Fig. 4NAC significantly neutralizes the catabolic effect of ROS. **a**, **b** NAC significantly abolished the induction of TNFα, MMP-3, and COX-2 expression and reduction of aggrecan expression in H~2~O~2~-treated **a** or BSO-treated **b** AF cells by real-time RT-PCR analysis. **c**, **d** Western blotting and densitometry clearly showed that NAC treatment inhibited the phosphorylation of p38 but not of JNK, ERK, and p65. NTC, nontreated control. Data presented as mean ± SD of three independent experiments performed in triplicate (*n* = 3); \**p* \<0.05. *BSO* buthionine sulfoximine, *COX* cyclooxygenase, *ERK* extracellular signal-regulated kinase, *H* ~*2*~ *O* ~*2*~ hydrogen peroxide, *JNK* c-Jun N-terminal kinase, *MMP* matrix metalloprotease, *NAC* N-acetyl cysteine, *ns* not significant, *TNF* tumor necrosis factorFig. 5Treatment of α-tocopherol significantly abolishes the catabolic effect of ROS. **a**, **b** Real-time RT-PCR analysis. Treatment with α-tocopherol significantly abolished the H~2~O~2~-mediated **a** or BSO-mediated **b** induction of TNFα and COX-2 mRNA expression, and reduction of aggrecan. H~2~O~2~-mediated induction of MMP-3 expression was not significant, but tended to be decreased. Data presented as mean ± SD of five independent experiments performed in triplicate (*n* = 5); \**p* \<0.05. *BSO* buthionine sulfoximine, *COX* cyclooxygenase, *H* ~*2*~ *O* ~*2*~ hydrogen peroxide, *HPRT* hypoxanthine phosphoribosyl transferase, *MMP* matrix metalloprotease, *ns* not significant, *TNF* tumor necrosis factor Treatment with antioxidant also significantly inhibited the effect of TNFα on AF cells {#Sec19} -------------------------------------------------------------------------------------- To assess whether intracellular ROS levels are regulated by inflammatory cytokines in AF cells, we treated cultured AF cells with TNFα and evaluated the level of intracellular ROS using MitoTracker Orange CMH2TMR. Flow cytometry showed that ROS levels were significantly increased in AF cells after treatment with TNFα (Fig. [6a](#Fig6){ref-type="fig"}). Recently, Oikawa et al. \[[@CR36]\] reported that an OKD48 construct (P(3 × ARE)TKbasal-hNrf2(1--433)-GL4-F) specifically responded to oxidative stress and was useful for monitoring stress in vitro and in vivo. We transfected the plasmids to AF cells and assessed the reporter activity with and without TNFα. As expected, the activity was significantly induced by treatment with TNFα in AF cells (Fig. [6b](#Fig6){ref-type="fig"}). These results suggest that TNFα upregulates the intracellular ROS level and induces oxidative stress in AF cells. In addition, real-time RT-PCR showed that treatment with NAC attenuated TNFα-mediated induction of MMP-3 and COX-2 expression and reduction of aggrecan in AF cells (Fig. [6c](#Fig6){ref-type="fig"}). In the case of α-tocopherol, significant abolishment of TNFα-mediated induction of MMP-3 and reduction of aggrecan was also observed. Concerning COX-2, α-tocopherol had the tendency to attenuate, but not significantly, the influence of TNFα (Fig. [6d](#Fig6){ref-type="fig"}). Moreover, western blotting clearly showed that NAC treatment inhibited the phosphorylation of p38, ERK, JNK, and p65 in TNFα-treated AF cells (Fig. [6e](#Fig6){ref-type="fig"}). These results indicated that in AF cells, the catabolic influence of TNFα was partly mediated by ROS signaling.Fig. 6Catabolic effect of TNFα is partially mediated by ROS in AF cells. **a** Flow cytometry showed that ROS levels were significantly increased in AF cells after treatment with TNFα. **b** Reporter activity of the OKD48 construct was significantly induced by treatment with TNFα (10 and 50 ng/ml) in AF cells. **c** Real-time RT-PCR analysis. Treatment of NAC significantly attenuated TNFα-mediated induction of COX-2 mRNA expression and reduction of aggrecan in AF cells. Data presented as mean ± SD of three independent experiments performed in triplicate (*n* = 3). **d** α-Tocopherol significantly reduced MMP-3 mRNA expression and restored aggrecan mRNA expression in the TNFα-treated cells. Data presented as mean ± SD of five independent experiments performed in triplicate (*n* = 5). **e** Western blotting. NAC treatment inhibited the phosphorylation of p38, ERK, JNK, and p65 in TNFα-treated AF cells. \**p* \<0.05. *COX* cyclooxygenase, *ERK* extracellular signal-regulated kinase, *HPRT* hypoxanthine phosphoribosyl transferase, *JNK* c-Jun N-terminal kinase, *MMP* matrix metalloprotease, *NAC* N-acetyl cysteine, *ns* not significant, *TNF* tumor necrosis factor Oral administration of NAC improved IVD degeneration in a rodent model {#Sec20} ---------------------------------------------------------------------- Finally, to investigate the efficacy of NAC on IVD degeneration in vivo, we administered NAC (1 g/l) orally to degenerative model rats 1 week before puncture and continued for another 1 week until RNA isolation (*n* = 9), for 1 month until protein isolation (*n* = 9), and for 2 months until MRI analysis and histological examination (*n* = 9) (Fig. [7a](#Fig7){ref-type="fig"}). Real-time RT-PCR clearly showed that NAC significantly abolished the induction of TNFα mRNA expression and reduction of aggrecan in the AF tissues of the degenerative model. The mRNA expression of MMP-3 and COX-2 had a tendency to be decreased with oral administration of NAC in the degenerative model (Fig. [7b](#Fig7){ref-type="fig"}). To determine whether NAC neutralize the progression of oxidative stress in degenerative discs, we assessed the protein expression level of nitrotyrosine in AF tissues 1 month after surgery. Western blotting showed that the expression of nitrotyrosine was clearly reduced by oral administration of NAC (Fig. [7c](#Fig7){ref-type="fig"}). The densitometry confirmed these observations (Fig. [7d](#Fig7){ref-type="fig"}). Moreover, we found that the phosphorylation of p38 was obviously attenuated by treatment of NAC, but not JNK and ERK (Fig. [7e](#Fig7){ref-type="fig"}). Furthermore, H&E staining showed improvements in the reduction of NP size and the disorganization of the AF by oral administration of NAC (Fig. [7f](#Fig7){ref-type="fig"}). Finally, mid-sagittal T2-weighted MRI findings showed the maintenance of T2 high intensity in discs of the degenerative models with NAC (Fig. [7g](#Fig7){ref-type="fig"}). The ratio of the T2-weighted high-intensity area to IVD was significantly improved by the administration of NAC in the degenerative group (Fig. [7h](#Fig7){ref-type="fig"}).Fig. 7Oral NAC administration improves IVD degeneration in the rodent model. **a** Schematic representation of oral NAC administration experiment for IVD degenerative model. **b** Real-time RT-PCR analysis. NAC significantly abolished the induction of TNFα mRNA expression and reduction of aggrecan in punctured AF tissues. Data presented as mean ± SD of nine IVD discs derived from three independent rats performed in triplicate (*n* = 9); \**p* \<0.05. **c** Western blotting. Protein expression of nitrotyrosine was clearly reduced by oral administration of NAC. **d** Densitometry confirmed that the protein level of nitrotyrosine was significantly abrogated by oral administration of NAC. **e** Western blotting. Phosphorylation of p38 was significantly attenuated by treatment of NAC. **f** Histological examination of H&E staining showed that oral administration of NAC improved the reduction of NP size and the disorganization of the AF in this degenerative model. Scale bars, 300 μm. **g** Mid-sagittal T2-weighted MRI. Area of T2 high intensity was increased by oral administration of NAC in the degenerative group. Representative data are shown. **h** The T2-weighted high-intensity area to IVD ratio was significantly improved by NAC administration. Data presented as mean ± SD; \**p* \<0.05. *COX* cyclooxygenase, *ERK* extracellular signal-regulated kinase, *H/E* hematoxylin and eosin, *JNK* c-Jun N-terminal kinase, *MMP* matrix metalloprotease, *MRI* magnetic resonance imaging, *NAC* N-acetyl cysteine, *ns* not significant, *TNF* tumor necrosis factor Discussion {#Sec21} ========== This study demonstrated that excessive ROS, which are induced in IVD degeneration, have a catabolic effect on AF cells via MAPK signaling. Our study also showed that TNFα induced oxidative stress with increased intracellular ROS levels in AF cells through MAPK and NF-κB signaling, indicating that a positive feedback loop was formed between excessive ROS and catabolic factors, including TNFα, in IVD degeneration. The second major observation was that antioxidant NAC significantly abrogated the catabolic effect of excessive ROS both in vitro and in vivo. These findings lend strong support to the hypothesis that excessive ROS are critical mediators in the pathogenesis of degenerative disc conditions and are therapeutic targets for disc diseases. In this study, we showed that nitrotyrosine, a product of tyrosine nitration mediated by ROS, was highly expressed in IVD in a rat degenerative model and in human degenerative samples. ROS also contribute to the onset and progression of osteoarthritis by inducing chondrocyte death and matrix degradation \[[@CR37]\]. Tomiyama et al. \[[@CR38]\] showed that compression on the cartilage induced the expression of nitrotyrosine. Furthermore, compared with healthy volunteers, plasma nitrotyrosine has been reported to be increased in osteoarthritis patients \[[@CR39]\]. These observations indicated that nitrotyrosine was useful as an indirect oxidative stress marker in musculoskeletal degenerative diseases. An immuno-spin trapping technique which was recently developed by Khoo et al. \[[@CR40]\] can be useful for direct detection of free radicals in future oxidative disc studies. Many animal models of IVD degeneration have been reported; however, there are few ideal animal models which precisely mimic a pathological state of human IVD degeneration. A rat degenerative model induced by needle puncture has been used in many studies and has been shown to create morphological and biochemical features similar to many of those of degenerative discs in humans \[[@CR41], [@CR42]\]. Because it is clinically difficult to obtain a sufficient number of human healthy disc samples, the rat model is helpful for comparative study between degenerative and nondegenerative disc. However, the punctured model still has some limitations that should be taken into consideration, including the possibility of traumatic changes. We are assessing the expression level of nitrotyrosine in a rat-tail static compression-induced disc degeneration model to clarify the interaction between oxidative stress and mechanical loading in IVD degeneration. Clinically, the characteristics of IVD degeneration such as ECM breakdown can be observed in both the AF and NP. The NP is known to have small chondrocyte-like cells and large vacuolated notochordal cells \[[@CR6], [@CR7]\]. Moreover, it has been reported that notochordal cells are rarely present after adolescence in humans \[[@CR6]\]. However, it has not been determined whether the number of notochordal cells in the NP declines after birth because of their slow transformation or replacement by chondrocyte-like cells \[[@CR43]\]. Which cells should be clinically targeted for the treatment of NP degeneration therefore remains unclear. On the other hand, although AF anatomically consists of inner and outer layers, the cells existing in both layers are known to originate from the mesenchyme and are homogeneous regardless of age and species. Therefore, it is reasonable to assume that alteration in the molecular phenotype of AF cells may be directly involved in IVD degeneration. From these viewpoints, in vitro analysis of AF cells was carried out to assess disc degeneration in this study. IVD degeneration is characterized by increases in levels of the proinflammatory cytokines TNFα, IL-1α, IL-1β, IL-6, and IL-17, secreted by IVD cells \[[@CR12], [@CR13], [@CR44], [@CR45]\]. In particular, it is known that TNFα and IL-1β play pivotal roles in the progression of these degenerative changes \[[@CR12], [@CR13]\]. Cytokines have been shown to upregulate chemokines and various catabolic mediators, including ADAMTS-4, ADAMTS-5, MMP-1, MMP-2, MMP-3, MMP-13, and syndecan-4, and to suppress the expression of important ECM genes through NF-κB and MAPK signaling pathways in degenerative disc cells \[[@CR14]--[@CR17], [@CR46], [@CR47]\]. In a previous study, Yoshimura et al. \[[@CR48]\] demonstrated that IL-1β induced cell death in rat primary chondrocytes and mouse chondrocytic ATDC5 cells via mitochondrial dysfunction in a ROS-dependent manner. Our flow cytometry analysis clearly showed that intracellular ROS levels were significantly induced by treatment with TNFα in AF cells. Furthermore, to evaluate oxidative stress conditions in TNFα-treated AF cells, a luciferase assay was carried out using OKD48 construct. Under oxidative stress conditions, Nrf2, a basic region leucine zipper transcription factor, is known to be stabilized at the posttranscriptional level \[[@CR49]\]. OKD48 consists of an oxidative stress-inducible promoter, luciferase, and an Nrf2 fragment that contributes to stress-dependent stabilization \[[@CR36]\]. This analysis clearly showed that oxidative stress was significantly induced by TNFα in AF cells. Vice versa, our results showed that the expression of catabolic factors, including TNFα, was induced by treatment with H~2~O~2~ or BSO in AF cells, which indicated that there was a reciprocal interaction between excessive ROS and TNFα in inflammatory AF cells (Fig. [8](#Fig8){ref-type="fig"}). We previously reported that ROS activated the ERK and p38 MAPK pathways but not the JNK pathway in ATDC5 cells \[[@CR50]\]. On the other hand, the present study showed that JNK signaling was also activated by treatment of AF cells with ROS, suggesting that the downstream pathway of ROS is cell or tissue specific.Fig. 8Schematic of relationship between oxidative stress and disc degeneration. Schematic showing positive catabolic feedback loop between excessive ROS and catabolic factors in AF cells. Excessive ROS induced expression of catabolic factors, which are upregulated in the degenerative state, and reduced cartilage ECM aggrecan via the signaling pathways of p38 in AF cells. Conversely, TNFα increased intracellular ROS levels in AF cells through p38, JNK, ERK, and p65. These pathways were neutralized by NAC. *COX* cyclooxygenase, *ERK* extracellular signal-regulated kinase, *JNK* c-Jun N-terminal kinase, *MAPK* mitogen-activated protein kinase, *MMP* matrix metalloprotease, *NAC* N-acetyl cysteine, *NF* nuclear factor, *TNF* tumor necrosis factor NAC is primarily a pharmaceutical drug for the management of acetaminophen overdose and is commonly used as a nutritional supplement. In addition, NAC is widely used for treatment of lung diseases such as chronic obstructive pulmonary disease \[[@CR51], [@CR52]\]. MRI and RT-PCR analysis in our in vivo study clearly showed that the oral administration of NAC prevented the progression of IVD degeneration. In analysis of molecular mechanisms, NAC treatment attenuated ROS-mediated or TNFα-mediated activation of MAPKs, including ERK1/2, p38, and JNK, and NF-κB signaling in AF cells in vitro. Previously, resveratrol in red wine and epigallocatechin 3-gallate in green tea were also reported to have an anti-inflammatory and anti-catabolic effect by modulating the signaling of p38, JNK, or NF-κB in IVD \[[@CR53], [@CR54]\]. Although several human clinical trials with anticytokine agents have investigated the alleviation of symptoms of back or radicular pain associated with IVD degeneration, there are no drugs that can directly prevent the progression of disc degeneration. Because the expression of p38 and p65 is ubiquitous, the inhibitors for targeting this signaling are often toxic to normal cells or tissue and have therefore been clinically unsuccessful \[[@CR55]\]. Meanwhile, since NAC is commonly used as a supplement and has less toxic side effects, it may serve as a therapeutic option for IVD degeneration. When the human clinical daily dosage of NAC was converted to rats normalized with body surface area according to the US Food and Drug Administration's guideline, they were almost equivalent doses. Experiments are in progress to determine the minimum effective concentration of NAC for the improvement of IVD degeneration. Recently, clinical use of another antioxidant, vitamin E, has been established for the treatment of nonalcoholic steatohepatitis in which oxidative stress was implicated \[[@CR56]\]. Our results showed that vitamin E also attenuated the ROS-mediated or TNFα-mediated catabolic effect in the cultured AF cells, which indicated that vitamin E as well as NAC has the potential for preventing disc degeneration. Conclusions {#Sec22} =========== Oxidative stress induced in IVD degeneration has a catabolic effect on AF cells via MAPK signaling. Elevated TNFα induces oxidative stress with increasing intracellular ROS levels through MAPK and NF-κB signaling, indicating that a positive feedback loop is formed between excessive ROS and TNFα in AF cells. Antioxidant NAC significantly abrogated the catabolic effect of excessive ROS in vitro and in vivo. These findings lead strong support for the hypothesis that NAC can be a therapeutic option for IVD degeneration. Additional files {#Sec23} ================ Additional file 1:**is Figure S1 showing molecular phenotype of the passaged rat AF cells.** Real-time RT-PCR analysis of the mRNA expression of type I collagen, type II collagen, and aggrecan in the second (P2) and fifth (P5) passaged AF cells. Data presented as mean ± SD of three independent experiments performed in triplicate (*n* = 3); \**p* \<0.05; ns, not significant. (PDF 873 kb)Additional file 2:**is Figure S2 showing treatment of MAPK inhibitors to AF cells with ROS.** Real-time RT-PCR analysis of the expression of TNFα and MMP-3 in AF cells. MAPK signaling inhibitors, including p38 inhibitor (SB), JNK inhibitor (SP), and ERK inhibitor (PD), were treated to AF cells with H~2~O~2~ (upper) or BSO (lower). Data presented as mean ± SD of three independent experiments performed in triplicate (*n* = 3); \**p* \<0.05; ns, not significant. (PDF 987 kb) ADAMTS : A disintegrin and metalloprotease with thrombospondin motifs AF : Annulus fibrosus AGE : Advanced glycation end product ANOVA : Analysis of variance BSO : Buthionine sulfoximine COX-2 : Cyclooxygenase-2 DMEM : Dulbecco's modified Eagle's medium ECM : Extracellular matrix ERK : Extracellular signal-regulated kinase FBS : Fetal bovine serum H~2~O~2~ : Hydrogen peroxide H&E : Hematoxylin and eosin HPRT : Hypoxanthine phosphoribosyl transferase HRP : Horseradish peroxidase IL : Interleukin IVD : Intervertebral disc JNK : c-Jun N-terminal kinase MAPK : Mitogen-activated protein kinase MMP : Matrix metalloprotease MRI : Magnetic resonance imaging NAC : *N*-acetyl cysteine NF : Nuclear factor NO : Nitric oxide NP : Nucleus pulposus O^2−^ : Superoxide anion OH : Hydroxyl radical ROS : Reactive oxygen species SD : Standard deviation TNFα : Tumor necrosis factor alpha TPER : Tissue Protein Extraction Reagent **Competing interests** The authors declare that they have no competing interests. **Authors' contributions** SS carried out the experiments, performed the statistical analysis, and drafted the manuscript. NF conceived of the study, participated in the design of the study, performed the statistical analysis, and drafted the manuscript. NH participated in the design of the study and coordination, performed the statistical analysis, and drafted the manuscript. KW participated in the design of the study, collected samples from human subjects, performed the statistical analysis, and helped to draft the manuscript. KI participated in study design and coordination, collected samples from human subjects, and helped to draft the manuscript. YT conceived of the study, participated in its design and coordination, and helped to draft the manuscript. KT carried out the experiments, performed the statistical analysis, and helped to draft the manuscript. KH participated in study design and coordination, interpreted the data, and helped to draft the manuscript. TM participated in its design and coordination, interpreted the data, and helped to draft the manuscript. MN participated in the design of the study, interpreted the data, and helped to draft the manuscript. MM participated in the design of the study, interpreted the data, and helped to draft and revise critically the manuscript for important intellectual content. All authors read and approved the final manuscript. Written informed consent was obtained from the patient for publication of their individual details and accompanying images in this manuscript. The consent form is held by the authors' institution and is available for review by the Editor-in-Chief. The authors would like to thank all participating individuals for their contribution to this study. They thank Dr Takao Iwawaki for providing OKD48 construct. The authors also thank Dr Suketaka Momoshima and Yoshinobu Nunokawa for MRI and Yui Sato for technical support. This work was supported by MEXT KAKENHI Grant Numbers 25861337 and 24791562, and in part by grants from The Nakatomi Foundation and the Japan Orthopaedic and Traumatology Foundation.

Cells without complete genomes {#s1} ============================== Cells have long been recognized as life\'s building blocks (e.g., Virchow\'s dictum "*omnis cellula e cellula*," Virchow, [@B31]). Specifically, a cell\'s genome is considered the repository of genetic information that pairs with the cellular machinery to determine the organism\'s phenotype. Except for rare circumstances, the majority of a genome is passed on from ancestor to descendant, although the acquisition of genes from organisms that are not direct ancestors is recognized to play an important role in evolution (Swithers et al., [@B27]). Jeffrey Lawrence, in discussing minimal genome size proposed a meta-cell model (Lawrence, [@B17]), in which many micelles (small vesicles containing resources, products, and genes) exchange genes frequently. Genes temporarily reside in a micelle and direct the synthesis of compounds important for replication. A micelle only can replicate when all compounds necessary for division have been generated. However, at each point in time only a fraction of the necessary genes are present in an individual micelle. This model relies on gene transfer being so frequent that each of the genes that encode necessary functions visits the individual micelles often enough to allow for sufficient synthesis of the necessary gene products for future micelle divisions. The meta-cell can be considered an organism, whose genome is divided into a network of micelles. Lawrence\'s meta-cell model is reminiscent of Woese\'s progenote (Woese, [@B34]) and Kandler\'s pre-cell populations (Kandler, [@B15]) that were postulated to have existed early in evolution before genes coalesced into genomes. The pan-genome as a shared genomic resource {#s2} =========================================== For most bacterial and archaeal species different strains contain non-overlapping gene sets. The pan-genome of a taxon or group refers to the sum of all genes that are present in members of the group (Tettelin et al., [@B28]; Lapierre and Gogarten, [@B16]). Pan-genomes comprise the core genome, i.e., the genes that are found in all members, and the accessory genome, i.e., genes that are present in only one or a few members of the group. Welch et al. ([@B33]) provided the first illustration that genome content in bacteria changes rapidly. Comparing three *Escherichia coli* strains they found the shared core to be less than 40% of the gene families present in all three genomes. More recently the size of this core was further reduced to only 6% of gene families present in 61 *E. coli* genomes (Lukjancenko et al., [@B21]). Baumdicker et al. ([@B3]) estimate that the *Prochlorococcus* pan-genome contains about 58,000 genes, whereas the individual genomes encode only about 2000 genes each. The pan-genome concept was originally developed to explore the fluidity of prokaryotic genomes (Tettelin et al., [@B28]). Because HGT is more frequent between close relatives (Andam and Gogarten, [@B1]), the pan-genome may also represent the set of genes that is potentially available via HGT to any member of the group. The function of the pan-genome may then be thought of as a shared resource. This is supported by the observation that genes encoding weakly selected functions are frequently lost from bacterial genomes, when they do not provide selective advantages, only to be re-acquired through HGT, when new conditions provide a selective advantage to carriers (Lawrence and Roth, [@B19]). The idea of the pan-genome of a population as a shared genomic resource is similar to the description of meta-cells and pre-cell populations. In particular, these concepts have in common that the individual genome of a cell or micelle does not represent a sufficient description of the genomic resources of the population. The following paragraphs discuss some factors that contribute to the large size of pan-genomes. The strong black queen hypothesis {#s3} ================================= The black queen hypothesis proposed by Morris et al. ([@B23]) is built on the premise of "leaky" common good functions, which cannot be restricted to benefit only the producer. The hypothesis suggests that these functions combined with selection for small genomes may lead to a situation in which these leaky functions are encoded in only a fraction of the genomes comprising the community. Under the black queen hypothesis a cell\'s evolution can follow one of two pathways (see Figure [1](#F1){ref-type="fig"}): (1) the cell can retain all genes encoding leaky functions (in the game of hearts, from which the name for the black queen hypothesis derives, this strategy is known as "shooting the moon"). The cost is a large genome maintaining and expressing many genes that are not essential to central metabolism, growth, and reproduction. Consequently, maintaining these genes and expression of extra proteins competes for cellular resources that could be put toward replication and results in a lower growth rate (Dong et al., [@B9]; Scott et al., [@B26]; Weiße et al., [@B32]). The advantage of the "shooting the moon" strategy is that following a population bottleneck all genes encoding leaky functions are available in the genome. These members of a community following this strategy may be thought of as analogous to a keystone species. (2) The cell looses some or all of its leaky functions and increases its growth rate (in hearts, this represents the usual strategy of taking as few point cards as possible). Traditionally this is described as cheating, as the second strategy relies upon other cells in the population for the leaky functions it has lost. If a bottleneck occurs, a single cheating cell is unlikely to survive on its own. A possible outcome of all cells in a population following strategy \#2 is that all members of a population cheat on some leaky functions. The members in the population then become mutually dependent on one another (Figure [1](#F1){ref-type="fig"}). In this scenario there are no keystone members providing all of the leaky functions. For the population to establish itself in a new environment several members of the population are required for the migration to be successful, as no single cell has all the components necessary to sustain itself. We term this the "strong" version of the black queen hypothesis. If all members of a population follow the second strategy, this may under some conditions lead to instability, the tragedy of the commons, and extinction of the population; however, experimental work by Morris et al. ([@B24]) has shown that partitioning of a leaky common-goods function can enable the stable co-existence of two very similar organisms that use the same resources. Additionally, under natural conditions cells do rarely exist in homogeneous mixture (Davey and O\'toole, [@B7]). Cells existing in biofilms or small aggregates are likely to be proximal to cells with which they share recent ancestry, and therefore proximal cells will have the same genotype with respect to shared functions. Drescher et al. ([@B11]) show that *Vibrio cholera* can avoid the public goods dilemma by strengthening relationships between cells of the same genotype through creation of a thick biofilm, thereby providing a local selective advantage to producers of a particular common good in case this good becomes an overall limiting resource. It seems likely that genes encoding common goods are under frequency dependent selection, leading to local feedback loops that contribute to a long-term co-existence of the different types of cheaters. ![**Schematic depiction of a strong version of the black queen hypothesis (Morris et al., [@B23])**. Each cell in the left panel contains all the genes that produce the four depicted common goods. Cells on the right, due to selection to minimize genome size, each produce only one of the common goods, and rely on the presence of other cells to produce the other ones. We consider this a strong version of the black queen hypothesis because it no longer contains exclusive helper strains; rather all individuals in the population described in the right hand panel are simultaneously helpers and beneficiaries. In this example dark open ovals represent individual cells; red diamonds: hydrolases that digest an extracellular polymer (e.g., phosphatase, sialidase, glucanase); green squares: siderophores that allow cells to acquire iron; black ovals: building blocks of the extracellular matrix (ECM) or enzymes that produce and assemble these building blocks; yellow triangles: enzymes that destroy oxygen radicals (e.g., catalase---reaction given; or peroxidase---reaction: ROOR\' + electron donor (2 e^−^) + 2H^+^ → ROH + R\'OH).](fmicb-06-00728-g0001){#F1} Black vs. the red queen {#s4} ======================= Bacteria are under severe predation by phage (Thurber, [@B29]). They need to constantly change to evade predation, hence the analogy to the red queen from Lewis Carroll\'s (Carroll and Gardner, [@B4]) *Through the Looking-Glass*, who needs to run as fast as she can just to stay in place (Van Valen, [@B30]). The analysis of phage metagenomes and rank abundance curves indicated that the phage predation follows the *kill the winner* strategy (Hoffmann et al., [@B14]), where successful strains are targeted more frequently. The surprising long term stability of species composition despite phage predation suggests that cycling between different susceptible target cells occurs within a population and not between populations from different species (Rodriguez-Brito et al., [@B25]). Consequently, within a population, host genes that encode receptors utilized by phage and virus to enter the cell are expected to turn over quickly, creating within population diversity (Chaturongakul and Ounjai, [@B5]). Random acquisition of genes {#s5} =========================== Genes are constantly acquired by genomes, and many of the transferred genes do not find a long term home in the recipient genome (Lawrence and Ochman, [@B18]). Among these genes are parasites (prophages) and selfish genetic elements. Most, but certainly not all (Lobkovsky et al., [@B20]), of the transferred genes are selectively neutral or nearly neutral to the recipient (Gogarten and Townsend, [@B12]; Baumdicker et al., [@B2]; Haegeman and Weitz, [@B13]). Though these genes may not find long term homes in the genomes they "visit," selfish genes especially can affect the rates of gene sharing and thus the size of the pan-genome in a population. Furthermore, many selfish elements induce genome rearrangements that can promote the loss and gain of genes, and thus may have a significant impact on the initiation of the loss of leaky functions. Generation of paralogs may play a role in facilitation of loss of leaky functions. Additional copies increase gene dosage, ameliorating the loss of function in other members of the population by providing more of the common good. However, the pressure to delete genes from genomes is much stronger than to duplicate them (Mira et al., [@B22]) and an increase in gene transcription can have a similar or greater effect on the overall expression level (Weiße et al., [@B32]). Regardless of whether the increased production comes from paralogy or regulation it would need to be countered by a greater decrease in production from other common good functions to overcome the cost of increased protein expression. Conclusion {#s6} ========== Random acquisition of genes and selfish genetic elements, selection by predators, and cheating on common goods, all undoubtedly play a role in generating diversity within populations of bacteria and archaea. The conjecture of the strong black queen hypothesis is that mutual cheating leads to mutual dependencies and therefore cooperation. Under this hypothesis individual cells would be integrated into a meta-organism, whose genome is the pan-genome of the population, similar to Lawrence\'s meta-cells whose genome is distributed over individual micelles. The pan-genome of a population as shared genomic resource could explain part of the "genome of Eden" paradox (Doolittle et al., [@B10]), where estimations of ancestral genomes are far larger and more complex than those of any extant individual genome. Large estimates of archaeal ancestors\' genome sizes (Csurös and Miklós, [@B6]; Wolf et al., [@B35]) could actually represent the pan-genome of the ancestral population rather than any single cell. If this is the case, the complexity of the progenitor cells in a lineage/population might often be at a similar level of complexity as their extant relatives. We hypothesize the large estimates of progenitor genome size might in part reflect a "strong" black queen scenario where genome variation creates a large pan-genome, but no single cell contains a "keystone genome" with all genes in the population represented. More extensive studies of individual and population genomes, and rates of within population transfer are needed to confirm that master genomes, encoding all the leaky functions needed for survival of the population, can be and often are absent from a population. If the hypothesis of the population pan-genome as a shared genomic resource is borne out, then the scientific community will need to continue to increase its appreciation for the import of pan- and meta-genomes. Likewise, we may need to more seriously consider populations as the operative units in which genes are selected in rather than exclusively individual organisms. Similar to how Richard Dawkins ([@B8]) advocated thinking of an organism as a collection of generally agreeable, but selfish, genes perhaps we should be thinking of lineages and populations as the collections of genes, i.e., pan-genomes, rather than the individual cells. Conflict of interest statement ------------------------------ The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank Michael Stephens, Joerg Graf, and Thane Papke for Discussion. Work in the Gogarten-lab was supported through grants from the National Science Foundation (AToL DEB0830024), the National Aeronautics and Space Agency (Exobiology NNX13AI03G), and the Bi-national Science Foundation (BSF 2013061). [^1]: Edited by: Luis Delaye, Centro de Investigación y de Estudios Avanzados - Unidad Irapuato, Mexico [^2]: Reviewed by: Luis David Alcaraz, Universidad Nacional Autónoma de México, Mexico; Luisa I. Falcon, Universidad Nacional Autónoma de México, Mexico [^3]: This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology

Questions concerning the health and well-being of older persons are becoming more important in relation to the growing population of older persons (Swedish National Institute of Public Health \[SNIPH\], [@CIT0041]). Laslett ([@CIT0026]) discussed the increased years of good health that many older persons experience. According to Björklund, Erlandsson, Lilja, and Gard ([@CIT0006]), more focus should be placed on resources for older persons that might promote health and well-being, instead of focusing on loss of function. *Health* has been defined by the World Health Organization as "a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity" (WHO, [@CIT0051], p. 100), which is necessary for the individual\'s possibility of living a good and satisfying life (WHO [@CIT0052], [@CIT0053]). *Well-being* can be referred to as a person\'s subjective experience of health (SOU, [@CIT0042]). According to Svensson, Mårtensson, and Muhli ([@CIT0043]), well-being in older persons can be found in past life experiences, as well as in the present. Spiritual or existential well-being is also considered to be vital for the health of human beings (Melder, [@CIT0032]). It appears that there is an emerging interest in including existential issues in caring for human beings although it can be a challenging aspect of professionals' daily work. For example Udo, Danielson, and Melin-Johansson ([@CIT0045]) found that some nurses caring for patients dying of cancer integrated their personal self in their care, handling existential issues, and that they were helped by collegial support through regular sessions. Existential issues are an inevitable part of people\'s life, even though the way in which we deal with these issues is unique for each individual (Van Deurzen, [@CIT0046]). Past experiences can also be understood in accordance with Firman and Russel\'s ([@CIT0014]) description of the inner child as including all of the past hidden ages that have made up one\'s life journey. Jung is often referred to as the originator of the phenomenon of the inner child in his descriptions of archetypes, in which he argued that we may extend the individual analogy of the divine child to the life of mankind (Jung & Kerényi, [@CIT0021]). Others have also used the concept of the inner child. For example, Kohut ([@CIT0024]) described the inner child in connection with the pains and strains of not being acknowledged. Winnicott ([@CIT0049]) focused on the communication between the child and the caretaker and on how being or not being acknowledged affects the inner child and hence the human being later in life. Cullberg Weston ([@CIT0009]) added the importance of the inner child\'s specific strengths that an individual brings into adulthood and knowledge about the contribution of the early years to the person we have become. Whether healthy or wounded, Firman and Russel ([@CIT0014]) argued that the inner child profoundly affects human beings' overall expressions of themselves in the world. According to Assagioli ([@CIT0003]), the inner child is a psychosynthesis of all ages, the transition from childhood to old age. Each developmental age is not left behind but forms one small part of all that we are. In addition, Assagioli holds that the psychosynthesis of the ages can be achieved by keeping the best aspect of each age alive. Firman and Russel ([@CIT0014]) also discuss the psychosynthesis of the ages and the importance of nurturing the child within. Lamagna ([@CIT0025]) studied the concept of self-relatedness and argued that an "internal attachment system" coordinates its activity in ways that experience, coordinates its activity in ways that best regulate the individual's affects, thoughts, perceptions and behavior. He proceeded to explain that the implicit memories associated with these various states also govern our perceptions of the world and our ways of being in it. This conclusion is in accordance with (Siegel [@CIT0037], Siegel [@CIT0039]), who argued that when people feel they have been listened to they are "feeling felt," which is about being seen heard and understood by another -- establishing an interpersonal connection of belonging and increasing the sense of well-being. Antonovsky ([@CIT0001]) studied the salutogenic process using the sense of coherence to shows how generalized resistance resources can contribute to help people handle problems and difficulties in life. According to Lindström and Eriksson ([@CIT0028]), the salutogenic approach could have a more central position in creating a theoretical framework for health promotion research; in particular it could contribute to the solution of mental health promotion. Lindström and Eriksson ([@CIT0029]) argued that people should be seen as active, participating subjects shaping their lives through their action competence. This approach is similar to the empowerment concept in health promotion explained in the Ottawa Charter (1986). Rennemark and Hagberg ([@CIT0034]) studied the sense of coherence among the elderly in relation to their perceived history and the Eriksonian development stages. The results indicated that the more positive an individual\'s evaluation of his or her life history, the stronger the sense of coherence. Further they concluded that the social network was sometimes used as compensation for shortcomings in an individual\'s concept of self. According to Heckhausen, Wrosch, and Schultz ([@CIT0019]), the challenges that individuals face as they develop from infancy into older age are selecting, adapting, and pursuing developmental and personal goals that reflect changing life course opportunities. Further, they highlighted the role of goal-setting processes in social relationships and interpersonal interactions throughout the lifespan. A similar social focus can be found in the World Health Organization\'s questionnaire about self-rated health and quality of life, which includes spirituality, religiousness, and personal beliefs (WHO, [@CIT0054]). Therefore, studies that track trajectories over the life course could be helpful in understanding older persons' health and well-being through childhood experiences. According to Svensson et al. ([@CIT0043]), narratives create an experience of renewal *via* dialogue about what older persons experienced earlier in life through social contact and can be compared to what Ricoeur ([@CIT0035]) referred to as *meaningful wholeness*. George ([@CIT0015]) suggested that if we are to do justice to the older persons whose interests we are attempting to promote, we must pay attention to how older persons feel about their lives and their sense of well-being throughout life. Therefore, the aim of this study was to describe and gain more knowledge about the phenomenon of the inner child, as reflected in events during childhood experienced by older persons. Methods {#S0002} ======= According to van Manen ([@CIT0047]), phenomenology is a systematic attempt to uncover and describe the structure of lived experience. The frame of reference in this study is based largely on van Manen\'s ([@CIT0047]) hermeneutical phenomenological approach to lived experience research, which focuses on collecting and analyzing other human beings' stories in order to understand a phenomenon. Participants and procedure {#S0002-S20001} -------------------------- We invited Swedish-speaking senior citizens without cognitive disabilities to participate in the study. The participants were recruited through an organization for senior citizens and were contacted by the first author. An oral presentation and a letter presenting the aim of the study were given at one of the organization\'s member meetings. Altogether 13 persons, all of them retired, agreed to participate in the study. The participants included seven men and six women. Eleven participants were interviewed on the premises of the organization, and two participants were interviewed in their own homes. Eight participants had grown up in the city and five had grown up in the countryside. The ages of the participating women ranged from 72 to 85 years. All of them had children and grandchildren. All of the women had been employed at some point in their lives, and two of them had started an educational program when their children became older. The ages of the participating men ranged from 70 to 91 years. All of them had children and six of them had grandchildren. All of the men had started working early in life and six of them had changed occupation and/or studied later in life. All the interviews were coded to protect the identities of the participants (see below). Data collection {#S0002-S20002} --------------- Data were collected through open-ended interviews conducted by the first author. A pilot interview was conducted to test the research questions, resulting in fewer questions. Instead of several detailed questions, the interview started with the broad question, *Can you describe significant events from your childhood that you have carried with you throughout life?* This approach made the interviews more open and left more space for the participants to choose which experiences to share. Supporting questions were asked, including the following examples: *Do you remember your feelings at the time? Has this affected you as an adult? Did you learn anything or change in any way after this event? Is there anything in what you have narrated that you have forwarded to your children?* The interviews lasted between 1 and 1.5 h and were tape recorded by the first author. The recorded interviews were transcribed verbatim by the first author and coded from A1 to A6 for the women and B1 to B7 for the men. The code list will be destroyed after the analysis is complete and the manuscript is published. No names, addresses, or identification numbers were collected. All through the analysis process the recordings were kept in a locked cupboard when not under the direct supervision of the researcher. The data will be archived for 10 years. Data analysis {#S0002-S20003} ------------- The data analysis employed a hermeneutic-phenomenological approach that was inspired by van Manen ([@CIT0047]), and all of the authors were involved throughout the process described below. The analysis process included three steps: seeking meaning, thematic analysis, and interpretations with reflections. According to van Manen ([@CIT0047]), a holistic approach entails seeking meaning on different levels. The analysis started with reading all of the transcribed interviews to capture the significance in each interview and simultaneously seek the meaning of the collective picture that included all of the interviews. The analysis employed a back-and-forth movement between the whole and the parts, as described by van Manen ([@CIT0047]). The second step of the process was thematic analysis, which involved striving to determine the experiential structures that made up the participant\'s experiences. The textual units from the interviews were then organized into different experiences in several steps and were finally reduced to the main themes and the themes of the participant\'s lived experiences. The third and final step consisted of interpretation with reflection, a process that van Manen ([@CIT0047]) described as recovering the embodied meanings in the text in a free and insightful way. Ethical considerations {#S0002-S20004} ---------------------- The persons at the organization for seniors were given oral and written information about the study, as outlined in the Helsinki Declaration (2008). In accordance with Swedish ethical law (SFS [@CIT0036]:460), participation was voluntary, and informed consent was collected from the participants. The participants received information about their role in the project, their autonomy, and confidentiality. Thus the participants had the option to withdraw from the project at any time. They were also informed that no one apart from the authors of this article would have access to the collected material. In addition, the ethical committee in Umeå approved the research project before it started (2013/342-31Ö). Findings {#S0003} ======== The analysis revealed two main themes and six themes that captured the participants' experiences of events during childhood, which were understood as reflecting the phenomenon of the inner child ([Table I](#T0001){ref-type="table"}). ###### Overview of the main themes and themes ----------------------------------------------------------------------------------------- Main themes Themes ------------------------------------------ ---------------------------------------------- The inner child becomes visible Feeling safe, loved, and supported\ Feeling alone, scared, and sad\ Creating space for fantasy and possibilities The inner child\'s presence through life When something bad becomes good\ A source for development\ How I became who I am ----------------------------------------------------------------------------------------- The inner child becomes visible {#S0003-S20001} ------------------------------- Based on the participants' narratives, the understanding of their experiences included both positive and negative feelings, as well as ways to be creative, through which the inner child became visible. ### Feeling safe, loved, and supported The participants described experiences related to feelings of being safe, which were often connected to family and the structure of the older generation\'s inclusion in the home. Different people and situations added to the feeling of safety. Feeling safe was also connected to everyday activities that created habits or routines, such as buying milk from the milk shop and playing with dolls when the mother was weaving or reading. Safety was also connected to knowing that someone was always there to listen. The participants talked about growing up in safe surroundings with a secure atmosphere, which made them feel safe at home and during leisure time. One participant said, "I am thinking of safety, a safe upbringing in every respect. We lived in the same house as my grandparents, who always were at home and had time to listen" (A3). The participants also shared experiences of feeling loved by a number of people and in many ways, even though times were hard and money was scarce. The participants remembered parents, relatives, and other adults as being loving, caring, and generous towards them. Siblings or other peers were also considered to be loving and empathetic. The participants shared stories of loving actions, for example, how bullies were confronted by siblings when they were being bullied or feeling a mother\'s love and generous caring in spite of hard work and poor circumstances. One participant said, "My father made an effort to take time off from his work so we could be together" (B7). The participants described experiences that were understood as being supported during childhood by parents, relatives, teachers, and other significant adults. Sometimes these adults became role models, and activities or situations served as inspiration that exemplified how to live a good life. For example, one participant described how "... an aunt with a handicap became a guide in how to reach my future goals, as far as studying was concerned, so I believed that I could do it" (A1). One other participant explained: "Thanks to my time as a scout, I learned how to be more outgoing and not to be afraid of strangers" (B2). ### Feeling alone, scared, and sad The participants talked about experiences during their childhood when they had felt alone, scared, and sad. With respect to their upbringing, the participants described how their parents did not encourage them or show them gratitude because at the time adults had the preconceived idea that these practices might spoil their children. Unfortunately, this lack of recognition was perceived by the youngsters as a feeling of being abandoned. One participant described it this way:"I remember when I was 13 years old and had to help my mother take care of the farm while my father was in the army... We had no milk machines and had to milk by hand... When my father came home, he didn\'t give me any compliments about running the farm, but rather criticized me for what I had done wrong... It was a trauma that I blamed my father for. (B3)" The participants described being scared. One participant remembered a funeral, as follows: "It was when my grandmother died. She was lying there in bed, and my mother lifted me up so I could see her. It was horrible and terrifying, and I hit my mother" (A2). The participants talked about the sadness they felt as a child when their parents did not engage in their upbringing. They described experiences with formal and distant relationships with their parents. One participant said to his parents when he got older: "When I get to work I can say 'you' to all of them immediality \[instead o 'ma\'am' or 'sir'\], but at home I never say that" (B2). In those days, parents did not interfere in the upbringing of the children very much. The sadness experienced by the participants was reinforced by not being able to talk about feelings with their parents and by the fact that the parents did not always explain matters to their children. Children were often left alone with their fantasies and misinterpretations. One participant remembered being sad when his mother became ill and how this feeling was worsened by not being able to talk about it:"I was not allowed to tell my younger siblings... This made it difficult for me to trust people... My mother disappeared, and this wasn\'t natural for a child; was she dead or had she just left us for some other reason? (B1)" Some participants described a feeling of sadness connected to having to work from a very early age, sometimes instead of going to school, leaving no time for play or spending time with friends. One participant said, "When I came home from school I had to start working at once. The schoolwork came second and had to be done in the evening even if I was exhausted" (B6). ### Creating space for fantasy and possibilities The participants did not remember playing with their parents very often; instead, their experiences of playing with friends and siblings were more frequent. This play was understood as something that had supported them in developing their fantasy and creativity. Some of the participants described being lonely as a child without siblings and friends but finding creative solutions by playing with domestic animals, dolls, and other toys. One participant said:"I had a lot of imagination, but I don\'t remember playing with my mother or any other adult, but they told stories and I loved that... I also had a playmate, and one of our adventures was a landfill where we could find things. (A2)" The participants had memories from the war, during which Sweden was isolated and feared military invasion. The participants remembered role-playing about what they heard on the radio. One person\'s memories from role-playing were described as follows: "We played funeral and wedding and made flowers of newspaper. My mother felt uncomfortable but let us go on playing" (A5). According to the participants' descriptions, another way to spend time during childhood was listening to stories and thereby gaining experiences that contributed to the evolution of fantasy. The participants developed their imaginations through adults' storytelling and were also inspired to read themselves. One participant remembered when a daughter of a neighbor came to look after them:"I have a fantastic memory of her because she could tell stories, and among other thing she told us the story of *The Children from the Frostmo Mountains* (a Swedish tale). Nobody had told stories to us children before. (B3)" One participant had memories from being a scout: "It was a bit of an adventure and we were a nice gang and stayed with the scouts until 15 years old" (B4). The inner child\'s presence through life {#S0003-S20002} ---------------------------------------- Our understanding of the participants' experiences indicates that the inner child is present throughout the lifespan, is found in times of challenge during life, and can turn something bad into something good. However, the presence of the inner child could also be a source for development through life and interact with the person that the individual truly is. ### When something bad becomes good The participants experienced challenges during childhood, which proved to be good experiences later in life or led to something positive in the future. The participants experienced difficult circumstances at times during their upbringing, with little support from surrounding family and friends; however, some adults became good role models despite hardship in early relationships. One participant remembered:"I didn\'t spend much time with my father playing or reading during childhood because he was always working hard and to give in didn\'t exist... My father was a good role model and taught me to work hard, be careful, and accomplish what I was supposed to do. (B3)" For example, the participants experienced how fear felt as a child turned out to be an asset or strength later on in life and could serve as an inspiration for others when they felt scared and did not know how to handle a situation. One participant explained:"My mother didn\'t talk about feelings and did not explain things to me, which made me scared of many things when I grew up... When my children were small, I didn\'t want them to be afraid of things like darkness or going to the hospital and getting a shot, so I explained how everything worked and what they had to expect... This also had an impact on my work, to give good explanations for why you do certain things and in what way. (A5)" ### A source for development The participants developed their own strength when growing up with strong or even authoritative parents, who guided them in how to cope with difficulties. In situations when their parents hindered them from doing something they wanted, their disappointment and pain could turn into an inner feeling of strength that helped in not giving up hope. One participant said, "If you are not satisfied with your situation, it is up to you to make a change. Everything is possible, even if you haven\'t got it all served on a silver platter" (B6). The participants described how they gained strength in the form of curiosity and eagerness to learn new things and welcomed new ideas that did not follow the same paths as their parents. One participant said, "I learned a lot on my own and I always had seven to eight books at home from the library. My mother was very proud when I went to technical college" (B5). ### How I became who I am According to the participants, strong desires or interests were acquired during childhood. The participants described how they possessed a strong will or interest, which made it easier for them to overcome difficulties and challenges. One participant explained:"My mother died early, and I started studying to become a nurse, as the first one in my family to get an education. As I was alone with my children, I could manage thanks to my employment as a nurse. (A4)" One participant explained:"One of my teachers wanted me to go to high school. My parents said that we would lose our friends---a working class child could not go to high school. Later when I was an adult I got an education and worked until my retirement. (A6)" The participants described how they became the people they are today due to relationships with their parents or other significant adults. The participants felt they had had a happy childhood during which they felt loved and accepted. The participants experienced how a safe upbringing with a supporting social network had helped them to feel secure, even in difficult situations. One participant said:"I have learned from my parents to be loyal towards those around me... I think it is important to be generous and helpful... Since childhood, I felt loyalty towards those around me... This has become a guiding star for me to help people in less fortunate circumstances in Sweden, as well as in many other countries. (B5)" Discussion of the method {#S0004} ======================== The method of this study was inspired by van Manen ([@CIT0047]), who argued that phenomenological research is not only concerned with what it means to live a life but is also attuned towards caring and thoughtfulness. The aim of this study was to describe and gain more knowledge about the phenomenon of the inner child, as reflected by events during childhood experienced by older persons. The experiences of the participants were gathered in the spirit of van Manen ([@CIT0047]) in an attempt to answer questions about the essential human experiences that represent the nature of the phenomenon that in this study was interpreted as the inner child. The problem of phenomenological inquiry is not always that we know too little about the investigated phenomenon but that we know too much (Lincoln & Guba, [@CIT0027]). To enhance the confirmability of our study, we needed to handle our pre-understandings. We made our beliefs explicit in the analysis phase, discussing our different points of view, personal experiences, backgrounds, and professions (psychologist, nurse, occupational therapist, and health educator). According to Guba and Lincoln ([@CIT0017]), credibility is strengthened when the authors come from different backgrounds. In addition, to test the research questions, a pilot interview was conducted. According to van Manen ([@CIT0047]), the interview serves the specific purpose of answering the question: "What is the nature of this phenomenon as an essentially human experience?" (p. 66). This approach, which we used, strengthens the trustworthiness of a study and may help to determine whether the interview questions are suitable for obtaining rich data (Guba & Lincoln, [@CIT0017]). Kahneman ([@CIT0022]) argued that there is a difference between participants telling their stories in a narrative way and answering more structured questions in a survey. To capture the phenomenon of the inner child, the participants in this study were able to narrate stories about events during childhood. According to Kahneman ([@CIT0022]), these stories will be more predictive and tell more of the person\'s life and how he or she handles challenges than a survey. One limitation of this study is that the older persons from the senior citizens' organization who agreed to participate in this research study may have had a more positive outlook than those who decided not to accept the invitation to participate in the study. In addition, older persons who do not participate in the senior citizens' organization may be less active than those who are involved in the organization. However, the findings show a wide range of narrative stories that include positive and negative experiences. Discussion of the findings {#S0005} ========================== The aim of the study was to describe and gain more knowledge about the phenomenon of the inner child, as reflected in childhood events experienced by participants. The interpretation of the participants' experiences was understood as the phenomenon of the inner child, which was illuminated in two main themes without any hierarchy: *the inner child becomes visible* and *the inner child\'s presence through life*. In this study, the meaning of the inner child, as reflected in childhood events described by the participants, appears to encompass positive and negative events during childhood. According to Assagioli ([@CIT0003]), the psychosynthesis of the ages involves the psychological parts of childhood, including spontaneous affection and joy, as well as uncontrolled impulsiveness and aggression. This notion is also consistent with Firman and Russel\'s ([@CIT0014]) thoughts on how the inner child, whether healthy or wounded, impacts how human beings express themselves. The participants felt loved by parents, siblings, and relatives when they were young. The participants described positive experiences that were understood as being safe, loved, and supported. Different people and situations added to the feeling of safety, which helped the children to not be afraid of strangers but instead to trust people. Parents, relatives, and teachers supported the children by being positive role models who guided them towards future goals, resulting in feelings of loyalty, which were carried into adulthood and passed on to children and grandchildren. Similar to Winnicott\'s ([@CIT0049]) description of the inner child, the early emotional communication between the child and caretaker is essential for developing confidence and determining how the child will relate to others later in life. This finding is also consistent with Rennemark and Hagberg ([@CIT0034]), who argued that positive life history evaluations regarding the periods of early childhood and adolescence are associated with a strong sense of coherence. Parallel to these positive experiences, the participants in this study also described negative experiences. The participants narrated experiences that had caused them to feel alone, scared, and sad. The participants were often left alone with their thoughts and their own ideas when young and often did not have things explained to them by their parents, which sometimes led to misinterpretations. The participants also experienced their relationships with their parents as formal and distant, which led to distrust of their parents as well as other people. This finding appears to be similar to Winnicott\'s (1987) notion about being wounded by not being seen and Kohut\'s ([@CIT0024]) description of "the none empathic responding world" (p. 18). Kohut explains that this experience can lead to the development of a survival personality, which Winnicott ([@CIT0049]) calls a *false self*. The positive and negative experiences described by the participants in this study can be interpreted as expressed by Firman and Russel ([@CIT0014]): that the inner child profoundly affects human beings' overall expressions of themselves in the world. Those authors also stress that both wounding and healing are a matter of empathic connection (in other words, creating health-promoting relationships). In addition, the participants in this study remembered feeling curious, adventurous, and strong. These experiences were understood as creating spaces for fantasies and possibilities. Often, these fantasies were linked to something that the children did not quite understand or to something that they felt was frightening. According to Kaplan ([@CIT0023]), the world of fantasy and play can be helpful as an opportunity to escape from reality, by creating distance from what is happening in the surroundings. Hjort ([@CIT0020]) viewed play as a source of "joie de vivre" that has an immediate value for the child in acting based on his or her needs and desires. In addition, she argues that play creates models for the future development of the child and models for what personality to become. According to Winnicott ([@CIT0049]), *good-enough-mothering* is what creates sufficient basic trust and security, with the help of a transitional object, to develop the ability of the child to represent himself in the world. Winnicott means that it is the creative locus between the inner and outer world where the child\'s experiences of play and fantasies can be expressed and where the child can develop as an individual. The concept of play is not only important in the life of the child but throughout the lifetime, as the inner child impacts our lives as adults (Cullberg Weston, [@CIT0009]). The participants in this study described the absence of adults in play situations. The participants did not mention what Newson and Newson ([@CIT0033]) argued is of importance, namely that the parent focus on the child, providing a "listening ear" and being the most important toy for the child. Similarly, Ginsburg ([@CIT0016]) noted the importance of maintaining strong bonds between parent and child through play and thereby promoting healthy child development. In contrast, the findings in this study show that the participants found playmates in peers and siblings, who were loving, supportive, and joyful and added to their feelings of adventure. Cutting and Dunn ([@CIT0010]) found that preschool children\'s successful communication and joint pretend play with siblings and friends were related to social cognitive skills. Their results showed the importance of cooperative pretend play, including shared affection in both child--friend and child--sibling conversations. The participants in this study described how they developed their imaginations through adults' storytelling and were inspired to read themselves. Bus and Ljzendoorn ([@CIT0007]) point out that interest in reading is not a natural phenomenon but is evoked by the pleasure of sharing a book with a parent. According to Aram and Shapiro ([@CIT0002]), parents have it in their power to contribute to their child\'s development of empathic skill by reading a book with content that can be interactive, inviting dialogue between the child and the parent. The fascination of reading was not only enjoyed during childhood but continued throughout life, promoting reading to children and grandchildren and adding to the feeling of well-being. Denham and Auerbach ([@CIT0012]) found that a mother\'s questions about emotional situations in stories predicted their children\'s helpfulness and concern towards others, which also meant that the children obtained a better understanding of their own emotions and well-being. In the main theme *the inner child\'s presence through life*, the participants in this study described how they became who they are today due to their inner strength, strong will, or interest, which made it easier for them to overcome difficulties and challenges. The participants also described how when they were young, they learned the importance of carrying things through to the conclusion, even under difficult circumstances. This lesson was due to being presented with situations early in life in which they had to take responsibility over their own lives and change something bad into something good. This finding can be compared to Dale, Söderhamn, and Söderhamn\'s ([@CIT0011]) findings that independence and the ability to control their own lives in accordance with their own preferences were the ultimate goals required for participants to achieve health and well-being. The participants in this study described living under very poor circumstances when young. In their experience, it was more important to help support the family by working than going to school. There was a gender difference in the participants' experiences from childhood that showed that the girls helped the mother in the kitchen, and the boys helped the father outdoors with the animals or other tasks that were considered heavier. Phenomenological research shows that men and women have different life-worlds, which may cause gender segregation (Martinsen, Dreyer, Haahr, & Norlyk, [@CIT0031]). In previous research, one can find examples of deprived children who experienced very bad conditions during childhood but still succeeded as adults in not only making themselves a future but also living healthy and prosperous lives (Lönnroth, [@CIT0030]; Werner & Smith, [@CIT0048]). The findings of this study show that the negative experiences that the participants carried with them from childhood had become an asset in adulthood. In connection to these hardships, the participants described how they were seen and heard by at least one person in the surrounding network. This is similar to a form of intrapersonal attunement of feeling felt suggested by Siegel ([@CIT0038]), which may promote well-being with physical and psychological dimensions. Firman and Russel ([@CIT0014]) argued that it is crucial for the development of a human being to be recognized, acknowledged, and understood. Winnicott ([@CIT0049], [@CIT0050]) calls this phenomenon *mirroring*. In addition, this notion can be connected to Cullberg Weston\'s ([@CIT0009]) argument that experiences from childhood that form the inner child have a profound effect on our lives as adults. According to Firman ([@CIT0013]), the dependent--independent paradox means that the authentic personality is not "childish" or "immature" but as a child *vis-à-vis* the deeper self. Firman and Russel ([@CIT0014]) argued that it is not until you can honestly be with the inner child with no criticism or pressure to change that you can connect to the authentic personality and the deeper self. This study describes how the participants tried to counteract what they experienced as negative in their parents' way of upbringing in relation to their own children, with respect to studies, interests, and ways of viewing things in life. The participants explained how they reflected on their experiences from childhood and made their own choices. For example, a very authoritative parent inflicted much pain for the child, but as an adult, that parent could serve as a role model for not giving in when facing challenges in life. When people manage to create consensus in their lives, they also achieve high narrative competence, which increases mental health (Havnesköld and Risholm Mothander, [@CIT0018]). In addition, this process of making one\'s own choices can be compared to Heckhausen et al. ([@CIT0019]), who argued for the importance of goal-setting processes in social relationships and interpersonal interactions throughout the lifespan. The participants gained strength from their curiosity to learn and welcome new ideas that did not always follow the same paths as their parents. The participants also experienced how their own strength became a source for development, helping them to not give up hope when they were hindered in fulfilling future aspirations. According to Clancy, Balteskard, Perander, and Mahler ([@CIT0008]), recollecting past experiences and sharing stories can create an orientation towards the future and be important for hope and a feeling of possibilities. The participants experienced how a safe upbringing with a supporting social network had helped them feel secure even in difficult situations. They had had a happy childhood when they felt loved and accepted. This finding is in accordance with the findings of Svensson et al. ([@CIT0043]), who emphasized the importance of factors such as well-being and the maintenance of a social network for experiencing a good quality of life. The results of this study show that the phenomenon of the inner child is reflected in events during childhood and that these experiences are remembered throughout life and relate to the well-being of older persons. This is in some way what Tornstam ([@CIT0044]) talks about: a reconciling of experiences and memories and the idea that we are all ages at the same time. However, his conclusions that older persons become less interested in social interactions and have a greater need for solitary meditation were not noted in this study. Instead, the findings of this study point in a somewhat complex direction, showing that some older persons became more contented, while others were weighed down by remembering their childhood experiences throughout life, which affected their interest in being socially active. According to Berg, Hassing, McClearn, and Johansson, McClearn, and Johansson ([@CIT0004], [@CIT0005]), social network quality, a sense of being in control of one\'s life, and decreased depressive symptoms were significantly associated with higher life satisfaction among the oldest individuals. According to Antonovsky ([@CIT0001]), a sense of coherence helps people handle problems and difficulties in life as some people stay healthy and develop a sense of meaning in their lives despite stressful and traumatic experiences. Even if he does not mention the inner child, Antonovsky ([@CIT0001]), talks about resistance resources as a sort of inner strength. However, he does not connect the resistance resources as strongly to the relation between the human being and his or her social network during childhood, which was described by the participants in this study. In conclusion, the findings of this study demonstrate new knowledge illuminating how human beings are influenced by the inner child throughout their lifespan. Experiences during childhood have an impact on the choice of profession and also on how we act in relation to the next generation. The participants in this study voiced a need to be recognized, acknowledged, and understood as unique persons living their own lives. This finding is consistent with Assagioli\'s ([@CIT0003]) argument that the inner child is a psychosynthesis of all ages and that the psychosynthesis of the ages can be achieved by keeping the best aspect of each age alive. In accordance with George ([@CIT0015]), we argue that attention should be paid to how older persons feel about their lives, as well as to the strategies that they need to sustain a sense of well-being. In other words, when caring for older persons keeping Firman and Russel\'s ([@CIT0014]) notion of nurturing the inner child in mind can be of great value for promoting health. The findings of this study point to the well-being dimensions of feeling safe, loved, and supported, as well as creating space for fantasy and possibilities. These dimensions can be compared to the physical, mental, social, and existential well-being dimensions that are found in the WHO\'s definitions of health (WHO, [@CIT0051], [@CIT0052], [@CIT0053]) and surveys (WHO, [@CIT0054]). Although the sample in this study was limited, we believe the findings are worthy of recognition as we suggest that a holistic approach be used when caring for older persons. We would also like to thank the department of Health Sciences of Luleå University of Technology for their support. (Instead of omitted for blind review). Conflict of interest and funding {#S0006} ================================ The authors declare that there is no conflict of interests concerning the research presented in this paper. This research has received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Introduction {#Sec1} ============ Existing guidelines recommend initiating enteral nutrition (EN) within the first 24--48 h after intensive care unit (ICU) admission if patients are unable to eat, not clearly defining reasons to delay EN \[[@CR1]--[@CR3]\]. The present guideline is issued by the Working Group on Gastrointestinal Function within the Metabolism, Endocrinology and Nutrition (MEN) Section of the European Society of Intensive Care Medicine (ESICM) and is endorsed by ESICM. Our objective was to provide evidence-based guidelines for early enteral nutrition (EEN) in critically ill patients, focusing on specific clinical conditions frequently associated with delayed EN. Caloric and protein requirements, time to reach targets, type and route of EN, and timing of supplemental or full parenteral nutrition (PN) were not addressed. A full version of the introduction with references is available in Supplement 1. Methods {#Sec2} ======= A full version of methods with references is available in Supplement 1. We performed a systematic review of "early" EN (EEN) vs. early parenteral nutrition (PN) and EEN vs. delayed EN in adult critically ill patients. After critical appraisal of identified studies and in accordance with current guidelines \[[@CR1]--[@CR3]\], we defined EEN as EN started within 48 h of admission independent of the type or amount. Thereafter, we predefined conditions in which EN is frequently delayed and performed a systematic review for each of these questions. If randomised controlled trials (RCT) were available, we gave an evidence-based recommendation; if not, our recommendations were based on expert opinion (very low quality evidence), as all observational studies evaluating EEN are intrinsically biased, because patients who are less severely ill are more likely to receive and tolerate EEN. General considerations {#Sec3} ====================== We focussed on specific conditions in which EN is frequently delayed and tolerance of EN might be impaired. Therefore, all our recommendations are based on general principles and precaution measures outlined in Table [1](#Tab1){ref-type="table"} \[[@CR4]--[@CR9]\]. All study questions and recommendations refer to adult critically ill patients.Table 1General principles and precautions for using EEN in critically ill patients at risk of intoleranceStarting and continuing EENStart EN at a slow rate (10--20 ml/h) while carefully monitoring abdominal/gastrointestinal symptomsIncrease EN slowly once previous symptoms are resolving and no new symptoms occurDo not increase EN in cases of intolerance or new symptoms, such as pain, abdominal distension or increasing intra-abdominal pressure. In these circumstances EN should be either continued at a slow rate or ceased depending on the severity of symptoms and suspected underlying sinister pathology (e.g. mesenteric ischaemia)Energy target during EENDo not aim to cover full energy target with EEN. The optimal energy and protein target in the early phase of acute critical illness is not known. EEN that exceeds actual energy expenditure appears harmful and should be avoided \[[@CR4], [@CR5]\], whereas hypocaloric EEN may be safe \[[@CR6]--[@CR8]\]Monitoring and protocolised management of GI dysfunction during EENIn case of gastric retention without other new abdominal symptoms use prokinetics and/or postpyloric feeding in a protocolised way \[[@CR9]\]During introduction and increasing the rate of EN, measurement of intra-abdominal pressure (IAP) provides an additional numeric value to detect negative dynamics of IAP during EN in patients with severe abdominal pathology, hypoperfusion or fluid overloadIndividualized approachFor patients with diminished consciousness and inadequate swallowing, precautions to prevent aspiration of gastric contents may be useful, including considering postpyloric feedingPremorbid health and course of the acute illness may differ between patients with similar diagnose; therefore an individual approach should always be applied Results {#Sec4} ======= All recommendations with the final agreed results are presented in Table [2](#Tab2){ref-type="table"}.Table 2RecommendationsRecommendationAgreement (%)Comments1. We suggest using EEN in critically ill adult patients rather than early PN (conditional recommendation based on low quality evidence = Grade 2C) or delaying EN (conditional recommendation based on low quality evidence = Grade 2C)1002. We suggest delaying EN if shock is uncontrolled and haemodynamic and tissue perfusion goals are not reached, but start low dose EN as soon as shock is controlled with fluids and vasopressors/inotropes (conditional recommendation based on expert opinion = Grade 2D)91.4Concern regards applying EN when very high doses of vasopressors (e.g. noradrenalin \>1 μg/kg/min) are required and hyperlactatemia is persisting or other signs of end organ hypoperfusion are present3. We suggest delaying EN in case of uncontrolled life-threatening hypoxaemia, hypercapnia or acidosis, but using EEN in patients with stable hypoxaemia, and compensated or permissive hypercapnia and acidosis (conditional recommendation based on expert opinion = Grade 2D)1004. We suggest that EN should not be delayed solely because of the concomitant use of neuromuscular blocking agents (conditional recommendation based on expert opinion = Grade 2D)91.4Concern regards very seldom patients in whom continuous infusion of neuromuscular blocking agents is needed, because these patients are in a very critical situation5. We suggest starting low dose EEN in patients receiving therapeutic hypothermia and increase the dose after rewarming (conditional recommendation based on expert opinion = Grade 2D)1006. We suggest using EEN in adult patients receiving extracorporeal membrane oxygenation (conditional recommendation based on expert opinion = Grade 2D)1007. We suggest that EN should not be delayed solely because of prone positioning (conditional recommendation based on expert opinion = Grade 2D).91.4Concern regards tolerance of EN8. We suggest using EEN in critically ill adult patients with traumatic brain injury (conditional recommendation based on expert opinion = Grade 2D)95.7No agreement regards strength of recommendation9. We suggest using EEN in critically ill adult patients with stroke (ischaemic or haemorrhagic) (conditional recommendation based on expert opinion = Grade 2D)10010. We suggest using EEN in critically ill adult patients with spinal cord injury (conditional recommendation based on expert opinion = Grade 2D)10011. We suggest using EEN in critically ill adult patients with severe acute pancreatitis (conditional recommendation based on low quality evidence = Grade 2C)10012. We suggest using EEN in critically ill adult patients after gastrointestinal surgery (conditional recommendation based on low quality evidence = Grade 2C)10013. We suggest using EEN in critically ill adult patients after abdominal aortic surgery (conditional recommendation based on expert opinion = Grade 2D)10014. We suggest using EEN in critically ill adult patients with abdominal trauma after the continuity of the GI tract is confirmed/restored (conditional recommendation based on expert opinion = Grade 2D)100Adequate gut perfusion needs to be confirmed15. We suggest delaying EN in critically ill adult patients with overt bowel ischaemia (conditional recommendation based on expert opinion = Grade 2D)10016. We suggest delaying EN in critically ill adult patients with high-output intestinal fistula if reliable feeding access distal to the fistula is not achievable (conditional recommendation based on expert opinion = Grade 2D)10017. We suggest using EEN in critically ill adult patients with an open abdomen (conditional recommendation based on expert opinion = Grade 2D)10018a. We suggest using EEN in patients with intra-abdominal hypertension without abdominal compartment syndrome, but consider temporary reduction or discontinuation of EN when intra-abdominal pressure values further increase under EN (conditional recommendation based on expert opinion = Grade 2D)87.1Concern regards impaired gut perfusion and tolerance of EN. Monitoring trend of IAH and tolerance of EN are essential18b. We suggest delaying EN in critically ill adult patients with abdominal compartment syndrome (conditional recommendation based on expert opinion = Grade 2D)10019. We suggest delaying EN in patients with active upper GI bleeding, and starting EN when the bleeding has stopped and no signs of rebleeding are observed (conditional recommendation based on expert opinion = Grade 2D)10020. We suggest starting low dose enteral nutrition when acute, immediately life-threatening metabolic derangements are controlled with or without liver support strategies, independent on grade of encephalopathy (conditional recommendation based on expert opinion = Grade 2D)10021. We suggest delaying EN in critically ill adult patients if gastric aspirate volume is above 500 ml/6 h (conditional recommendation based on expert opinion = Grade 2D91.4Single large gastric aspirate volume should trigger administration of prokinetics and reassessment, but not prolonged withholding of EN22. We suggest using EEN in critically ill adult patients regardless of the presence of bowel sounds unless bowel ischaemia or obstruction is suspected (conditional recommendation based on expert opinion = Grade 2D)10023. We suggest using EEN in critically ill adult patients presenting with diarrhoea (conditional recommendation based on expert opinion = Grade 2D)95.7Uncertainty regards volume and persistence of diarrhoeaResponse rate was 100% in both Delphi rounds (all co-authors responded, methodologist did not participate). Agreement is calculated as percentage of "agree" answers from total A flow chart with evidence identification process (Supplement 2), number of identified abstracts and assessed full texts for each study question (Supplement 3), Pubmed search formulas (Supplement 4), evidence tables for each question with respective references (Supplement 5), evidence profiles for questions with meta-analyses (Table [3](#Tab3){ref-type="table"}), evidence profiles for additional meta-analyses for Question 1 and 11 (Supplement 6), Forest plots for meta-analyses (Figs. [1](#Fig1){ref-type="fig"}, [2](#Fig2){ref-type="fig"} and Supplement 7) are provided.Table 3Evidence profiles for the questions where meta-analyses were performed*EN* enteral nutrition, *PN* parenteral nutrition, *CI* confidence interval, *RR* risk ratio, *GI* gastrointestinal Fig. 1Forest plots (**a** mortality; **b** infections) Question 1A: early EN (EEN) vs. early PN (EPN) in unselected critically ill patients Fig. 2Forest plots (**a** mortality; **b** infections) Question 1B: early EN (EEN) vs. delayed EN (DEN) in unselected critically ill patients **[Question 1:]{.ul}** **Should we use EEN in critically ill adult patients?** The methodology is described in Supplement 1. **[Question 1A:]{.ul}** **Should we use EEN rather than early PN?** Eight trials fulfilled the criteria and were included in meta-analyses (Supplement 5, Table 1A). Results are presented in Fig. [1](#Fig1){ref-type="fig"}. For *mortality*, we included seven RCTs (2686 patients). EEN did not reduce mortality compared to early PN (RR 0.95; 95% CI 0.76--1.19; *P* = 0.64; *I* ^2^ = 9%). The certainty of evidence was **moderate**. We rated down for imprecision (Table [3](#Tab3){ref-type="table"}). For *infection*, we included seven RCTs (2729 patients). EEN reduced the risk of infections compared to early PN (RR 0.55; 95% CI 0.35--0.86; *P* = 0.009; *I* ^2^ = 65%). The certainty of evidence was **low**. We rated down for risk of bias and inconsistency (Table [3](#Tab3){ref-type="table"}). Adding 11 additional studies identified during searches for questions in specified patient groups did not significantly change our results (included studies are presented in Supplement 5, Table 1C; evidence profiles in Supplement 6 and Forest plots in Supplement 7, Fig. 3). **[Question 1B:]{.ul}** **Should we use EEN rather than delay nutritional intake?** Fourteen studies fulfilled the criteria and were included in the meta-analysis (Supplement 5, Table 1B). Results of the meta-analyses on EEN vs. delayed nutritional intake (including delayed EN, oral diet or PN) are presented in Fig. [2](#Fig2){ref-type="fig"}. For *mortality*, we included 12 RCTs (662 patients). EEN did not reduce mortality compared to delayed nutritional intake (RR 0.76; 95% CI 0.52--1.11; *P* = 0.149; *I* ^2^ = 0%). For *infection*, we included 11 RCTs (597 patients). EEN reduced risk of infection compared to delayed EN (RR 0.64; 95% CI 0.46--0.90; *P* = 0.010; *I* ^2^ = 25%). The certainty of evidence was **low**. We rated down for risk of bias and imprecision (Table [3](#Tab3){ref-type="table"}). In one study it was not possible to determine whether early PN was also used in some patients in the EEN group \[[@CR10]\]. Adding eight additional studies identified via specific searches did not significantly change the results (included studies are presented in Supplement 5, Table 1D; evidence profiles in Supplement 6 and Forest plots in Supplement 7, Fig. 4). Recommendation 1. We suggest using EEN in critically ill adult patients rather than early PN (Grade 2C) or delaying EN (Grade 2C). {#Sec17} ---------------------------------------------------------------------------------------------------------------------------------- **[Question 2:]{.ul}** **Should we delay EN in patients with shock receiving vasopressors or inotropes?** No RCTs were retrieved. We identified and analysed four prospective cohort studies, four case series/retrospective cohort studies and two reviews (Supplement 5, Table 2). There is concern that EN in shock further jeopardizes the already impaired splanchnic perfusion. Non-occlusive bowel necrosis or non-occlusive mesenteric ischaemia (NOMI) has been reported in fewer than 1% of patients \[[@CR11], [@CR12]\], without evidence for causal relationship between shock, vasopressors, EN and NOMI \[[@CR11]--[@CR14]\]. In a large observational study, EEN (\<48 h) in patients with 'stable' haemodynamics after fluid resuscitation, whilst receiving at least one vasopressor, was associated with reduced mortality compared to late EN (\>48 h) \[[@CR15]\]. These results suggest that the use of concomitant vasopressors (especially with stable or decreasing doses) should not preclude a trial of EN, despite a high prevalence of feeding intolerance \[[@CR16]\]. In very unstable patients, EN may not have priority and potential positive effects of EN are unlikely to help improve instability. Persisting lactic acidosis may help identify uncontrolled shock. Recommendation 2. We suggest delaying EN if shock is uncontrolled and haemodynamic and tissue perfusion goals are not reached, but start low dose EN as soon as shock is controlled with fluids and vasopressors/inotropes (Grade 2D). {#Sec18} -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **[Question 3:]{.ul}** **Should we delay EN in patients with:** A.**Hypoxaemia;**B.**Hypercapnia;**C.**Acidosis?** We found no direct evidence on these subquestions in the literature, and RCTs in this population are unlikely to become available. The rationale to withhold EN in patients with hypoxaemia, hypercapnia and acidosis is to limit oxygen consumption and CO~2~ production. However, the process of starving mobilises endogenous stores and is energy-consuming \[[@CR17]\]. Acidosis may represent persistent shock and possibly contribute to gut dysfunction. Identifying and treating the cause of shock has priority over the initiation of EN. Similarly, in uncontrolled life-threatening hypoxaemia and hypercapnia, EN should be delayed until the symptoms are resolving. In patients with acute lung injury, an RCT comparing trophic to full EN for up to 6 days was associated with less gastrointestinal intolerance when compared to full EN, without affecting ventilator-free days, infectious complications, physical function, or survival \[[@CR7], [@CR18]\]. There are no data suggesting EN in patients with chronic, subacute, compensated or permissive hypercapnia is unsafe or not feasible. Recommendation 3. We suggest delaying EN in case of uncontrolled life-threatening hypoxaemia, hypercapnia or acidosis, but using EEN in patients with stable hypoxaemia, and compensated or permissive hypercapnia and acidosis (Grade 2D). {#Sec19} ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **[Question 4:]{.ul}** **Should we delay EN in patients receiving neuromuscular blocking agents?** One prospective study was identified (Supplement 5, Table 4), reporting similar gastric emptying as measured by gastric residual volume (GRV) in sedated patients with or without concomitant use of neuromuscular blocking agents \[[@CR19]\]. The critical condition necessitating the use of neuromuscular blocking agents always needs to be considered, but these agents per se should not preclude EN. Analgosedation is known to slow gastric emptying \[[@CR20]\]. Increased rate of EN intolerance is expected in deeply sedated patients with/without concomitant use of neuromuscular blocking agents. Recommendation 4. We suggest that EN should not be delayed solely because of the concomitant use of neuromuscular blocking agents (Grade 2D). {#Sec20} --------------------------------------------------------------------------------------------------------------------------------------------- **[Question 5:]{.ul}** **Should we delay EN in patients receiving therapeutic hypothermia?** One case series study addressing EN during therapeutic hypothermia was identified \[[@CR21]\] (Supplement 5, Table 5). During therapeutic hypothermia, energy metabolism might be markedly reduced \[[@CR22], [@CR23]\] when shivering is prevented. The rationale to withhold EN during therapeutic hypothermia is based on the presumed decrease in gut motility due to hypothermia \[[@CR24], [@CR25]\] and required analgosedation \[[@CR20]\]. It has been suggested that EN could be successfully administered to these patients \[[@CR21]\]. Tolerance to enteral feeding was impaired during hypothermia, but improved during rewarming \[[@CR21]\]. Recommendation 5. We suggest starting low dose EEN in patients receiving therapeutic hypothermia and increase the dose after rewarming (Grade 2D). {#Sec21} -------------------------------------------------------------------------------------------------------------------------------------------------- **[Question 6:]{.ul}** **Should we delay EN in patients receiving extracorporeal membrane oxygenation (ECMO)?** No RCTs and no prospective cohort studies were identified. Four case series in adult patients with ECMO were assessed (Supplement 5, Table 6), suggesting that EN is feasible during ECMO. Recommendation 6. We suggest using EEN in patients receiving ECMO (Grade 2D). {#Sec22} ----------------------------------------------------------------------------- **[Question 7:]{.ul}** **Should we delay EN during prone position?** One prospective cross-over, one cohort and three case series studies were identified (Supplement 5, Table 7). Data on tolerance of EN in prone position are controversial. Observational studies found similar GRVs in prone and supine position \[[@CR26]\], whereas poor feeding tolerance was improved with semi-recumbent position during supine periods and prokinetics \[[@CR27], [@CR28]\]. Although no RCTs on EN tolerance during prone position are available, reported studies do not support withholding EN in prone position. Gastric emptying seems not to be significantly influenced by prone position and adverse events in most studies not increased. Recommendation 7. We suggest that EN should not be delayed solely because of prone positioning (Grade 2D). {#Sec23} ---------------------------------------------------------------------------------------------------------- **Remark:** We suggest considering early use of prokinetics followed by post-pyloric feeding in case of persisting gastric retention. **[Question 8:]{.ul}** **Should we delay EN in patients with traumatic brain injury?** We identified a Cochrane review with two updates and one recent meta-analysis, comparing early vs. late feeding, independent on the route of nutrition (EN or PN) (Supplement 5, Table 8C). We identified three RCTs comparing EEN vs. early PN, three RCTs comparing EEN vs. delayed EN (one with restricted randomisation), and one RCT comparing early PN vs. delayed EN (Supplement 5, Table 8A). **[Question 8A:]{.ul}** **EEN vs. early PN** Three RCTs (116 patients) were included. EEN compared to early PN in patients with traumatic brain injury did not affect mortality (RR 1.91; 95% CI 0.59--6.18; *P* = 0.279; *I* ^2^ = 0%) or the risk of pneumonia (RR 1.23; 95% CI 0.79--1.90; *P* = 0.36; *I* ^2^ = 0%). The certainty of evidence for mortality outcome was **low**, for pneumonia it was **very low**. We rated down for risk of bias and imprecision (Table [3](#Tab3){ref-type="table"}). Supplement 7, Fig. 5. **[Question 8B:]{.ul}** **EEN vs. delayed EN** For *mortality*, two RCTs (86 patients) were included. EEN did not affect mortality compared to delayed EN (RR 0.66; 95% CI 0.18--2.45; *P* = 0.53; *I* ^2^ = 0%). The certainty of evidence was **low**. We rated down for imprecision (Table [3](#Tab3){ref-type="table"}). For *pneumonia*, three RCTs (118 patients) were included. EEN did not affect the risk of pneumonia compared to delayed EN (RR 0.86; 95% CI 0.55--1.35; *P* = 0.51; *I* ^2^ = 0%). The certainty of evidence was **very low**. We rated down for risk of bias and imprecision (Table [3](#Tab3){ref-type="table"}). Supplement 7, Fig. 6. In addition to RCTs, five cohort studies addressing this question were identified (Supplement 5, Table 8B). Existing evidence did not allow determining or excluding any benefit or harm of EEN, therefore our recommendation is based on expert opinion. Recommendation 8. We suggest using EEN in patients with traumatic brain injury (Grade 2D). {#Sec24} ------------------------------------------------------------------------------------------ **[Question 9:]{.ul}** **Should we delay EN in patients with stroke (haemorrhagic or ischaemic)?** We identified two RCTs in patients with ischaemic stroke and one retrospective study in patients with hypertensive intracerebral haemorrhage (Supplement 5, Tables 9A, B). One small RCT compared early vs. delayed EN and reported amelioration of cell-mediated immunity \[[@CR29]\]; however, both groups received PN to meet caloric targets from day 1. A large RCT compared EEN ("as soon as possible") to no nutrition within 7 days and reported a trend towards reduction of long-term mortality (6 months) with EN, with an increased risk of poor neurologic outcome in survivors \[[@CR30]\]. An observational study reported reduction in infectious complications with EEN vs. delayed EN \[[@CR31]\]. Recommendation 9. We suggest using EEN in patients with stroke (ischaemic or haemorrhagic) (Grade 2D). {#Sec25} ------------------------------------------------------------------------------------------------------ **[Question 10:]{.ul}** **Should we delay EN in patients with spinal cord injury?** One RCT addressed EEN (\<72 h) vs. delayed EN in cervical spinal injury \[[@CR32]\]. No differences in outcome variables were identified. One retrospective cohort study addressed safety of EN early after spinal cord injury and reported no major complications \[[@CR33]\] (Supplement 5, Tables 10A, B). Recommendation 10. We suggest using EEN in patients with spinal cord injury (Grade 2D). {#Sec26} --------------------------------------------------------------------------------------- **[Question 11:]{.ul}** **Should we delay EN in patients with severe acute pancreatitis (SAP)?** We identified five systematic reviews with meta-analyses comparing EN to PN while not considering timing (Supplement 5, Table 11B). All meta-analyses concluded that EN was beneficial in reducing infections and three reported reduced mortality \[[@CR3], [@CR34], [@CR35]\]. We identified five RCTs addressing EEN ("early" as defined by the authors) vs. early PN in SAP whereas only two studies defined "early" as \<48 h. Three further RCTs addressed EEN vs. early PN and one RCT EEN vs. delayed EN in "predicted SAP". Two RCTs addressing acute pancreatitis independent of severity and one RCT studying mixed patients undergoing abdominal surgery were not included. Supplement 5, Table 11A. We performed three separate meta-analyses all comparing EEN vs. early PN: (A) SAP and "early" as defined by the authors of the original study; (B) predicted SAP and "early" as defined by the authors of the original study; (C) predicted SAP and early defined as \<48 h. **[Question 11A:]{.ul}** **SAP (as stated by the authors). Early ("early" as defined by the authors) EN vs. PN** For *mortality* we included five RCTs (283 patients). EEN did not reduce the risk of death compared to PN (RR 0.57; 95% CI 0.23--1.38; *P* = 0.21; *I* ^2^ = 35.1%). The certainty of evidence was **low**. We rated down for imprecision (Table [3](#Tab3){ref-type="table"}). For *any infections* we included five RCTs (283 patients). EEN reduced the risk of infections compared to PN (RR 0.48; 95% CI 0.23--0.98; *P* = 0.045; *I* ^2^ = 76%). The certainty of evidence was **low**. We rated down for inconsistency and imprecision (Table [3](#Tab3){ref-type="table"}). For *pancreatic infections* we included four RCTs (233 patients). EEN reduced the risk of pancreatic infections compared to PN (RR 0.33; 95% CI 0.21--0.52; *P* \< 0.0001; *I* ^2^ = 0%) The certainty of evidence was **low**. We rated down for risk of bias and imprecision (Table [3](#Tab3){ref-type="table"}). Supplement 7, Fig. 7. **[Question 11B:]{.ul}** **Predicted SAP. Early ("early" as defined by the authors) EN vs. PN** For *mortality* we included eight RCTs (417 patients). EEN did not reduce the risk of death compared to PN (RR 0.50; 95% CI 0.22--1.13; *P* = 0.09; *I* ^2^ = 38%). The certainty of evidence was **low**. We rated down for imprecision (Supplement 6). For *any infections* we included eight RCTs (417 patients). EEN reduced the risk of infections compared to PN (RR 0.53; 95% CI 0.30--0.91; *P* = 0.023; *I* ^2^ = 63.5%). The certainty of evidence was **low**. We rated down for risk of bias and inconsistency (Supplement 6). For *pancreatic infections* we included five RCTs (202 patients). The use of EEN reduced the risk of pancreatic infections compared to PN (RR 0.35; 95% CI 0.24--0.52; *P* \< 0.0001; *I* ^2^ = 0%). The certainty of evidence was **low**. We rated down for risk of bias and imprecision (Supplement 6). Supplement 7, Fig. 8. **[Question 11C:]{.ul}** **Predicted SAP. Early (\<48 h) EN vs. PN** For *mortality* we included five RCTs (232 patients). EEN (\<48 h) did not reduce the risk of death compared to PN (RR 0.61; 95% CI 0.15--2.55; *P* = 0.50; *I* ^2^ = 41%). The certainty of evidence was **low**. We rated down for imprecision (Supplement 6). For *any infections* we included five RCT (232 patients), EEN (\<48 h) reduced the risk of infections compared to PN (RR 0.49; 95% CI 0.28--0.83; *P* = 0.008, *I* ^2^ = 9%). The certainty of evidence was **low**. We rated down for risk of bias, inconsistency and imprecision (Supplement 6). For *pancreatic infections* we included three RCTs (167 patients). EEN (\<48 h) reduced the risk of pancreatic infections compared to PN (RR 0.40; 95% CI 0.22--0.73; *P* = 0.003; *I* ^2^ = 0%). The certainty of evidence was **low**. We rated down for risk of bias and imprecision (Supplement 6). Supplement 7, Fig. 9. Taken together, the studies in different subpopulations have demonstrated a reduction of infections but no convincing effect of EEN on mortality. Recommendation 11. We suggest using EEN in patients with severe acute pancreatitis (Grade 2C). {#Sec27} ---------------------------------------------------------------------------------------------- **[Question 12:]{.ul}** **Should we delay EN in patients after GI surgery?** Out of three published meta-analyses \[[@CR36]--[@CR38]\] addressing early postoperative feeding including early oral diet, the two more recent papers \[[@CR36], [@CR37]\] reached different conclusions: reduced mortality and length of stay (LOS) but increased risk of vomiting analysing 15 RCTs \[[@CR37]\] vs. no difference in mortality and LOS, but reduced complications in early group from 13 RCTs \[[@CR36]\]. We identified three RCTs comparing early vs. delayed EN after emergency GI surgery and six RCTs in elective GI surgery. Two RCTs compared EEN vs. early PN in patients after elective GI surgery (Supplement 5, Table 12). **[Question 12A:]{.ul}** **Emergency GI surgery. EEN vs delayed EN** Three RCTs (343 patients) were included. EEN did not affect mortality compared to delayed EN (RR 0.80; 95% CI 0.46--1.40; *P* = 0.44; *I* ^2^ = 0%). EEN reduced the risk of infections compared to delayed EN (RR 0.61; 95% CI 0.40--0.93; *P* = 0.02; *I* ^2^ = 0%). The certainty of evidence was **low**. We rated down for risk of bias and imprecision (Table [3](#Tab3){ref-type="table"}). Supplement 7, Fig. 10. **[Question 12B:]{.ul}** **Elective GI surgery. EEN vs. delayed EN** For *mortality* three RCTs (346 patients) were included. EEN did not affect mortality compared to delayed EN in patients after elective GI surgery (RR 0.83; 95% CI 0.25--2.81; *P* = 0.77; *I* ^2^ = 17%). The certainty of evidence was **low**. We rated down for imprecision (Table [3](#Tab3){ref-type="table"}). For *any infections* six RCTs (432 patients) were included. EEN reduced the risk of infections compared to delayed EN (RR 0.43; 95% CI 0.23--0.82; *P* = 0.01; *I* ^2^ = 46%). The certainty of evidence was **low**. We rated down for risk of bias and imprecision (Table [3](#Tab3){ref-type="table"}). Five RCTs (404 patients) reported *anastomotic leak*. EEN reduced the risk of surgical leak compared to delayed EN (RR 0.43; 95% CI 0.20--0.93; *P* = 0.03; *I* ^2^ = 0%). The certainty of evidence was **low**. We rated down for imprecision (Table [3](#Tab3){ref-type="table"}). Supplement 7, Fig. 11. **[Question 12C:]{.ul}** **Elective GI surgery. EEN vs early PN** Two RCTs (440 patients) were included. EEN did not reduce the risk of pneumonia compared to early PN (RR 0.59; 95% CI 0.31--1.14; *P* = 0.12, *I* ^2^ = 0%), but reduced the risk of anastomotic leak compared to early PN (RR 0.42; 95% CI 0.19--0.95; *P* = 0.04; *I* ^2^ = 63%). The certainty of evidence was **low**. We rated down for risk of bias, inconsistency and imprecision (Table [3](#Tab3){ref-type="table"}). Supplement 7, Fig. 12. Recommendation 12. We suggest using EEN in patients after GI surgery (Grade 2C). {#Sec28} -------------------------------------------------------------------------------- **[Question 13:]{.ul}** **Should we delay EN in patients after abdominal aortic surgery?** No RCTs but two cohort studies were identified (Supplement 5, Table 13). Cohort studies both in elective \[[@CR39]\] and emergency repair \[[@CR40]\] did not compare EEN with any of our comparators, but showed that EEN was successful in a minority of patients. A multimodal approach has been proposed \[[@CR41]\], including early removal of nasogastric tubes, immediate postoperative mobilisation early oral or enteral feeding, accepting GRV up to 500 ml and use of prokinetics. Although these patients are at risk of bowel ischaemia with prevalence reported between 7 and 17% \[[@CR42], [@CR43]\], the risk itself should not lead to withholding EN, unless bowel ischaemia is suspected (see also Recommendation 15). Recommendation 13. We suggest using EEN in patients after abdominal aortic surgery (Grade 2D). {#Sec29} ---------------------------------------------------------------------------------------------- **[Question 14:]{.ul}** **Should we delay EN in patients with abdominal trauma?** Ten RCTs and ten cohort studies addressing EEN in trauma patients (RCTs: within 6--48 h; cohort studies: within 12--96 h) were identified, but abdominal trauma specifically was addressed in six RCTs, four of them compared EEN to early PN and two EEN to delayed EN (Supplement 5, Table 14A). **[Question 14A:]{.ul}** **EEN vs early PN** For *mortality* two RCTs (142 patients) were included. EEN did not affect mortality compared to early PN (RR 0.49; 95% CI 0.09--2.69; *P* = 0.41; *I* ^2^ = 0%). The certainty of evidence was **very low**. We rated down for risk of bias and imprecision (Table [3](#Tab3){ref-type="table"}). For *any infection* four RCTs (219 patients) were included. EEN did not affect the risk of infections compared to early PN (RR 0.59; 95% CI 0.24--1.42; *P* = 0.24; *I* ^2^ = 59%). The certainty of evidence was **very low**. We rated down for risk of bias, inconsistency and imprecision (Table [3](#Tab3){ref-type="table"}). Supplement 7, Fig. 13. **[Question 14B:]{.ul}** **EEN vs delayed EN** Two RCTs (101 patients) were included. EEN did not affect mortality compared to delayed EN (RR 0.74; 95% CI 0.18--3.11; *P* = 0.708). The certainty of evidence was **very low**. We rated down for risk of bias and imprecision (Table [3](#Tab3){ref-type="table"}). EEN did not affect the risk of infections compared to delayed EN (RR 0.83; 95% CI 0.41--1.70; *P* = 0.837). The certainty of evidence was **very low**. We rated down for risk of bias, inconsistency and imprecision (Table [3](#Tab3){ref-type="table"}). See Supplement 7, Fig. 14. Of note, earlier studies in this patient group almost exclusively used surgical jejunostomy for EN. Existing evidence did not allow verifying or excluding any benefit or harm of EEN; therefore our recommendation is based on expert opinion. In addition to RCTs, nine observational studies were identified (Supplement 5, Table 14B). An earlier meta-analysis in adult trauma patients in ICU (not specifically abdominal trauma) showed survival benefit in EEN commenced within 24 h after trauma \[[@CR44]\]. Recommendation 14. We suggest using EEN in patients with abdominal trauma when the continuity of the GI tract is confirmed/restored (Grade 2D). {#Sec30} ----------------------------------------------------------------------------------------------------------------------------------------------- **[Question 15:]{.ul}** **Should we delay EN in patients with bowel ischaemia?** We identified no clinical studies, but physiological knowledge and common sense support withholding EN in patients with overt bowel ischaemia. However, patients with endoscopic evidence of mild to moderate large bowel mucosal ischaemia, without signs of transmural ischaemia or bowel distension, might profit from low dose EN. In this case we support considering EN. In a recent retrospective study, survivors were more often fed enterally before the diagnosis of acute mesenteric ischaemia, but no independent association between EN and mortality was demonstrated \[[@CR45]\]. Recommendation 15. We suggest delaying EN in patients with overt bowel ischaemia (Grade 2D). {#Sec31} -------------------------------------------------------------------------------------------- **[Question 16:]{.ul}** **Should we delay EN in critically ill adult patients with intestinal fistula?** We identified one retrospective cohort study and two case series, all showing outcome benefit of "early" EN (Supplement 5, Table 16). However, "early" was defined as EN started within 7 days or 14 days of admission. Retrospective design further diminishes the importance of these studies. Intolerance of EN or increasing fistula output causing skin breakdown or fluid/electrolyte imbalance are evident reasons to decrease or discontinue EN \[[@CR46]\]. Recommendation 16. We suggest delaying EN in patients with high-output intestinal fistula if reliable feeding access distal to the fistula is not achievable (Grade 2D). {#Sec32} ------------------------------------------------------------------------------------------------------------------------------------------------------------------------ **[Question 17:]{.ul}** **Should we delay EN in patients with an open abdomen?** Seven observational studies (one prospective cohort study, three retrospective cohort studies and four case series) were identified; two studies compared EEN (different definitions) vs delayed EN and reported higher rate of early abdominal closure, less fistula formation and lower incidence of ventilator-associated pneumonia in the "early" EN group (Supplement 5, Table 17). The largest study comparing EN to no EN in patients with open abdomen after abdominal trauma reported independent associations between EN and ultimate fascial closure and decreased mortality rate in patients without bowel injury, but no difference in a subgroup of patients with bowel injury \[[@CR47]\]. Recommendation 17. We suggest using EEN in patients with open abdomen (Grade 2D). {#Sec33} --------------------------------------------------------------------------------- **[Question 18:]{.ul}** **Should we delay EN in patients with intra-abdominal hypertension?** Four observational studies were identified (Supplement 5, Table 18), only one addressed early vs. delayed EN \[[@CR48]\]. All studies reported high incidence of feeding intolerance associated with intra-abdominal hypertension, but data are not conclusive regarding causality. A recently published study demonstrated that EEN did not increase intra-abdominal pressure, but values exceeding 15 mmHg were associated with higher rates of feeding intolerance in patients with severe acute pancreatitis \[[@CR48]\]. No prospective study addressing EN in patients with abdominal compartment syndrome \[[@CR49]\] was identified. As abdominal compartment syndrome is an immediately life-threatening condition with jeopardized splanchnic perfusion, we suggest to withhold or stop EN and try to lower intra-abdominal pressure. Recommendation 18a. We suggest using EEN in patients with intra-abdominal hypertension without abdominal compartment syndrome, but consider temporary reduction or discontinuation of EN when intra-abdominal pressure values further increase under EN (Grade 2D). {#Sec34} ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Recommendation 18b. We suggest delaying EN in patients with abdominal compartment syndrome (Grade 2D). {#Sec35} ------------------------------------------------------------------------------------------------------ **[Question 19:]{.ul}** **Should we delay EN in patients with upper GI bleeding?** No studies addressing EEN were identified. One RCT in bleeding due to gastric or duodenal ulcer reported shorter hospital stay (4.2 ± 1.2 vs. 5.9 ± 1.4 days, *P* \< 0.001) in the early oral feeding group \[[@CR50]\]. EN as protection against stress ulceration and GI bleeding is suggested in one meta-analysis \[[@CR51]\], one retrospective study in burns \[[@CR52]\] and several reviews \[[@CR53]--[@CR55]\]. An RCT comparing ranitidine and sucralfate reported EN as an independently protective factor against GI bleeding \[[@CR56]\]. The main rationale to prohibit eating/EN is based on fear for disturbed visibility in a further endoscopy/intervention due to rebleeding. Therefore, delaying EN for 48--72 h in patients with a high risk of rebleeding has been suggested \[[@CR57]\]. Considering the absence of evidence to support this time frame, we suggest starting EN during the first 24--48 h after bleeding has been stopped; prolonged postponement of EN is unnecessary or even harmful because of increased risk of stress ulceration. Importantly, there is no evidence that fine-bore nasogastric tubes cause variceal bleeding \[[@CR57]\]. Recommendation 19. We suggest delaying EN in patients with active upper GI bleeding, and starting EN when the bleeding has stopped and no signs of rebleeding are observed (Grade 2D). {#Sec36} -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **[Question 20:]{.ul}** **Should we delay EN in patients with acute liver failure?** We could not identify any study in acute or acute-on-chronic liver failure patients. Some benefits of EN have been shown in patients with alcoholic hepatitis, malnourished patients with cirrhosis and patients with liver transplantation \[[@CR58]--[@CR60]\], where glycogen stores may be depleted after an overnight fast and metabolic conditions resemble prolonged starvation in healthy individuals \[[@CR61]\]. EN in fulminant acute liver failure has never been studied. These patients often present with hypoglycaemia, which should be corrected with intravenous glucose, sometimes together with insulin. Fulminant liver failure is associated with increased serum amino acid concentrations, especially glutamine \[[@CR62], [@CR63]\]. It seems likely that a failing liver is unable to provide effective metabolic support required for nutrition. The pathophysiological rationale to delay EN in fulminant hepatic failure would be to "spare" the severely injured liver from the duties of metabolising and storing nutrition during a period of stress and also to avoid additional increases in ammonia. Intravenous provision of nutrients except correction of hypoglycemia and appropriate provision of vitamins and trace elements may be futile or harmful early in the clinical course \[[@CR64]\]. Recommendation 20. We suggest starting low dose EN when acute, immediately life-threatening metabolic derangements are controlled with or without liver support strategies, independent on grade of encephalopathy (Grade 2D). {#Sec37} ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ **Remark:** Arterial ammonia levels should be monitored. **[Question 21:]{.ul}** **Should we delay EN in patients with large gastric aspirate volumes (GAV)?** We identified no study addressing this question. Based on existing evidence from two RCTs comparing the threshold volumes to stop already started EN \[[@CR65], [@CR66]\], a clear threshold volume (in ranges up to 500 ml) that increased the risk of ventilator-associated pneumonia was not identified. Measurements of GAV/GRV are not a gold standard and alternative methods (like ultrasound) can be applied to diagnose overfilling of the stomach. Gross distension of the stomach is likely to be undesirable and therefore we suggest that EN should be delayed when GAV/GRV is \>500 ml/6 h \[[@CR65]\], either for a limited time period or until administration of prokinetics. For patients with persistently large GAV/GRVs the use of postpyloric feeding should be considered rather than withholding EN, unless bowel ischaemia or obstruction is suspected (see also Recommendation 15). Recommendation 21. We suggest delaying EN if gastric aspirate volume is above 500 **ml/6** h (Grade 2D). {#Sec38} -------------------------------------------------------------------------------------------------------- **[Question 22:]{.ul}** **Should we delay EN in patients with absent bowel sounds?** One cohort study was identified \[[@CR67]\] (Supplement 5, Table 22). Bowel sounds are frequently absent in mechanically ventilated patients and this is associated with impaired outcome \[[@CR68]\]. The concept that bowel sounds must be present before initiation of enteral feeding is not based on evidence and should be abandoned \[[@CR69]\]. After laparotomy small intestinal motility is frequently preserved despite gastric and colonic paresis. The small intestine may contract silently (absence of gas), while feeding is well tolerated \[[@CR69]\]. Gastric and colonic paresis may effectively be treated with prokinetics \[[@CR70]\]. Initiation of EN in absence of bowel sounds might be associated with earlier return of bowel sounds, fewer episodes of vomiting, and shorter ICU and hospital stay \[[@CR67]\]. Recommendation 22. We suggest using EEN regardless of the presence of bowel sounds unless bowel ischaemia or obstruction is suspected (Grade 2D). {#Sec39} ------------------------------------------------------------------------------------------------------------------------------------------------- **[Question 23:]{.ul}** **Should we delay EN in patients with diarrhoea?** There were no studies testing delay of EN in case of diarrhoea, but diarrhoea is often considered as a reason to delay EN \[[@CR71]\]. Prevalence of diarrhoea in unselected ICU population is between 14 and 21% \[[@CR72], [@CR73]\]. Causes include impaired digestion/absorption, bacterial overgrowth or infection such as *Clostridium difficile*. Observational studies \[[@CR74], [@CR75]\] suggest that diarrhoea can be effectively managed with protocolised measures other than immediate cessation in EN. We recommend analysing the causes of diarrhoea and treat appropriately (e.g. *C. difficile* colitis). We also suggest considering treating bacterial overgrowth by selective decontamination, fibre-enriched or semi-elementary diet or digestive enzymes to reduce diarrhoea. Recommendation 23. We suggest using EEN in patients with diarrhoea (Grade 2D). {#Sec40} ------------------------------------------------------------------------------ Conclusions {#Sec15} =========== We suggest using EEN, initiated at a low rate, in the majority of critically ill patients; however, the evidence is weak. Beneficial effects in terms of infection prevention have been demonstrated in unselected critically ill patients, as well as in patients with severe acute pancreatitis and after GI surgery. However, we suggest delaying EN in patients with uncontrolled shock (haemodynamic and tissue perfusion goals are not met despite of fluids and vasopressors), uncontrolled hypoxaemia and acidosis, uncontrolled GI bleeding, overt bowel ischaemia (occlusive or non-occlusive), bowel obstruction (mechanical ileus), abdominal compartment syndrome, gastric aspirate volume \>500 ml/6 h or high-output fistula if reliable distal feeding access is not achievable. Electronic supplementary material ================================= {#Sec16} Below is the link to the electronic supplementary material. Supplement 1. Introduction and methods (DOC 85 kb) Supplement 2. Flow chart literature search and creation of evidence tables. (TIFF 1140 kb) Supplement 3. Search results summary (DOC 75 kb) Supplement 4. Search codes (DOC 36 kb) Supplement 5. Evidence tables (DOC 603 kb) Supplement 6. Evidence profiles for additional analyses (Q1C, Q1C, Q11B, Q11C) (DOC 86 kb) Supplement 7. Forest plots for Questions 1C, 1D, 8, 11, 12 and 14 (DOC 1516 kb) Supplement 8. Conflicts of interest (DOC 35 kb) Supplement 9. PRISMA Checklist (DOC 68 kb) Supplement 10. PRISMA study selection diagram for Question number 1. (DOC 29 kb) **Take-home message:** The administration of early EN appears to reduce infections and should be used for the majority of critically ill patients. However, there are certain situations when we recommend EN be delayed. Collaborators in ESICM Working Group on Gastrointestinal Function: Claudia Spies, Klinik für Anästhesiologie mit Schwerpunkt operative Intensivmedizin der Charité-Universitätsmedizin Berlin, Campus Virchow Klinikum, Berlin; Pietro Vecchiarelli, Intensive Care Unit, Ospedale Belcolle, Strada Sammartinese, Belcolle Hospital, Viterbo, Italy; Anne Berit Guttormsen, Department of Anesthesia and Intensive Care, Haukeland University Hospital, Bergen, Norway. The costs covering the open access publication of this article were covered by the International Fluid Academy (IFA). The IFA is integrated within the not-for-profit charitable organization iMERiT (International Medical Education and Research Initiative) under Belgian Law, and IFA website (<http://www.fluidacademy.org>) is an official SMACC (Social Media and Critical Care) affiliated site, based on the philosophy of FOAM (Free Open Access Medical Education). Conflicts of interest {#FPar1} ===================== See Supplement 8.

One of the visual system\'s most extraordinary feats is its ability to function across a billion-fold range of ambient illumination, from starlight to midday sun. To achieve this ability, the retina and downstream visual pathways have mechanisms in place that actively respond to the prevailing conditions. This is evident in the transition from rod- to cone-based vision, as well as sensitivity normalization of individual photoreceptors. Changes in the retinal network as a function of ambient light also optimize sensitivity or acuity according to current conditions through effects at the level of photoreceptors, inner retina, and ganglion cell layer. Such changes can be readily measured in a field potential measurement of retinal activity, the electroretinogram (ERG).^[@i1552-5783-57-14-6305-b01]^ An accurate independent measurement of ambient light intensity (irradiance) would be an obvious aid in defining the state of network adaptation. Intrinsically photosensitive retinal ganglion cells (ipRGCs), which express the photopigment melanopsin, encode ambient irradiance levels in their firing activity.^[@i1552-5783-57-14-6305-b02][@i1552-5783-57-14-6305-b03]--[@i1552-5783-57-14-6305-b04]^ Several lines of evidence link ipRGCs to regulation of the retinal network. Knocking out melanopsin in mice reduces circadian variation in cone ERG,^[@i1552-5783-57-14-6305-b05]^ while elimination of the M1 class of ipRGCs impairs growth of the cone ERG b-wave that is normally associated with light adaptation.^[@i1552-5783-57-14-6305-b06]^ Stimuli biased towards activating melanopsin reduce b-wave implicit time^[@i1552-5783-57-14-6305-b07]^ and b-wave amplitude^[@i1552-5783-57-14-6305-b08],[@i1552-5783-57-14-6305-b09]^ in the light-adapted ERG in humans and mice, respectively. An open question is how far upstream in the visual pathway do these effects reach? As the ERG b-wave is a read out of bipolar cell activity, the published data indicate that ipRGCs can influence the activity of the first postreceptoral cells; however, it remains unknown whether they also target the activity of photoreceptors themselves. Light-adapted ERG do not clarify this point, as the direct read out of photoreceptor activity (the a-wave) is typically unmeasurably low amplitude. Another limitation of previous studies is that, owing to the use of bright light as a modulator of melanopsin activity, previous data have preferentially examined the influence of ipRGCs over cone pathways. ipRGCs are responsive to dim light thanks to input from rods and cones, allowing the possibility that their influence on retinal circuits extends also to rod-evoked or dark-adapted responses in the retina across lower light intensities. Therefore, two outstanding questions are: 1) Can ipRGCs modulate retinal network all the way to the photoreceptor level? 2) Can ipRGCs affect retinal response at low light levels? Up to now, addressing these questions remained a challenge. In order to examine the ipRGCs\' contribution to retinal adaptation in visually intact animals, we previously used a silent-substitution approach in which spectrally distinct stimuli were used to differentially excite melanopsin (but remain equivalent for cone photoreceptors). This experimental approach requires light-adapted conditions and so precludes its use in answering the questions posed above. To this end, we adopted a chemogenetic approach^[@i1552-5783-57-14-6305-b10]^ in which hM3Dq Gq-coupled receptor is exclusively expressed in ipRGCs, allowing for their selective activation upon administration of clozapine *N*-oxide (CNO). We previously used this strategy in vivo to selectively excite ipRGCs.^[@i1552-5783-57-14-6305-b11]^ Using that approach in this study enabled us to specifically activate ipRGCs, independently of any light stimulation, and hence examine the impact of ipRGC activity on the scotopic ERG. We found that selective activation of ipRGCs suppressed both a- and b-wave amplitude and caused a shift in b-wave sensitivity. This reveals for the first time that ipRGCs are capable of modulating retinal activity all the way to the photoreceptors, affecting both rod and cone pathways. Materials and Methods {#s2} ===================== Animals {#s2a} ------- Animal care was in accordance with the UK Animals, Scientific Procedures Act of 1986,^[@i1552-5783-57-14-6305-b12]^ and the study was approved by the University of Manchester ethics committee. All experiments complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were kept in a 12L:12D cycle at a temperature of 22°C with food and water available ad libitum. Experiments were performed in adult (3--6 months old) *Opn4^Cre/+^* mice.^[@i1552-5783-57-14-6305-b13]^ Unilateral intravitreal injections of AAV2-hSyn-DIO-hM3Dq-mCherry vector (2.3 × 10^13^ genomic particles/mL; the Vector Core, University of North Carolina at Chapel Hill, NC, USA) were performed as previously reported^[@i1552-5783-57-14-6305-b11]^ and used hyaluronan lyase and heparinase III (200 U each) to maximize retinal penetration (total volume, 2.5 μL injected over 1 minute).^[@i1552-5783-57-14-6305-b14]^ Control mice underwent the same procedure, with injections including glycosydic enzymes and the virus-lacking hM3Dq receptor AAV2-hSyn-DIO-mCherry vector (1.2 × 10^13^ genomic particles/mL; UNC Vector Core). Mice were allowed at least 6 weeks to recover before being used in in vivo studies. Administration of CNO (Abcam, Cambridge, UK) was performed using an intraperitoneal (ip.) route at a dose of 5 mg/kg. Immunohistochemistry {#s2b} -------------------- Immunohistochemistry was performed as previously described^[@i1552-5783-57-14-6305-b11]^ in retinal wholemounts in methanol-free 4% paraformaldehyde. The primary antibodies used in these studies included rabbit anti-dsRed (product 632496; 1:1000 dilution; Clontech, Saint-Germain-en-Laye, France) and chicken anti-GFP (product ab13970; 1:1000 dilution; Abcam). The secondary antibodies were Alexa 488 conjugated donkey anti-chicken (Jackson Immunoresearch, Bar Harbor, ME, USA) and Alexa 546 conjugated donkey antirabbit (Life Technologies, Corp., Carlsbad, CA, USA) at 1:200 dilution. Images were collected using a model TCS SP5 AOBS inverted confocal microscope (Leica, Wetzlar, Germany), using a 40×/0.50 plan Fluotar objective and 1.5× confocal zoom. Pupillometry {#s2c} ------------ Pupils were filmed in the dark under infrared illumination (light-emitting diode \>900 nm), using a video camera (RoleraXR; QImaging, Surrey, Canada) in unanaesthetized and gently restrained mice before and after CNO injection (5 mg/kg, ip.). Videos were analyzed using ImageJ software (<http://imagej.nih.gov/ij/>; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). The areas of the pupil after CNO administration were expressed relative to the area of the pupil before CNO injection. One-sample *t* test was performed to determine whether pupil constriction was significant after administration of CNO. Electroretinography {#s2d} ------------------- Electroretinograms were recorded from *Opn4^Cre/+^* mice. Anesthesia was induced with an intraperitoneal injection of ketamine (100 mg/kg; Narketan-10, 100 mg/mL; Vetoquinol, Buckingham, UK) and xylazine (10 mg/kg; Rompun; 2% w/v, Bayer, Germany). A topical mydriatic (tropicamide 1%; Chauvin Pharmaceuticals, Kingston-upon-Thames, Surrey, UK) and mineral oil (Sigma-Aldrich, Dorset, UK) were applied to the recording eye before placement of a corneal contact lens-type electrode. Mice were placed in a stereotaxic frame to keep a fixed head position; a bite bar was also used for head support and acted as a ground. A needle reference electrode (Ambu Neuroline; Ambu, Ballerup, Denmark) was inserted approximately 5 mm from the base of the contralateral eye. Electrodes were connected to a PC (Windows operating system; Microsoft, Redmond, WA, USA) through a signal conditioner (model 1902 Mark III; Cambridge Electronic Design, Cambridge, UK), which differentially amplified (×3000) and filtered (band-pass filter cutoff of 0.5--200 Hz) the signal, and a digitizer (model 1401; Cambridge Electronic Design, Cambridge, UK). Core body temperature was maintained at 37°C throughout recording with a homeothermic heat mat (Harvard Apparatus, Cambridge, UK). Heart rate was monitored during the ERG recording and found no differences between hM3Dq *Opn4^Cre/+^* and control *Opn4^Cre/+^* mice (average heart rate, 442 ± 76 and 420 ± 44 beats per second in hM3Dq and control mice, respectively; unpaired two-tailed *t* test; *P* = 0.81). Dark-adapted ERGs were measured in response to a 15-ms flash from a warm white light-emitting diode light source (Thorlabs Inc, Newton, NJ, USA). Neutral density filters (Edmund Optics Inc., UK) placed in the light path attenuated light in 1-log-unit increments, producing corneal irradiances spanning a 9-log-unit range (6.60--14.60 log photons/cm^2^/s). Interflash intervals ranged from 1.5 seconds at the dimmest to 30 seconds at brightest intensities. The ERG a-wave amplitude and b-wave amplitudes and implicit times were measured relative to baseline values (time of flash onset). Oscillatory potentials were extracted with a band-pass filter (80--200 Hz). The peaks of the first four oscillatory potentials were measured and summed to generate the total oscillatory potential (OP) amplitude. 2-way ANOVAs were used to test for differences in a- and b-wave amplitude, latency, or total OP amplitudes, with post hoc Bonferroni corrections, to compare responses between hM3Dq and control mice, either with vehicle or CNO treatments. For a- and b-wave amplitudes and total OP amplitudes, sigmoidal dose-response curves were fitted to irradiance response relationships. An *F* test comparison was then used to determine whether functions were best fitted with the same or two separate sigmoidal curve(s). Paired-flash ERG recording was done at the brightest light intensity (14.60 log photons/cm^2^/s), separating the two 10-ms flashes with 700-ms interstimulus interval. 2-way ANOVAs were used to test for differences in b-wave amplitudes of first and second flash, as well as in Δb-wave amplitude between first and second flash, with post hoc Bonferroni corrections, to compare responses between hM3Dq and control mice, either with vehicle or CNO treatments. Results {#s3} ======= Using Chemogenetics To Acutely and Selectively Activate ipRGCs {#s3a} -------------------------------------------------------------- Using viral delivery techniques, we aimed to drive targeted expression of excitatory hM3Dq in ipRGCs and thus modulate their activity in vivo (this approach is validated in reference 11). In *Opn4^Cre/+^* mice, which express cre recombinase only in melanopsin-expressing cells, we have driven cell-specific gene expression of the hM3Dq receptor via intravitreal delivery of "double-floxed" inverted open reading frame (DIO) AAV2 vector with hM3Dq-mCherry sequence. The hM3Dq receptor is a Gq-coupled receptor derived from the human muscarinic M3 acetylcholine receptor carrying two-point mutations, making it insensitive to endogenous acetylcholine but potently activated by a pharmacologically inert drug, CNO (10). Targeted expression of hM3Dq is shown in [Figure 1](#i1552-5783-57-14-6305-f01){ref-type="fig"}A, where mCherry is an hM3Dq receptor\'s tag and GFP is a marker for ipRGCs (transduction efficiency of ∼30%). {#i1552-5783-57-14-6305-f01} To validate the functional consequences of hM3Dq expression in ipRGCs in vivo, we used a well-known function of ipRGC activity, the pupillary light reflex. We predicted that an intraperitoneal injection of CNO under dark-adapted conditions would result in ipRGC activation and, in turn, pupillary constriction. In mice that had received a unilateral injection of the hM3Dq-mCherry viral vector, we recorded the pupil size of injected eyes in complete darkness. We found that significant pupil constriction was detected as soon as 10 minutes after intraperitoneal injection of CNO (5 mg/kg; one-sample *t*-test; *P \<* 0.0001). This effect plateaued and lasted for more than an hour (one-sample *t*-test; *P \<* 0.0001), with a partial recovery ∼4 hours later ([Fig. 1](#i1552-5783-57-14-6305-f01){ref-type="fig"}B, [1](#i1552-5783-57-14-6305-f01){ref-type="fig"}C). This time frame was sufficient to examine the impact of ipRGC activity on ERG recordings used in this study. No such constriction was apparent in control *Opn4^Cre/+^* (mCherry only-expressing) mice ([Fig. 1](#i1552-5783-57-14-6305-f01){ref-type="fig"}B). Impact of Chemogenetic Activation of ipRGCs on Scotopic Flash ERG {#s3b} ----------------------------------------------------------------- In order to examine the effect of selective activation of ipRGCs on photoreceptors, we recorded dark-adapted flash ERGs from mice that had received unilateral intravitreal injections with virus-carrying hM3Dq-mCherry (hM3Dq *Opn4^Cre/+^* mice; *n* = 4) or mCherry sequence alone (control *Opn4^Cre/+^* animals; *n* = 5). Following 12 hours of dark adaptation, we proceeded to record dark-adapted ERGs from these mice in response to a 15-ms full-field flash across a 9-fold range of irradiances (6.60--14.60 log photons/cm^2^/s). Importantly, the pupil constriction caused by chemogenetic activation of ipRGCs was antagonized by artificially dilating the pupils with mydriatic eye drops (1% tropicamide; pupil size was measured after completion of all experiments and showed no confounding influence of pupil size on our ERG results) ([Fig. 1](#i1552-5783-57-14-6305-f01){ref-type="fig"}D, [1](#i1552-5783-57-14-6305-f01){ref-type="fig"}E). Prior to CNO injection, both a- and b-wave amplitudes showed typical irradiance response functions ([Fig. 2](#i1552-5783-57-14-6305-f02){ref-type="fig"}A--C), which were statistically similar in hM3Dq *Opn4^Cre/+^* and control *Opn4^Cre/+^* animals (a-wave: 2-way ANOVA with post hoc Bonferroni correction; *P* \> 0.99; *F* test: *P* = 0.96; b-wave: 2-way ANOVA with post hoc Bonferroni correction; *P* \> 0.99; *F* test: *P* = 0.67). Next, while maintaining dark-adapted conditions, we injected mice with CNO (5 mg/kg) to excite ipRGCs. Following a period of 30 minutes for stabilization/further dark adaptation, we repeated the above protocol ([Fig. 2](#i1552-5783-57-14-6305-f02){ref-type="fig"}A). Following CNO injection, we found a marked difference between the ERGs recorded in hM3Dq *Opn4^Cre/+^* and control *Opn4^Cre/+^*animals. Statistical analyses revealed a significant decrease in a-wave amplitudes in hM3Dq *Opn4^Cre/+^* mice compared to that in controls injected with CNO ([Fig 2](#i1552-5783-57-14-6305-f02){ref-type="fig"}D) (2-way ANOVA finds significant effect of irradiance (*P* \< 0.0001), treatment (*P* \< 0.0001), and interaction (*P* \< 0.05); *F* test comparing sigmoidal dose response curves finds data are better fit with two separate functions; *P \<* 0.0001). These effects were found to be significantly divergent at irradiances \>10^13^ photons/cm^2^/s (post hoc Bonferroni correction, *P \<* 0.01). Similarly, there was a significant reduction in b-wave amplitudes in hM3Dq *Opn4^Cre/+^* mice compared to controls following CNO injection ([Fig. 2](#i1552-5783-57-14-6305-f02){ref-type="fig"}E) (2-way ANOVA finds significant effect of irradiance (*P* \< 0.0001), treatment (*P* \< 0.0001), and interaction (*P* \< 0.05); *F* test, comparing sigmoidal dose response curves: *P* \< 0.0001). Bonferroni post hoc analyses revealed these effects were significantly different at irradiances \>10^10^ photons/cm^2^/s. {#i1552-5783-57-14-6305-f02} To assess whether there was also a shift in sensitivity in hM3Dq *Opn4^Cre/+^* mice upon CNO treatment, we also examined the irradiance-response curve of a- and b-wave amplitudes normalized to the maximum response ([Fig. 2](#i1552-5783-57-14-6305-f02){ref-type="fig"}F, [2](#i1552-5783-57-14-6305-f02){ref-type="fig"}G). Normalized irradiance-response functions for a-wave amplitude could be fitted with the same sigmoidal curve ([Fig. 2](#i1552-5783-57-14-6305-f02){ref-type="fig"}F) (*F* test, *P* \> 0.05). However, those for b-wave amplitude could not ([Fig. 2](#i1552-5783-57-14-6305-f02){ref-type="fig"}G) (*F* test, *P* = 0.004). Thus, although chemogenetic activation of ipRGCs had no significant impact on a-wave sensitivity, b-wave sensitivity was substantially reduced, with more than a log difference in half-maximal effective irradiance between the conditions (3.52 × 10^9^ and 5.21 × 10^10^ photons/cm^2^/s for vehicle and CNO, respectively). We also examined the effects of CNO on the timing of the ERG. Interestingly, there were no differences in a- and b-wave implicit times between conditions, either before or after CNO injection ([Fig. 2](#i1552-5783-57-14-6305-f02){ref-type="fig"}G--J) (2-way ANOVA with post hoc Bonferroni correction, *P* \> 0.05). Flash ERGs also drive high-frequency, low-amplitude wavelets that were superimposed on the ascending b-wave of ERG, termed OPs.^[@i1552-5783-57-14-6305-b15]^ Although the exact origin of OPs is still unclear, it is generally believed that they are mainly generated in the inner retina by neural interactions among bipolar, amacrine, and ganglion cells. We examined the amplitude of total OPs (sum of the first four oscillatory potentials) before (vehicle) and after CNO administration ([Fig. 3](#i1552-5783-57-14-6305-f03){ref-type="fig"}A). Prior to CNO injection, total OP amplitudes were statistically similar in hM3Dq *Opn4^Cre/+^* and control *Opn4^Cre/+^* animals (2-way ANOVA with post hoc Bonferroni correction, *P* \> 0.99; *F* test *P* = 0.64) ([Fig. 3](#i1552-5783-57-14-6305-f03){ref-type="fig"}B). After CNO injection, we found a reduction in total OP amplitude in hM3Dq *Opn4^Cre/+^* mice compared to controls (2-way ANOVA finds significant effect of irradiance, *P \<* 0.0005) and treatment (*P \<* 0.0005; *F* test, *P \<* 0.005) ([Fig. 3](#i1552-5783-57-14-6305-f03){ref-type="fig"}C). {#i1552-5783-57-14-6305-f03} Effects on Dark-Adapted Cone ERG {#s3c} -------------------------------- Published work provides evidence that ipRGCs regulate light-adapted cone ERG.^[@i1552-5783-57-14-6305-b07],[@i1552-5783-57-14-6305-b09]^ To examine whether a similar influence was apparent under dark-adapted conditions, we recorded paired-flash ERGs. This approach exploits the differing recovery kinetics of rods and cones to separate their relative contribution to the ERG.^[@i1552-5783-57-14-6305-b01]^ Here, we presented two flashes of equal intensity (14.60 log photons/cm^2^/s) separated by 700 ms. The first flash should excite both rods and cones, but due to the poorer recovery kinetics of rods, the second flash should be predominantly cone driven, thus allowing for isolation of a cone response. Comparing the amplitude of the responses to the first and second flashes, therefore, revealed changes arising with cone or both rod and cone responses ([Fig. 4](#i1552-5783-57-14-6305-f04){ref-type="fig"}A). {#i1552-5783-57-14-6305-f04} Prior to CNO injection, we saw a characteristic decrease in amplitude of the second flash compared to the first, consistent with the goal of isolating cone responses in the second flash ([Fig. 4](#i1552-5783-57-14-6305-f04){ref-type="fig"}A). b-Wave amplitudes were statistically similar between hM3Dq *Opn4^Cre/+^* and control *Opn4^Cre/+^* animals for both first and second flashes (first flash: 2-way ANOVA with post hoc Bonferroni correction, *P* \> 0.99; second flash: 2-way ANOVA with post hoc Bonferroni correction, *P* = 0.89) ([Fig. 4](#i1552-5783-57-14-6305-f04){ref-type="fig"}A, [4](#i1552-5783-57-14-6305-f04){ref-type="fig"}B). Following CNO injection, for the first flash, we observed a reduction in b-wave amplitude in hM3Dq *Opn4^Cre/+^* mice compared to controls, as we observed in response to single flashes (2-way ANOVA with post hoc Bonferroni correction, *P* = 0.035) ([Fig. 4](#i1552-5783-57-14-6305-f04){ref-type="fig"}A, [4](#i1552-5783-57-14-6305-f04){ref-type="fig"}B). Responses to the second flash also showed a significant difference in amplitude between hM3Dq *Opn4^Cre/+^* and control *Opn4^Cre/+^* mice (2-way ANOVA with post hoc Bonferroni correction, *P* = 0.004), indicating an influence of CNO on cone pathways ([Fig. 4](#i1552-5783-57-14-6305-f04){ref-type="fig"}A, [4](#i1552-5783-57-14-6305-f04){ref-type="fig"}C). In addition, we analyzed the rod response by assessing the differences in b-wave amplitude between first and second flash. Unsurprisingly, following CNO injection, we observed a significant reduction of Δb-wave amplitude in hM3Dq *Opn4^Cre/+^* mice compared to control animals (2-way ANOVA with post hoc Bonferroni correction, *P* = 0.025). Excitation of ipRGCs thus has substantial effects on the activity of both rod and cone pathways. Discussion {#s4} ========== By combining a targeted chemogenetic approach with electroretinography, we were able to examine the impact of selective activation of ipRGC activity on upstream retinal neurons under dark-adapted conditions. We found that the amplitude, but not timing, of photoreceptor (a-wave), bipolar cell (b-wave), and inner-retinal responses (OPs) were all reduced by acutely and selectively exciting ipRGCs. We further showed that selective activation of ipRGCs adjusted the amplitude of both rod- and cone-derived ERGs. The reduction in amplitude of the scotopic a-wave raises the exciting possibility that the influence of ipRGCs extends as far as rod (and maybe cone) photoreceptors. This is the first indication that ipRGCs could impact activity of the outer retina. The a-wave is typically regarded as reflecting the activity of the photoreceptors and, although a change in photoreceptor activity it is not the only plausible event that could reduce a-wave amplitude, is the most straightforward. If we accept that the a-wave reflects photoreceptor hyperpolarization and that the efficiency of photon capture is constant, then activation of ipRGCs could produce a smaller a-wave by either raising the floor or lowering the ceiling of the photoreceptor response. In other words, the influence of ipRGCs could be reducing the dark current (e.g., lowering resting membrane potentially by closure of cGMP-gated cation channels in the outer segment and/or the activity of a Na^+^/Ca^2+^,K^+^ (NCKX) exchange in the outer-segment or the activity of Na^+^/K^+^ pumps in the inner segment), or alternatively, by decreasing the magnitude of light response (by modulating the activity of some aspect of the phototransduction cascade).^[@i1552-5783-57-14-6305-b16][@i1552-5783-57-14-6305-b17]--[@i1552-5783-57-14-6305-b18]^ Interestingly, a very recent study showed that rod ion conductance is modulated by melanopsin phototransduction.^[@i1552-5783-57-14-6305-b19]^ In the mouse, under dark-adapted conditions and across the sensitivity range we used, the flash ERG is dominated by rods. Our data therefore imply an effect on rod photoreceptors, although a similar impact on cones is not excluded. Given that chemogenetic activation of ipRGCs modulates the activity of photoreceptors, it is not surprising that we also saw significant reductions in the amplitude of the b-wave and OPs. Thus, the most parsimonious explanation for these latter effects is that they are inherited from the reduced activity of the photoreceptors themselves. This could explain the reduced amplitude of the single-flash b-wave and OPs, although we noted that there was no change in a-wave sensitivity. Similarly, as we do not know whether the cone flash response is impacted in the same way by ipRGCs, it is not clear whether changes in the outer retina could also explain the reduced cone b-wave in our paired flash protocol. It thus remains entirely possible that ipRGCs impact the retinal network at multiple locations. ipRGC activation has been shown previously to modulate the light-adapted cone b-wave amplitude in mice^[@i1552-5783-57-14-6305-b08],[@i1552-5783-57-14-6305-b09]^ and implicit time in humans.^[@i1552-5783-57-14-6305-b07]^ Allen et al.^[@i1552-5783-57-14-6305-b09]^ found that the reduction in b-wave amplitude was correlated with a reduction in response amplitude in the mouse dorsal lateral geniculate nucleus (dLGN) in response to a full field flash. That result was explained by a change in spatial frequency tuning of a subset of dLGN neurons, which shifted to prefer higher spatial frequencies. That study proposed that the change in b-wave amplitude may therefore reflect the same event and be a consequence of more complex changes in the retinal network. In a similar way, the origins of the changes in these downstream responses we observe here could also have more complex origins that relate to higher-level retinal processes. Identifying the route(s) by which ipRGCs influence retinal physiology to produce these changes in the ERG is a fruitful area of future research. There are a number of examples of ipRGCs contacting other retinal neurons, both chemically and electrically.^[@i1552-5783-57-14-6305-b06],[@i1552-5783-57-14-6305-b20][@i1552-5783-57-14-6305-b21][@i1552-5783-57-14-6305-b22][@i1552-5783-57-14-6305-b23]--[@i1552-5783-57-14-6305-b24]^ However, to date this has been restricted to contacts with inner retinal neurons suggesting that such direct contacts are not sufficient to produce the range of effects we observe. An alternative explanation for our observed effects is a more global impact on the neuromodulatory environment of the retina, such as changes in inhibitory neuromodulators, signals of light adaptation, or even changes in the retinal pH, that could extend up to the photoreceptor level.^[@i1552-5783-57-14-6305-b25],[@i1552-5783-57-14-6305-b26]^ Although the intrinsic light response of ipRGCs (driven by melanopsin) has a low sensitivity, ipRGCs also receive signals from rods and cones.^[@i1552-5783-57-14-6305-b02]^ In this regard, it is perfectly feasible for ipRGCs to provide dynamic modulation of rod function even though this should occur over irradiances below the sensitivity threshold of melanopsin. The scotopic ERG is dominated by rods, especially at low flash intensities. The differences in ERG amplitude we saw here across the intensity range thus represent strong evidence that rod pathways are indeed targets of ipRGC signaling. Our paired flash protocol confirms that cone pathways are also affected (although in the absence of a measurable a-wave we cannot be sure that the effects extend to cone photoreceptors themselves). One is tempted to conclude that ipRGCs drive progressive changes in retinal circuits across a wide range of background light intensities. However, the chemogenetic manipulation, while allowing analytical examination of ipRGCs\' influence on the retinal network, also leaves the retina in an unnatural state in which ipRGCs are highly excited but the rest of the retina is kept under dark-adapted conditions. It may thus be that the changes we observe are reflections of the similar shift in the retinal physiology occurring in the natural conditions over a more limited range of irradiances. To summarize, our data add to the growing body of evidence that ipRGCs and melanopsin drive modulatory effects within the retina according to their independent measure of brightness. Using a chemogenetic approach, we have for the first time been able to reveal the impact of those effects in the scotopic ERG, demonstrating that the changes in retinal activity extend up to the photoreceptor level. Supported by European Research Council Grant 268970 (RJL) and Medical Research Council Confidence in Concept (MRC, CiC) (RJL, JC-K). Bioimaging facility microscopes used in this study were purchased with grants from Biotechnology and Biological Sciences Research Council (BBSRC), Wellcome Trust, and University of Manchester Strategic Fund. Disclosure: **N. Milosavljevic**, None; **A.E. Allen**, None; **J. Cehajic-Kapetanovic**, None; **R.J. Lucas**, None

1. Introduction {#sec1-jcm-09-00758} =============== Frequent malignancies like myeloma, kidney, breast or prostate cancer are known to have a high incidence of developing bony metastasis during the disease and the survival of these patients has improved steadily due to targeted treatment options. The proximal femur is the most frequent localization for bone metastases besides the spine. The risk of pathological fractures at the proximal femur is high due to weight-bearing and biomechanical conditions \[[@B1-jcm-09-00758],[@B2-jcm-09-00758],[@B3-jcm-09-00758]\]. Osteosyntheses by a locking plate, dynamic hip screw (DHS) or intramedullary nail, with or without cement augmentation, are still used most frequently for palliative treatment. Despite additional radiotherapy complications following intralesional procedures like persistent pain due to instability and local tumor progression, especially at the proximal femur, may often appear \[[@B4-jcm-09-00758],[@B5-jcm-09-00758],[@B6-jcm-09-00758]\]. Overall, there seem to be higher implant failure rates after osteosynthesis compared to endoprosthetic reconstruction already within the first year, further increasing with longer survival \[[@B7-jcm-09-00758],[@B8-jcm-09-00758]\]. In contrast to intralesional procedures, en-bloc resection of the metastasis and endoprosthetic reconstruction by modular devices offers the possibility to prevent local complications and can be performed for both curative and palliative treatment \[[@B9-jcm-09-00758],[@B10-jcm-09-00758],[@B11-jcm-09-00758]\]. Nevertheless, surgical treatment of metastasis, especially in palliative patients, is discussed controversially. It includes less invasive treatment by intralesional procedures as well as aggressive en-bloc resection with wide margins. Consecutively, the variety of implants ranges from osteosynthesis to modular tumor systems. Less data is available for a larger series of patients treated operatively with en-bloc resection of bone metastasis of the proximal femur and reconstruction with a modular tumor system. The aim of this study was to evaluate whether aggressive en-bloc resection and reconstruction by modular tumor prosthesis are effective and reliable even in advanced tumor stages. Survivorship data of the implant, clinical outcome and complication rates were therefore analyzed in a retrospective study with respect to a patient's survival in this cohort. 2. Patients and Methods {#sec2-jcm-09-00758} ======================= A consecutive series of 45 patients with en-bloc resection of metastasis of the proximal femur and reconstruction with a modular tumor endoprosthesis between 1997 and 2010 was evaluated retrospectively. 2.1. Inclusion Criteria and Surgical Algorithm {#sec2dot1-jcm-09-00758} ---------------------------------------------- All patients were treated according to a vote after presentation at an interdisciplinary tumor board preoperatively. Patients with the decision of surgery were treated with respect to individual patient's specifics according to the following surgical algorithm. Inclusion criteria for the current cohort with en-bloc resection and reconstruction with a modular tumor device were the presence of a tumor at the proximal femur with extension distally to the intertrochanteric line. Additional postoperative radiotherapy was planned in patients with intralesional procedures dependent on the pathological status of the specimen. As survival of the patients was not significantly different for patients with a solitary and disseminated disease we included all cases. In patients presenting a fracture or at a high risk of fracture (Mirels score of at least 9) and very poor prognosis of less than approximately 3--6 months, either a simple osteosynthesis with use of an intramedullary nail was performed when the tumor was located below the intertrochanteric zone (meta- and/or diaphysis) and the region of the femoral neck was intact. In localizations proximal to the intertrochanteric line and/or femoral neck region and in tumors with a good response to radiotherapy, a cemented primary stem after curettage was used. 2.2. Indication for Surgery and Methods {#sec2dot2-jcm-09-00758} --------------------------------------- 26 patients were female and 19 male with a total of 22 surgical procedures on the left side and 23 on the right. At the time of surgery, the mean age was 58.7 (34.0--85.0) years. The mean follow-up period was 16.4 (0.6--74.7) months and data was available for all cases (*n* = 45). The entity of the underlying malignancy, staging at the time of index operation and indication for operation are listed in [Table 1](#jcm-09-00758-t001){ref-type="table"} and [Table 2](#jcm-09-00758-t002){ref-type="table"}. A metastasis of the proximal femur had been stabilized operatively before the index operation in 5 patients. A failed osteosynthesis was causative for index surgery in four and a total hip arthroplasty (THA) in one patient ([Figure 1](#jcm-09-00758-f001){ref-type="fig"} and [Figure 2](#jcm-09-00758-f002){ref-type="fig"}). The mean Mirels score of the patients presenting with impending fractures (*n* = 23) was 9 (7--11) points. All patients were evaluated clinically and radiologically. Pre and postoperative activity and general condition were measured using the Karnofsky index \[[@B12-jcm-09-00758]\]. Function was evaluated according to the MSTS score \[[@B13-jcm-09-00758]\]. Local complications like infection or dislocation with or without revision or consecutive implant failure were collected and statistically evaluated. Migration analysis was performed according to the technique proposed by Callaghan et al. \[[@B14-jcm-09-00758]\]. Statistical analysis was performed with JMP 10 for MAC (SAS Institute Inc., Cary, NC). A time-to-event analysis was performed using the Kaplan--Meier method with death, removal of the stem for any cause, aseptic loosening of the stem and worst case (removal of the stem for any cause and/or aseptic loosening and/or lost to follow up) and dislocation as failure criteria. A 95% confidence interval was applied to all survivorship data and *p*-values for comparing survival curves were calculated with the log-rank test. One-, three- and five-year survival data were calculated for patient survival. Associations or correlations between continuous and/or discrete variables were tested by the Fisher, Student's t-, Paired t- or Chi square-test depending on the underlying empirical distribution. All tests were two-sided and *p* ≤ 0.05 was considered significant. 2.3. Surgical Technique and Postoperative Care {#sec2dot3-jcm-09-00758} ---------------------------------------------- A modular arthroplasty system for reconstruction of the proximal femur was used (MUTARS^®^, Implantcast, Buxtehude, Germany). A Trevira tube for protection of the dislocation and refixation of the muscles at the implant was applied in 28 patients. In 17 remaining cases without a tube, the tendons of the pelvitrochanteric muscles were intact and could be attached to the quadriceps (vastus lateralis, intermedius and/or rectus) or in the case of a resected vastus lateralis, to the maximus insertion (dorsal parts) and/or fascia. The psoas tendon was fixed around the prosthesis by non-resorbable sutures. To protect the dislocation of bipolar heads the capsule of the hip joint was preserved, and retention sutures were sewn for a tight closure of the capsule after repositioning the prosthesis. Bone resection length was assessed during surgery by X-ray validation according to the preoperative planning. The mean resection length of the proximal femur was 14.5 (7--25) cm with a median of 14 cm. The histopathological outcome is described in [Table 3](#jcm-09-00758-t003){ref-type="table"}. The mean reconstruction length of the prosthesis measured 14.4 (7.6--25.6) cm with a median of 14.0 cm. A bipolar head was inserted in 32 operations. In 12 cases an acetabular component was implanted; 11 of them were treated by a cemented PE cup, one with a cementless screw-in cup. The stable shell of the patient with a pre-existing THA was left in situ ([Figure 1](#jcm-09-00758-f001){ref-type="fig"} and [Figure 2](#jcm-09-00758-f002){ref-type="fig"}). Pre- and/or postoperative radiotherapy of the tumor region occurred in 22 patients and pre- and/or postoperative radio- and/or chemotherapy in 28 patients. Four out of eight patients received postoperative radiotherapy after intralesional surgery. 3. Results {#sec3-jcm-09-00758} ========== 3.1. Complications and Reoperations {#sec3dot1-jcm-09-00758} ----------------------------------- Two patients died within four weeks after surgery due to tumor-related general weakness. One hip joint was removed due to low-grade infection 34.5 months, postoperatively. In one patient, the revision of postoperative hematoma with additional debulking of the left soft-tissue tumor mass was performed. Open reduction was necessary in four patients, one with a bipolar head and three with a THA. Consecutively, the cumulative risk of revision without removal of the prosthesis (*n* = 5) was 11.6% at 74.7 months. A total of 2 out of 32 hips with bipolar heads and four of the 13 THAs dislocated. Recurrent dislocation was more likely in patients with severe soft tissue resections treated additionally by a Trevira tube than in patients with simple suture of remaining muscles and tendons as described earlier (6 out of 28 vs. 0 out of 17; Fisher test: *p* = 0.0463). Dislocation was more likely in total hip arthroplasties (Fisher test: *p* = 0.0488) with a significantly higher cumulative risk for dislocation compared to hemiarthroplasties (4 out of 13 with 31.6% at 49.5 months; 95% CI 5.8--57.4 vs. 2 out of 32 9.6% at 74.7 months; 95% CI 0.7--23.0; log-rank test: *p* = 0.0167; [Figure 3](#jcm-09-00758-f003){ref-type="fig"}). Resection/reconstruction length ([Figure 4](#jcm-09-00758-f004){ref-type="fig"} ) or local radiotherapy showed no influence on the risk of dislocation. Cumulative risk of reoperation without removal of the prosthesis (*n* = 6) was 11.6% at 74.7 months. A temporary lesion of the peroneal nerve with complete remission was observed in one patient. 3.2. Functional Evaluation {#sec3dot2-jcm-09-00758} -------------------------- Before full weight-bearing was reached, three patients died. The direct postoperative data of these patients were included in the functional scores. The mean Karnofsky index improved from 49.4 (20%--90%) preoperatively to 54.9 (10%--90%) and the MSTS score from 26.4 (0.0%--96.7%) preoperatively to 51.3 (6.7%--93.3%). The mean pain score improved from 1.1 (0--5) points preoperatively to 3.9 (0--5) points postoperatively. Of the 45 patients, 28 had no or mild pain (four or five points) with NSAIDs as the only pain medication ([Figure 5](#jcm-09-00758-f005){ref-type="fig"}). The use of a Trevira tube did not influence the functional outcome. The MSTS score with its subitems especially the use of walking aids, walking ability and gait were not influenced significantly. 3.3. Radiographic Evaluation {#sec3dot3-jcm-09-00758} ---------------------------- No subsidence or migration of the stems was seen, and all stems remained stable until the last follow-up. Postoperatively, leg length discrepancy of more than 1 cm could be measured in nine patients with a mean difference of 0.2 (−2.5--2) cm. 3.4. Survival Analysis {#sec3dot4-jcm-09-00758} ---------------------- Within six months after surgery, 16 patients died. Survival of all patients was 6.6% (95% CI: 0--14.9) at 74.7 months, six patients were alive at the last evaluation including one patient with implant failure. The one-year and three-year survival rates were 52.9% (95% CI: 38.2--67.6) and 13.2% (95% CI: 2.6--23.7). Patients with disseminated disease (*n* = 30) had an overall survival rate at 74.7 months of 3.5% (95% CI: 0--10.3). One-year and three-year survivorship rates were 45.7% (95% CI: 27.7--63.8) and 7.0% (95% CI: 0--16.4). The survival rate of patients with a solitary metastatic disease at the time of operation (*n* = 15) was 13.5% (95% CI: 0--36.0) at 57.0 months. One-year and three-year rates were 66.7% (95% CI: 42.8--90.5) and 27.0% (95% CI: 4.6--49.4), respectively. Nevertheless, a log-rank test showed no significance between survival rates of patients with solitary or disseminated disease (*p* = 0.1214) ([Figure 6](#jcm-09-00758-f006){ref-type="fig"}). Patients with impending (*n* = 24) or an apparent fracture (*n* = 21) showed a survival rate of 5.2% after 57.0 months or 8.4% after 74.7 months. Survival was not significantly different between the two groups (log-rank *p* = 0.8265). The worst-case survival rate of hip arthroplasty was 80.0% (95% CI: 44.9--100) at 74.7 months with one removal of the stem 34.5 months postoperatively due to low-grade infection ([Figure 7](#jcm-09-00758-f007){ref-type="fig"}). No patient was lost to follow up. 4. Discussion {#sec4-jcm-09-00758} ============= 4.1. Background, Principle Considerations for En-Bloc Resection and Functional Outcome {#sec4dot1-jcm-09-00758} -------------------------------------------------------------------------------------- Quality of life can be improved by the surgical intervention of bony metastases in most patients. Even in an advanced stage of tumor disease with a limited life expectancy of only a few months, patients may benefit from at least partial restoration of function \[[@B15-jcm-09-00758]\]. En-bloc resection of solitary metastasis is well established and obligatory to pursue a curative regimen. However, aggressive surgery of a metastasis of the proximal femur in patients with disseminated disease is discussed controversially. For decades, surgical treatment has not changed relevantly and less invasive procedures like intralesional stabilization by osteosynthesis or cemented endoprosthetic reconstruction after curettage are still performed most frequently. Only three decades ago, overall survival rates of patients presenting bony metastasis were very limited and, therefore, longer-lasting reconstructions were most often not necessary \[[@B16-jcm-09-00758],[@B17-jcm-09-00758]\]. In contrast to surgical therapy, a patient's survival of most entities has improved steadily due to a targeted systemic treatment during the same period. Failure of osteosynthesis and/or arthroplasty after intralesional surgery of lesions of the proximal femur was an indication for surgery in this cohort in nine respectively 2% (osteosynthesis *n* = 4; arthroplasty *n* = 1). Failure occurred at a mean of four (0.1--8.6) months after osteosynthesis. Comparable studies have described a rate of collapse of osteosyntheses in the proximal femur of up to 23% within the first year \[[@B4-jcm-09-00758],[@B5-jcm-09-00758],[@B6-jcm-09-00758],[@B18-jcm-09-00758]\]. Additionally, the risk for failure may increase with a patient's survival. Overall, there are significantly higher survival rates following arthroplasty compared to osteosynthetic stabilization at the proximal femur \[[@B5-jcm-09-00758],[@B10-jcm-09-00758],[@B19-jcm-09-00758],[@B20-jcm-09-00758]\]. In contrast to osteosynthesis, reconstruction with arthroplasty offers the possibility of an extralesional (marginal or wide) resection of the tumor that is not only required in patients with a curative regimen in primary tumors or solitary metastatic disease but also in disseminated stages for local tumor control \[[@B9-jcm-09-00758],[@B10-jcm-09-00758]\]. Additional postoperative radiotherapy is not necessary for tumor control or pain reduction. In patients with a limited life expectancy of less than three months, radiotherapy for pain reduction was ineffective in more than half of the patients \[[@B21-jcm-09-00758]\]. Accordingly, marginal or wide resection may also be favorable for pain control especially for patients with longer life expectancy. This finding is similar to other studies and should especially be considered for tumors with poor response to radiotherapy like kidney cancer \[[@B4-jcm-09-00758],[@B22-jcm-09-00758],[@B23-jcm-09-00758]\]. In this series, 9% had no or mild pain preoperatively (MSTS pain score of 4--5 points) but more than 62% postoperatively, which could be treated sufficiently by NSAID as a single medication. Nevertheless, the postoperative MSTS score of 51.3% was less compared to other series with scores of 67% to 87% \[[@B24-jcm-09-00758],[@B25-jcm-09-00758],[@B26-jcm-09-00758],[@B27-jcm-09-00758]\]. Inclusion of patients with primary bone tumors in these studies and incorporation of many patients in an advanced stage in this series are limiting factors \[[@B1-jcm-09-00758],[@B24-jcm-09-00758],[@B25-jcm-09-00758],[@B26-jcm-09-00758],[@B27-jcm-09-00758]\]. All data were included despite the death of 16 patients within six months after surgery. Although functional results were not significantly different between the two groups (follow-up greater or less than six months), differences in pain scores were less than in all other aspects of the MSTS score (pre- vs. postoperatively). At the last evaluation 10 out of the 16 patients (59%) with a limited life expectancy of less than six months had no or mild pain (more than four points) vs. 18 out of 28 of the group with longer follow-up (64%). Therefore, surgical resection of the metastasis and endoprosthetic reconstruction seems to be efficient for local pain control even in patients with a very limited survival. Our study has limitations. The comparatively small number of patients (*n* = 45) is one disadvantage. Additionally, the follow-up period is limited due to the disease of the patients. Furthermore, a control group with patients treated by simple osteosynthesis, intralesional curettage and cemented primary stems or revision devices is missing. Nevertheless, there is adequate data available to compare the results of the current cohort and to draw the described conclusions. There are also limitations due to the study design as it is a retrospective study. Prospective randomized studies may be necessary in the future. 4.2. Complications and Survivorship of Patients and Implants {#sec4dot2-jcm-09-00758} ------------------------------------------------------------ At current, survival rates of 90% at one year or 20% at five years are described in patients with favorable conditions with respect to prognostic factors of entities showing a high risk of developing bony metastases during follow-up \[[@B4-jcm-09-00758],[@B28-jcm-09-00758],[@B29-jcm-09-00758]\]. After surgical treatment of bone metastases of the lung, prostate, and kidney or breast cancer, patient survival rates of 40% after one year and 20% after three years are reported \[[@B1-jcm-09-00758]\]. In our series, the one-year, three-year and five-year survival rates were 52.9%, 9.9% and 6.6% respectively. Consecutively, this cohort consisted mainly of patients with a limited life expectancy but a survival of more than one year has to be considered in at least half of the patients. Endoprosthetic treatment of patients with metastatic disease of the proximal femur in this series showed a survival rate of the reconstruction of 80.0 (44.9%--100%) at 74.7 months with one removal of the stem 34.5 months postoperatively due to low-grade infection. Of the few studies discussing arthroplasty in metastases of the proximal femur, comparable survival rates of the prosthesis at five years between 83% and 100% were reported \[[@B19-jcm-09-00758],[@B30-jcm-09-00758]\]. Revision rates with the removal of the endoprosthesis after tumor reconstruction due to infection between 1.8% and 21% have been published \[[@B19-jcm-09-00758],[@B24-jcm-09-00758],[@B25-jcm-09-00758],[@B27-jcm-09-00758],[@B31-jcm-09-00758],[@B32-jcm-09-00758],[@B33-jcm-09-00758]\] further increasing the patients treated by neo-/adjuvant, intra-operative or direct adjuvant radiotherapy \[[@B31-jcm-09-00758]\]. There was one implant failure due to infection despite pre- and/or postoperative radio- and/or chemotherapy in 28 patients in our investigation. Dislocation rates of megaprostheses of up to 34% have been described \[[@B9-jcm-09-00758],[@B27-jcm-09-00758]\]. A dislocation rate of 13.3% (six hips) in this cohort is comparable with available data in tumor and revision surgery \[[@B5-jcm-09-00758],[@B6-jcm-09-00758],[@B26-jcm-09-00758],[@B34-jcm-09-00758],[@B35-jcm-09-00758]\]. Nevertheless, instability is a major local complication in this series with a time-dependent risk of 16.2% at 74.7 months. As discussed in other studies a significantly higher risk could be seen in THA than hemiarthroplasties (Fisher test: *p* = 0.0488) \[[@B36-jcm-09-00758]\]. None of the patients with attachment of intact soft tissue by single sutures dislocated while 6 out of 28 patients with deficient soft tissue and additional use of a tube presented instability during follow up. Nevertheless, clear data showing the improvement of dislocation rates by using certain devices for soft tissue refixation are missing \[[@B34-jcm-09-00758],[@B36-jcm-09-00758],[@B37-jcm-09-00758],[@B38-jcm-09-00758]\]. The resection length of the proximal femur and consecutive implant-related parameters like reconstruction length did not influence the dislocation rate in this study. However, the extent of the extra-osseous tumor mass might influence and explain the dislocation rate. In extensive tumors with a large extra-osseous tumor mass involving the joint capsule and stabilizing muscles, successful soft tissue attachment is difficult to address. There was no difference in the functional outcome (MSTS score with sub-items), which should be improved, as it is the other aim of using the tube due to tight fixation of the muscles at the prosthesis. In the case of dislocation, the tube may hinder closed reposition as it was the reason for two of four open reductions. Therefore, in cases with a preserved capsule as the best prevention for instability, we would not routinely recommend the use of a tube and, in our experience, its benefit is questionable. Consecutively, a time-dependent revision rate without removal of the prosthesis of 12.6% could be further reduced but seems to be acceptable and is lower compared to revision rates due to mechanical failure of osteosyntheses. The use of primary stems or cemented long stems would have been an option in some cases \[[@B39-jcm-09-00758]\]. From a biomechanical point of view, en-bloc resection of a highly insufficient proximal femur is identical to an intralesional procedure with a cemented primary or long-stemmed prosthesis. The length of anchorage in intact bone is more or less similar with both procedures. Due to these considerations and the topics mentioned above, the more aggressive treatment in this selective cohort was performed. A cementless Wagner SL revision stem was implanted in a similar group of patients with comparable results \[[@B22-jcm-09-00758]\]. Obviously, both systems allow accurate therapy in these patients. Our procedure in the operative treatment of secondary bone malignancies at the proximal femur can be summarized as follows:Simple osteosynthesis by intramedullary nail is justified in cases with a very poor prognosis of a few months (≈ less than six) and a tumor localization clearly below the intertrochanteric region (meta- and/or diaphysis).Intralesional curettage and implantation of a primary stem is justified in patients with a very limited life expectancy and a localization proximal to the intertrochanteric line/femoral neck in tumors with a good response to radiotherapy.Intralesional curettage and implantation of a long-stemmed, cemented prosthesis can be performed in patients with very limited life expectancy in biomechanical situations not suitable for osteosynthesis and/or a cemented primary stem and in patients with additional metastasis more distally.All other cases should be treated by en-bloc resection (wide or marginal) and endoprosthetic reconstruction.A cemented reconstruction should be performed in patients with necessary potential postoperative radiotherapy (R1, intralesional) and/or additional metastasis distally; all other cases can be treated with cementless devices.A tube for soft tissue attachment and prevention of dislocation is not necessary when the capsule can be preserved. 5. Conclusions {#sec5-jcm-09-00758} ============== En-bloc resection and endoprosthetic reconstruction of secondary malignancies of the proximal femur with the modular devices used showed reliable results with respect to implant survival, local tumor and pain control even in advanced tumor stages as represented in this cohort. The use of a Trevira tube did not avoid dislocation or increase functional outcome in patients with severe soft tissue damage. Statistical analysis was supported by T. Bruckner, Biometrics Consulting and Project Management, University Heidelberg, Germany. Conceptualization, O.E.B.; methodology, P.M.B. and O.E.B.; software, O.E.B.; validation, J.B.S. and A.J.S.; formal analysis, O.E.B.; investigation, B.L., A.J.S. and O.E.B.; resources, R.G.B. and O.E.B.; data curation, O.E.B.; writing---original draft preparation, O.E.B.; writing---review and editing, O.E.B., J.B.S. and A.J.S.; visualization, O.E.B.; supervision, P.M.B.; project administration, O.E.B. All authors have read and agreed to the published version of the manuscript. This research received no external funding. Author R.G.B. has received a speaker honorarium from Johnson & Johnson (DePuy/Synthes), B. Braun (Aesculap), Waldemar Link, Heraeus Medical, Implantcast and Zimmer/Biomet. This honorarium was not related directly or indirectly to the subject of this article. All other authors declare that they have no competing interests. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. {#jcm-09-00758-f001} {#jcm-09-00758-f002} {#jcm-09-00758-f003} {#jcm-09-00758-f004} {#jcm-09-00758-f005} {#jcm-09-00758-f006} {#jcm-09-00758-f007} jcm-09-00758-t001_Table 1 ###### Diagnosis and staging at the time of index operation. Diagnosis and Staging at Index Operation Number of Patients (45), Solitary/Disseminated ------------------------------------------ ------------------------------------------------ Bronchial Carcinoma 9 (5/4) Leiomyosarcoma 1 (1/0) Liposarcoma 1 (0/1) Melanoma 1 (0/1) Breast Carcinoma 13 (1/12) Plasmacytoma 1 (0/1) Oral Carcinoma 2 (0/2) Renal Carcinoma 9 (4/5) Paraganglioma 2 (0/2) Pheochromocytoma 1 (1/0) Prostate Cancer 1 (1/0) Colorectal Cancer 1 (1/0) Thyroidal Carcinoma 1 (0/1) Urothelial Carcinoma 2 (1/1) jcm-09-00758-t002_Table 2 ###### Indication for operative treatment. Indication for Operative Treatment No. of Cases (*n* = 45) ---------------------------------------------- ------------------------- Impending Fracture ^1^ 23 Impending Fracture/Loosening after THA ^2^ 1 Pathologic Fracture 17 Pathologic Fracture after Osteosynthesis ^3^ 4 ^1^ Mean Mirels Score (*n* = 23): 9 (7--11) pts. ^2^ 36 months after primary THA. ^3^ Proximal femoral nail (*n* = 3), double plate (*n* = 1); failure of osteosynthesis after a mean of 3.7 (0.1--8.6) months. jcm-09-00758-t003_Table 3 ###### Surgical margins and histopathological outcome. Surgical Margins/Histopathology Number -------------------- --------------------------------- -------- ---- En-bloc Resection 37 4/1 42 Curettage Distally 0 2/1 3 Total 37 8 45

Although wax-models played a significant part in the making of natural knowledge, only recently have historians started to devote systematic attention to them. Nick Hopwood\'s *Embryos in wax* reconstructs the story of wax-models of embryos from the end of the eighteenth-century to the days in which new experimental agendas and the wider political events of the twentieth century resulted in the quiet withdrawal of the models to museums and institute stores. Documenting the models of the Ziegler studio, and discussing a variety of aspects associated with their making and use, this very well-crafted work sheds light on a practice and a set of objects that for more than half a century lay at the very heart of embryology. Reproducing Friedrich Ziegler\'s last catalogue of the models as well as a rich and lavish selection of photographs and colour plates, this study brings together fine scholarship and unexplored source material. At the same time, it also allows readers to navigate with great ease across both verbal and non-verbal domains. From the end of the eighteenth century those who engaged in the modelling of embryos could build on the achievements of anatomical ceroplastics. Yet, the representation of embryos gave rise to new problems. Models of embryos were supposed to track the early stages of life. As embryology moved from miniature representation of children to the investigation of progressive development, wax-modellers were charged with the task of visualizing processes that took place over time and out of sight. *Embryos in wax* reconstructs how models contributed to the conceptualization of embryos as isolated objects of investigation that were defined independently of the body of the mother. It elucidates how the choices that underlay the three-dimensional representations of embryos had social and political as well as theoretical implications. Thus, for instance, embryos in wax not only stimulated medical debates between evolutionary and mechanical approaches to embryology, and informed views of normal embryonic development, they also lay at the centre of forms of expropriation and exploitation of the female body, corroborated more or less élitist views of society, epitomized visions of progress, and substantiated eugenic anxieties. Associated with "the lower-status activities of teaching and popularisation" (p. 3), models of embryos have long lingered at the margins of historical investigations. Along with other objects, they have borne the consequences of an enduring divide between things, traditionally characterized as mute, silent and opaque, and words, typically fashioned as the privileged medium of communication. Placing models at the centre of a complex interplay between things, people and words, Hopwood\'s work shows that, in fact, models of embryos made sense of people (and words) at least as much as people made sense of models. While magnifying microscopic structures, wax-models of embryos hardly resembled anything that one could come to recognize as part of one\'s experience. Yet, they became powerful means of scientific communication. In order to illustrate how this happened, Hopwood takes readers back from the models to the busy laboratories in which they were made, the scientific practices and teaching methods they brought about, the forms of business and division of labour they supported, and the professional alliances and academic contempt they generated. Particular relevance is also given to the codification of the view that wax-models of embryos could be published; the relationship between "plastic publishing" and printed culture; and the bearing this relationship had on the publicity, credibility, and circulation of the models. The discussion of all these aspects cogently addresses "the historical challenge" of getting "behind the finished products" (p. 2). Hopwood\'s reconstruction of the role of models in defining what embryos and embryology were all about makes a powerful case for a more integrated historical analysis of the different media of science. His assertion that this book is, among other things, "about wax" (p. 5) may whet the appetite for further systematic discussion of the part played by specific material domains---in this case wax---in the objectification of scientific tenets and values. But readers will find in this engaging study both a valuable source and new directions for research. At a time in which interest in the history of three-dimensional anatomical waxworks is growing rapidly, I would not be surprised if *Embryos in wax* came to be regarded as a model.

{#sp1 .33} {#sp2 .34}

1. Introduction {#sec1-ijerph-16-02582} =============== Obesity has become a major public health problem in the past three decades, especially in children and adolescents \[[@B1-ijerph-16-02582]\]. Specifically, the prevalence of obesity in this age group in 2016 was 5.6% in girls and 7.8% in boys \[[@B2-ijerph-16-02582]\]. In Croatia, the data from the Health Behavior in School Aged Children 2009/2010 survey \[[@B3-ijerph-16-02582]\] showed the prevalence of those overweight being 23.0% and 10.0% in boys and girls, respectively. Obesity is associated with many non-communicable diseases \[[@B4-ijerph-16-02582]\], and higher levels of excessive fat in childhood and adolescence lead to negative health consequences in adulthood \[[@B2-ijerph-16-02582]\]. Similar to being overweight or suffering from obesity at one end, thinness also represents a potential public health problem at the other end, and is associated with higher rates of mortality and several diseases \[[@B5-ijerph-16-02582],[@B6-ijerph-16-02582]\]. Physical fitness represents a significant part of physical activity \[[@B7-ijerph-16-02582]\]. It is defined as 'a measure of the capacity to perform physical activity and/or physical exercise that integrates the majority of the bodily functions involved in body movement' \[[@B7-ijerph-16-02582]\]. Similarly just like obesity, the level of physical fitness in childhood/adolescence often persists later on in adulthood \[[@B8-ijerph-16-02582]\]. Moreover, it has been shown that higher levels of physical fitness in children and adolescents lead to better overall health later on in life \[[@B9-ijerph-16-02582]\]. Several previous studies have examined the association between overweight/obesity (by using the body-mass index indicator) and physical fitness \[[@B10-ijerph-16-02582],[@B11-ijerph-16-02582],[@B12-ijerph-16-02582],[@B13-ijerph-16-02582],[@B14-ijerph-16-02582],[@B15-ijerph-16-02582]\]. Of those, only a few have calculated a quadratic association including both thinness and obesity in the equation \[[@B13-ijerph-16-02582],[@B15-ijerph-16-02582],[@B16-ijerph-16-02582],[@B17-ijerph-16-02582]\]. Results of those studies were consistent with each other, showing a non-linear association between the body-mass index and several fitness tests; that is both thin and overweight/obese participants scored lower on the physical fitness tests. According to the aforementioned, more studies have been using the body-mass index as an indicator of nutritional status, yet the body-mass index cannot differ fat mass from free-fat mass, and may lead to a misclassification \[[@B18-ijerph-16-02582]\]. Thus, some other indicators of obesity, like waist circumference, have been proposed \[[@B19-ijerph-16-02582]\]. Fewer studies have examined the association between waist circumference \[[@B11-ijerph-16-02582],[@B12-ijerph-16-02582],[@B14-ijerph-16-02582]\] and physical fitness. After an extensive literature review, there has been a lack of studies examining combined non-linear associations between body-mass index and waist circumference with physical fitness. Therefore, the main purpose of the study was to explore the body-mass index and the waist circumference associated with physical fitness in a large sample of Croatian adolescents aged 15--18 years by gender. We hypothesized that the association between all anthropometric indices and physical fitness would be non-linear with a parabolic shape. 2. Materials & Methods {#sec2-ijerph-16-02582} ====================== 2.1. Participants {#sec2dot1-ijerph-16-02582} ----------------- In this cross-sectional study, participants were secondary-school students. A detailed study protocol has been described elsewhere \[[@B20-ijerph-16-02582]\]. In brief, we randomly selected 11 (8 grammar and 3 vocational) out of 86 secondary-schools in the city of Zagreb, after which we randomly selected one class representing each grade within the school (from 1st to 4th). Each class had ≈25 students. All students were considered healthy, and were not affected by diseases. The selection criteria were: (1) An active participation in physical education classes and (2) an absence of injuries. According to the Croatian Bureau of Statistics for the year 2017 \[[@B21-ijerph-16-02582]\], there were 36,350 secondary-school students in total. Our sample size was estimated to be 1030, by using a 95% confidence level and a 3% margin of error. All procedures performed in this study were in accordance with the Declaration of Helsinki, and were approved by the Institutional Review Board of the leading author (code: 02/2019). Also, all of the participants and their parents/guardians provided written informed consent for participation in the study. 2.2. Anthropometric Measures {#sec2dot2-ijerph-16-02582} ---------------------------- Body height was measured to the nearest millimeter in bare or stocking feet with the adolescent standing upright against a stadiometer (Seca, Japan). The result was given in meters. Body weight was measured to the nearest 0.1 kg, and the participant wore light clothes with no shoes (Seca, Japan). The result was given in kilograms. BMI (kg/m^2^) was calculated as weight (in kilograms) divided by the square of height (in meters). WC was measured for each participant remaining still in a standing position. We used anthropometric tape placed horizontally midway between the lower rib margin and the iliac crest at the end of normal expiration \[[@B22-ijerph-16-02582]\]. The association between BMI and WC was 0.84 in boys and 0.73 in girls (*p* \< 0.001). 2.3. Objectively Estimated Physical Fitness {#sec2dot3-ijerph-16-02582} ------------------------------------------- We used a EUROFIT Battery Fitness Test to assess the level of physical fitness in adolescents. These tests are considered reliable and valid instruments to measure the level of physical fitness in children and adolescents \[[@B23-ijerph-16-02582]\]. Standing long jump, sit-ups for 1 min and a sit-and-reach test were chosen because of their mutual independence to the other \[[@B24-ijerph-16-02582]\]. Data were collected by two trained researchers in order to guarantee the standard measurement methodology \[[@B24-ijerph-16-02582]\]. A brief explanation of each test is presented below: Standing long jump: Each subject performed distance jumps from a standing start. While performing the jumps, the subjects were asked to bend their knees with their arms in front of them, parallel to the ground, then to swing both arms, push off vigorously and jump forward as far as possible, trying to land with their feet together and to stay upright. The best out of two attempts was taken as the final score (expressed in centimeters) \[[@B25-ijerph-16-02582]\]. Sit-ups in 1 min: Trunk strength was assessed as the maximum number of sit-ups achieved in one minute. Children were seated on the floor, backs straight, hands clasped behind their neck, knees bent at 90°, with their heels and feet flat on the mat. They then lay down on their backs, shoulders touching the mat, and returned to the sitting position with their elbows out in front to touch their knees, keeping the hands clasped behind their neck the whole time. The total amount of correctly performed sit-ups in 60 s was the score \[[@B25-ijerph-16-02582]\]. Sit-and reach test: Sitting on the floor or upon a mat, legs straight under the angle of 90°, the person being tested reached forward with the arms (hands overlapping). The distance of reach was measured in centimeters using a measuring non-elastic tape attached to the floor \[[@B26-ijerph-16-02582]\]. 2.4. Data Analysis {#sec2dot4-ijerph-16-02582} ------------------ Data are presented as mean (SD). All variables were grouped according to sex (boys vs. girls). First, we calculated the differences between the sexes in variables which were analyzed using univariate analysis of variance. Previous studies have shown sex differences in perceived and estimated physical fitness \[[@B27-ijerph-16-02582]\]. Therefore, we presented sex-stratified analyses. Next, we calculated quadratic associations between the anthropometric indices and each physical fitness test, using linear regression analyses. Finally, we also checked for multicollinearity between these physical fitness tests using the variance inflation factor (VIF). The VIF value was \<2, indicating no multicollinearity between the physical fitness tests. Significance was set up at *p* ≤ 0.05, and it was two sided (2-sided). All of the analysis were performed in the Statistical Package for Social Sciences Software, ver. 22 (IBM Corp., Armonk, NY, USA). 3. Results {#sec3-ijerph-16-02582} ========== Basic descriptive statistics of the study participants are presented in [Table 1](#ijerph-16-02582-t001){ref-type="table"}. Boys had higher body-mass index (Effect size (ES) = 0.66) and waist circumference (ES = 0.93) values, compared to girls (*p* \< 0.001). They also performed better in 1 min sit-ups (ES = 0.81) and standing long jump (ES = 1.69) tests (*p* \< 0.001), while girls obtained higher values in the sit-and-reach test (ES = 0.73, *p* \< 0.001). Quadratic associations between anthropometric indices and physical fitness tests and overall physical fitness for both boys and girls are presented in [Table 2](#ijerph-16-02582-t002){ref-type="table"} with a graphical presentation in [Figure 1](#ijerph-16-02582-f001){ref-type="fig"}. Among boys, a quadratic regression showed that their body-mass index was associated with 1 min sit-ups and a standing long jump, while no significant association with the sit-and-reach test was observed. Waist circumference was also only associated with 1 min sit-ups and standing long jump. In girls, their waist circumference was associated with 1 min sit-ups and the standing long jump, while the body-mass index was only inversely associated with the standing long jump. 4. Discussion {#sec4-ijerph-16-02582} ============= The main question of the study was as to whether body-mass index and waist circumference were associated with some aspects of physical fitness. This study shows that both body-mass index and waist circumference are associated with physical fitness tests, especially 1-min sit-ups and the standing long jump. Our results of the association between body-mass index and physical fitness are in line with previous studies conducted among a sample of children \[[@B13-ijerph-16-02582],[@B15-ijerph-16-02582],[@B17-ijerph-16-02582]\] and adolescents \[[@B13-ijerph-16-02582],[@B15-ijerph-16-02582],[@B16-ijerph-16-02582]\]. Most recently, Lopes et al. \[[@B16-ijerph-16-02582]\] showed a non-linear (curvilinear) association between body-mass index and standing long jump among boys aged 10--11 and 14--17 years and girls aged 10--13 years, while among girls aged 14--17 years, the association was linear. The same group of authors also found a non-linear association between body-mass index and push-ups and a multistage shuttle run in both boys and girls \[[@B16-ijerph-16-02582]\]. A study conducted among Croatian untrained boys aged 14--16 years showed a non-linear association between several anthropometric and physical fitness variables \[[@B17-ijerph-16-02582]\]. Although previous studies have found a decline in fitness or motor performance with higher body-mass index values \[[@B28-ijerph-16-02582]\], a study by Chen et al. \[[@B27-ijerph-16-02582]\] found that lower levels of body-mass index were associated with a better aerobic capacity in Taiwanese children aged 7--8 years, pointing out that different ranges of body-mass index are not similarly associated with physical fitness tests. Our study also shows a non-linear association between waist circumference and standing long jump and overall physical fitness score in boys, and overall physical fitness score in girls. Only a few previous studies have examined the aforementioned association \[[@B11-ijerph-16-02582],[@B12-ijerph-16-02582],[@B14-ijerph-16-02582]\]. However, none of those studies used a quadratic analysis to calculate the association between waist circumference and physical fitness. Nevertheless, similar results were observed showing an inverse association between waist circumference and physical fitness components \[[@B11-ijerph-16-02582],[@B12-ijerph-16-02582],[@B14-ijerph-16-02582]\]. Similar to body-mass index, a higher level of waist circumference often means excess body fat acting like an extra load during physical fitness test performances \[[@B12-ijerph-16-02582]\]. Also, obese individuals tend to have a lack of motor learning and reduced activation in motor units, which may explain poorer lower extremity strength \[[@B29-ijerph-16-02582]\]. 4.1. Study Strengths {#sec4dot1-ijerph-16-02582} -------------------- This study has several strengths. First, we conducted our study on a relatively large sample of secondary-school students (*N* = 1036). Second, we used both body-mass index and waist circumference as indicators of obesity. Third, we presented non-linear regression results, regarding anthropometric indices and some aspects of physical fitness. 4.2. Study Limitations {#sec4dot2-ijerph-16-02582} ---------------------- Our study has several limitations. First, by using a cross-sectional design, we cannot conclude the causality of the correlation. Second, to assess objectively estimated physical fitness, we only used musculoskeletal fitness. Previous studies have used aerobic capacity as the measure of physical fitness \[[@B10-ijerph-16-02582],[@B11-ijerph-16-02582],[@B12-ijerph-16-02582],[@B13-ijerph-16-02582],[@B14-ijerph-16-02582],[@B15-ijerph-16-02582],[@B16-ijerph-16-02582],[@B17-ijerph-16-02582]\]. However, a most recent meta-analysis of longitudinal studies has revealed a moderate-large negative association between muscular fitness in childhood/adolescence and adiposity and cardiometabolic parameters in adulthood, pointing out that muscle-strengthening activities have beneficial effects on health during lifespan and might be of greater importance than aerobic capacity \[[@B30-ijerph-16-02582]\]. Third, we did not collect information about biological maturity status, since individual specific height and weight growth might have led to different associations. Finally, although we conducted the study on a large sample of urban secondary-school students, and the data cannot be used for a rural or mixed population, our results can be used for comparisons in adolescents with different nationalities. 5. Conclusions {#sec5-ijerph-16-02582} ============== Our study shows that anthropometric indices are associated with some aspects of physical fitness tests in a large sample of Croatian adolescents. Although overweight and obesity have become a major public health problem worldwide, our study also shows that thin adolescents might also be a group at risk of performing poorly on physical fitness tests. Therefore, screening for both thinness and obesity to predict the level of physical fitness should be of great interest for future longitudinal research, and those findings might serve as certain recommendations for optimal nutritional status and better physical fitness performance in adolescents. We would like to thank all the participants for their enthusiastic participation in the study. Conceptualization, L.Š.; Methodology, L.Š.; Software, L.Š.; Validation, M.Z. and M.K.; Formal Analysis, L.Š.; Investigation, L.Š.; Resources, L.Š.; Data Curation, M.Z. and M.K.; Writing---Original Draft Preparation, M.Z., M.K. and L.Š.; Writing---Review & Editing, M.Z., M.K. and L.Š.; Visualization, L.Š.; Supervision, M.Z. and M.K. This paper was self-funded. Non-financial Conflicts of Interest. {#ijerph-16-02582-f001} ijerph-16-02582-t001_Table 1 ###### Basic descriptive statistics of the study participants, (Croatia, 2019). Study Variables Total Sample Boys Girls *p*-Value --------------------------- -------------- -------------- -------------- ----------- Mean (SD) Mean (SD) Mean (SD) Min-Max Min-Max Min-Max Age (yrs) 16.3 (1.1) 16.4 (1.1) 16.2 (1.1) \<0.001 15--18 15--18 15--18 Anthropometric Indices Body-mass index (kg/m^2^) 21.3 (2.9) 22.0 (3.2) 20.1 (2.6) \<0.001 14.3--40.9 15.9--40.9 14.3--40.8 Waist circumference (cm) 73.3 (9.4) 77.6 (9.6) 69.7 (7.5) \<0.001 35.0--138.0 35.0--138.0 40.0--109.0 Physical Fitness 1 min sit-ups (\#) 51.2 (11.7) 56.1 (11.9) 47.3 (10.0) \<0.001 20.0--112.0 20.0--112.0 20.0--82.0 Standing long jump (cm) 186.1 (32.9) 209.6 (29.2) 167.1 (21.4) \<0.001 72.0--280.0 102.0--280.0 72.0--225.0 Sit-and-reach test (cm) 67.1 (13.5) 62.0 (12.0) 71.2 (13.2) \<0.001 15.0--112.0 25.0--112.0 15.0--105.0 ijerph-16-02582-t002_Table 2 ###### Quadratic regression associations between anthropometric indices and physical fitness tests in boys and girls, Croatia (2019). Study Variables Boys (*N* = 463) Girls (*N* = 573) --------------------------- ---------------------------------------- ---------------------------------------- ---------------------------------------- ---------------------------------------- ---------------------------------------- ---------------------------------------- 1 min sit-ups (\#) Standing long jump (cm) Sit-and-reach test (cm) 1 min sit-ups (\#) Standing long jump (cm) Sit-and-reach test (cm) Anthropometric Indices Standardized β coefficient (*p*-value) Standardized β coefficient (*p*-value) Standardized β coefficient (*p*-value) Standardized β coefficient (*p*-value) Standardized β coefficient (*p*-value) Standardized β coefficient (*p*-value) Body-mass index (kg/m^2^) −0.62 (0.047) −0.89 (0.004) −0.12 (0.701) −0.69 (\<0.001) −0.18 (0.530) −0.54 (0.063) Waist circumference (cm) −0.30 (\<0.001) −0.42 (\<0.001) −0.04 (0.635) −0,20 (\<0.001) −0.24 (\<0.001) −0.13 (0.032) *p* \< 0.05.

Introduction ============ Wheat is a major staple of the diet worldwide and wheat allergy is defined as an adverse immunologic reaction to wheat protein \[[@B1]\]. Depending on the route of exposure and the underlying immunologic mechanisms, wheat allergy are diagnosed as different forms of allergic diseases such as classic food allergy, food-dependent exercise-induced anaphylaxis (FDEIA), occupational asthma, rhinitis or contact urticaria \[[@B2]\]. IgE mediated allergy to wheat may be presented with various severity from simple urticaria to anaphylaxis. In Korea, only a few studies on wheat-induced anaphylaxis (WIA) have been performed \[[@B3],[@B4]\]. We herein report six cases of severe wheat allergy including WIA and wheat-dependent exercise-induced anaphylaxis (WDEIA). Case ==== We reviewed three cases of WIA and three cases of WDEIA. All the patients visited our allergy clinic for the evaluation of symptoms such as urticaria, dyspnea, or syncope. History taking revealed that they ate wheat-containing foods before their symptoms had started. The clinical characteristics of the patients are shown in [Table 1](#T1){ref-type="table"}. Their ages ranged from 29 to 65 years and mean ± standard deviation (SD) of age was 45 ± 14 years. The mean ± SD of the patient\'s age at the onset of the symptoms was 38 ± 10 years (from 27 to 55 years). One patient with WIA and two patients with WDEIA were males and the others were females. All patients with WIA had combined allergic diseases and only one patient with WDEIA had allergic rhinitis. There was no patient who took aspirin or NSAIDs which might enhance food induced anaphylaxis. The patient\'s symptoms of anaphylaxis are presented in [Table 2](#T2){ref-type="table"}. All six patients had skin manifestation, such as urticaria (100%) and 4 of 6 had angioedema. Five patients had respiratory symptoms such as wheezing or dyspnea, and only one patient with WIA had gastrointestinal symptoms. All patients showed hypotension during the events of anaphylaxis and 4 patients had experienced loss of consciousness. The mean ± SD of the level of total IgE was 512.20 ± 668.86 IU/mL. The levels of total serum IgE were higher than normal range in 4 patients. Total serum IgE was not evaluated in one patient (No. 3) and another patient showed normal level of total serum IgE (No. 5, 94 IU/mL). Wheat-specific IgE detected by ImmunoCAP (UniCAP PHAD®, Pharmacia & Upjohn, Uppsala, Sweden) showed class 1 in two patients and class 2 in the other two patients. Gluten-specific IgE was increased as class 2 in 3 patients (2 WIA and 1 WDEIA) and as class 3 in 2 patients (1 WIA and 1 WDEIA). ω-5 gliadin-specific IgE was increased in 4 patients, class 3 in 3 patients (1 WIA and 2 WDEIA) and class 4 in one patient with WIA. The levels of gluten or ω-5 gliadinspecific IgE were higher than wheat-specific IgE in all six patients. The details are shown in [Table 3](#T3){ref-type="table"}. Skin prick test was conducted in 3 patients with WDEIA. The patients showed a positive response in skin prick test with wheat as shown in [Table 4](#T4){ref-type="table"}. All of them had positive results for skin prick tests for other foods: one patient (No. 4) also had a history of urticaria with seafood. Another patient (No. 5) was diagnosed as oral allergy syndrome. In addition, those with WDEIA had positive skin prick test for common inhalant allergens. While 5 patients were diagnosed on the basis of repeated histories and specific IgE to wheat proteins, one patient (No. 5) suspected as having WDEIA underwent an exercise provocation test 30 minutes after eating wheat-containing food. Within 5 minutes after 6 minutes free running, the patient developed generalized itching and urticaria. At 15 minutes, the patient complained of dyspnea and presented with hypotension (systolic blood pressure 63 mmHg). Symptoms were resolved gradually after treatment, with restoration of normal vital signs and physical examination findings. The three patients with WIA were advised the life-long avoidance of wheat containing products and the others with WDEIA were taught to avoid wheat-containing food, and importantly to avoid exercise for at least six hours when they accidentally ate wheat-containing food. They were prescribed Epipen® by the allergists in our clinic. Discussion ========== Food allergy is increasing and the prevalence of food allergy is estimated to be 6-8% of children and 1-3% of adults \[[@B5]\]. Wheat is an important part of human diet and one of the six most common foods causing allergy \[[@B6]\]. Depending on the route of allergen exposure and the underlying immunologic mechanisms, wheat allergy may appear as classic food allergy affecting the skin, gut, or respiratory tract; exercise-induced anaphylaxis; occupational asthma and rhinitis; or contact urticaria. In addition, ingestion of wheat gluten may cause celiac disease (T-cell-mediated intestinal inflammation), or dermatitis herpetiformis (blistering skin eruption). With the exception of the latter two, specific IgE to wheat proteins play a central role in all of the above \[[@B1]\]. Despite the high prevalence of wheat allergy in children, relatively little is known about its natural history. The natural course of only a few patients with wheat allergy has been reported in the literature, and in these studies 25% to 35% of patients became tolerant in a 1- to 2-year period \[[@B6]\]. In the study by Keet et al. \[[@B7]\], 35% of the children who had wheat allergy remained allergic during their teenage years. In some cases, it may lead to exacerbation of atopic dermatitis and gastrointestinal symptoms. A unique form of allergy, WDEIA, develops predominantly in young adults and adolescents \[[@B8]\]. A few cases of WIA, which were enhanced by aspirin or nonsteroidal anti-inflammatory drugs, have been reported in adult patients \[[@B9]\]. Recently, it has also been reported that FDEIA could be dependent on the temperature \[[@B10]\]. In this study, all of the cases of WIA were adult-onset and 4 (67%) of the patients had co-morbid allergic diseases. Most of them experienced urticaria, respiratory symptoms and hypotension during the events, but only one patient had gastrointestinal symptom. Four of the 6 patients showed increased levels of total IgE than normal range and 3 patients with WDEIA had positive responses to skin prick test with common food allergen and aeroallergens. Because of the difficulty of strict wheat avoidance, complete diagnosis for the patients who were suspected wheat allergy is critical. To diagnose WIA or WDEIA, food challenge test with wheat or exercise challenge test following wheat ingestion should be performed. However, the provocation test is time-consuming and has a risk of inducing anaphylaxis. On top of that, the amount of food and the degree of exercise vary. Thus, *in vitro* diagnostic test for predicting severe systemic reactions is desirable. Considering the risk of food challenge tests in patients with a history of anaphylaxis, *in vitro* tests using ImmunoCAP could be a useful method in clinical practice with definite history of anaphyalxis \[[@B4]\]. Wheat proteins are classified depending on the basis of their solubility in a series of solvents: water (albumins), dilute salt solutions (globulins), aqueous alcohol (gliadins), and dilute alkali or acid (glutenins) \[[@B2]\]. Classic food allergy and atopic dermatitis are related to a wide range of water/salt-soluble and insoluble wheat proteins \[[@B11]\]. WDEIA is usually known to be associated with gluten, particularly ω-5 gliadin and high-molucular-weight (HMW) glutenin subunits \[[@B12]\]. Although most recent studies suggested major roles of HMW glutenin subunits and ω-5 gliadin in WIA and WDEIA \[[@B13]\], low molecular weight (LMW) glutenin subunits are also known to be involved in immediate type childhood and adult wheat allergies, including WDEIA \[[@B11]\]. In the study by Park et al., 17 subjects with WIA or WDEIA showed strong positive specific IgE response to ω-5 gliadin allergens, but not to wheat, indicating that ω-5 gliadin is the causative allergen in adults with WIA \[[@B4]\]. Using the log-transformed specific IgE ratio of ω-5 gliadin to wheat, they found 100% sensitivity and specificity distinguishing patients with severe wheat allergy, such as WDEIA or WIA, from patients with other forms of wheat allergies in the 27 subjects. All of the patients in our cases also showed stronger specific IgE response to ω-5 gliadin than the response to wheat. In Korea, only a few studies reported the cases of wheat-induced anaphylaxis in adults. We report six cases of wheat-induced anaphylaxis diagnosed on the basis of clinical history and specific IgE of wheat proteins or provocation test. For immunologic evaluation of severe wheat allergy including WIA and WDEIA, it is important to measure specific IgE to each component of wheat including gluten and ω-5 gliadin rather than measuring wheat-specific IgE. ###### Clinical characteristics of the patients  WIA: wheat-induced anaphylaxis, BA: bronchial asthma, AR: allergic rhinitis, CU: chronic urticaria, WDEIA: wheat-dependent exercise-induced anaphylaxis. ###### Symptoms and signs of anaphylaxis  Y: yes, N: no. ###### Total serum IgE and specific IgE to wheat proteins using ImmunoCAP  WIA: wheat-induced anaphylaxis, WDEIA: wheat-dependent exercise-induced anaphylaxis, ND: not done. ###### Skin prick test with wheat and common allergens (food and inhalant) in the patients with WDEIA  WDEIA: wheat-dependent exercise-induced anaphylaxis, SPT: skin prick test, HDM: house dust mite.

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Introduction {#sec1} ============ Advanced therapy medicinal products belong to an emerging field in medical science. The field includes gene therapy, somatic cell therapy, tissue engineering, or combinations thereof. To develop such new approaches, several issues, such as target identification, determination and optimization of adequate methods, transport and implementation of new therapies, and advanced monitoring of the effect of the complete product, have to be addressed. For gene therapy, implementing the transfer of therapeutic nucleic acids, virus-based and non-viral delivery strategies have been explored. Adenovirus (Ad) is a widely used vector for gene transfer in gene therapy, which has been thoroughly studied regarding the structure and replication cycle.[@bib1], [@bib2] In terms of vector usage, Ads have experienced a long-time development that is still ongoing.[@bib3], [@bib4] From the first attempts to exploit virtual wild-type Ad, progress led to the development of the third-generation Ad vector that lacks almost all original viral DNA sequence and is termed high-capacity adenoviral (HCAdV) or helper-dependent adenoviral (HDAdV) vector.[@bib5], [@bib6] The vector production procedure, which is also applied in this study, is established to current good manufacturing practices standard (ClinicalTrials.gov: [NCT01433133](ctgov:NCT01433133){#intref0010} and [NCT00542568](ctgov:NCT00542568){#intref0015}).[@bib7], [@bib8], [@bib9] This and other advantages like the ability to transduce a large variety of dividing and nondividing cell types, lack of viral protein expression, capacity of up to 36 kb of foreign DNA, and a very low risk of insertional mutagenesis, because of an episomal status of vector genome in transduced cells, make it a very attractive delivery system.[@bib8] Attractive genetic cargos to be delivered for gene therapeutic approaches are designer nucleases like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), both acting as a protein dimer and requiring two separate expression cassettes, or the CRISPR/Cas9 system.[@bib10] Designer nucleases as tools for precise editing of DNA are handled as a new milestone in science, and efficient delivery is an inevitable issue to be solved. Several studies exemplified successful application of Ad vectors for the delivery of designer nucleases. For instance, ZFNs were delivered by adenoviral vectors.[@bib7], [@bib11], [@bib12], [@bib13] Similarly, intact TALEN sequences could be maintained as individual expression cassettes in first- and second-generation adenoviral vectors,[@bib14], [@bib15], [@bib16] as well as a complete TALEN pair in HCAdV.[@bib7], [@bib17] The CRISPR/Cas9 system was packed in early generation adenoviral vectors, too, including the expression cassettes of Cas9 and guide RNA (gRNA) apart[@bib18] or in combination.[@bib19], [@bib20], [@bib21], [@bib22] The latest development in the field of hepatitis B virus (HBV) gene therapy was the use of recombinant single-stranded adeno-associated viruses (ssAAVs) for co-delivery of Cas9 expression units, which was derived from *Staphylococcus aureus* (SaCas9) in combination with one gRNA.[@bib23] Here, the manufacturing of a multi-compound CRISPR/Cas9 construct harbored in one HCAdV was established and tested on its feasibility. The chronic HBV infection serves as an exemplary disease for the application of designer nucleases targeting multiple DNA sequences in a virus genome ([Figure 1](#fig1){ref-type="fig"}A). The HBV is an enveloped DNA virus, and the replication includes the formation of covalently closed circular DNA (cccDNA), a persistent DNA species in the nucleus of infected cells.[@bib24] The cccDNA is the major hurdle in chronic HBV infection. If the immune system fails to eliminate the virus during acute infection, the virus nests itself within the infected cells as episomal cccDNA ([Figure 1](#fig1){ref-type="fig"}B), leading to a chronic state of infection. Current therapies of chronic HBV infection target the process of viral replication or inhibit the inflammatory process to prevent continuing liver damage. Both strategies have in common that they require long-lasting medication with strict adherence to prevent reactivation of the infection.[@bib25] In this sense, a strategy to attack the persistent HBV DNA using gene-editing tools to induce mutations or even complete destruction of the HBV genome would provide a basis for advanced therapeutics to cure chronic HBV infection.Figure 1Overview of HBV Target Sites and Mode of Action of Anti-HBV Strategy(A) Schematic representation of the HBV genome with the unique EcoRI restriction enzyme site adjusted to the top, showing the location of the target sites of designer nucleases used in this study. The four viral open reading frames (ORFs) polymerase, surface antigen, X-protein, and core antigen are indicated in the inner circle. The S-/X-/C-TALEN labels on the outer circle mark the target sites designed for TALENs, which are based on No. 2, No. 5 and No. 6 RNAi target sites reported by McCaffrey et al.[@bib30] The target sites for the CRISPR/Cas9 system were adopted from Kennedy et al.[@bib26] (HBV-RT) and Lin et al.[@bib27] (P1 and XCp). (B) Illustration of the anti-HBV strategy against the background of the hepatitis B virus replication cycle. Upon entry of enveloped virions into liver cells, the capsid with the relaxed circular (rc) DNA is released into the cytoplasm and the DNA enters uncoated the nucleus. Here, the rc DNA is completed to form the covalently closed circular (ccc)DNA. The cccDNA can be eventually cleared out, silenced, or integrated into the host genome. Usually it is episomally maintained in the nucleus and is transcribed and translated by the host cell machinery. Subsequently, newly formed viral genomes are encapsulated together with the translated viral polymerase (P) through the translated capsid proteins (C). The nucleocapsid either migrates back to the nucleus to increase the pool of cccDNA or it is internalized by the endoplasmatic reticulum. In the latter process, it is enveloped with ER-membrane that already harbors translated viral surface proteins (S). Finally, it buds and is released from the cell. The cccDNA is the target for designer nucleases, which cut double-stranded DNA. The aim is to induce mutations or even degrade the cccDNA completely. RNAi acts on a later stage of replication, namely on the transcribed RNA. The RNA is marked for degradation through the host cell machinery, including the RNA-induced silencing complex (RISC), and, in this way, viral replication is shut down. (C) Enlargement of the target sites in the surface antigen ORF. TALEN-binding sites (TALEN S1 and TALEN S2), which were based on the previously published RNAi target site (HBVU6no.2) and Cas9 localization site (gRNA HBV RT), lie in close proximity and can be easily compared. In this study, complete nuclease systems with newly conceived TALEN arrays targeting the HBV genome or published chimeric gRNA sequences for the CRISPR/Cas9 system from Kennedy et al.[@bib26] and Lin et al.[@bib27] were introduced into HCAdVs. Both elements of a TALEN pair or the CRISPR/Cas9 system, including three gRNAs expressed from the Pol-III *U6* promoter and a Cas9 expression cassette, were cloned into a shuttle vector and then transferred into the HCAdV production plasmid. TALEN expression was driven from the liver-specific human alpha-1-antitrypsin (hAAT) promoter in combination with the HCR-1 (hepatic locus control region 1) of gene locus ApoE. Expression of the Cas9 endonuclease was controlled by a chicken β-actin short promoter (CBh). Activity of nucleases was either tested on cells transiently transfected with the 1.3× HBV genome containing plasmid pTHBV2,[@bib28] in HepG2.2.15 cells with a stably integrated HBV genome,[@bib29] or in HBV-infected HepG2-sodium-taurocholate cotransporting polypeptide (NTCP) cells (C. Ko, A. Chakraborty, W.-M. Chou, J.M. Wettengel, D. Stadler, R.Bester, T. Asen, K. Zhang, J.A. McKeating, W.-S. Ryu, and U.P., unpublished data). Treated cells were evaluated in terms of viral protein production, viral DNA transcription, genome copy numbers by qPCR, and integrity by mutation detection assays. Results {#sec2} ======= Characterization of Designer Nucleases {#sec2.1} -------------------------------------- The TALENs used in this study were designed on the basis of RNAi target sequences published by McCaffrey and colleagues.[@bib30] The three target sites in the HBsAg-open reading frame (ORF) (S-TALEN), protein X-ORF (X-TALEN), and HBcAg-ORF (C-TALEN), respectively, were chosen based on the performance in the RNAi experiments on the one hand and on the other hand on the location far apart from each other on the HBV genome. Therefore, all HBV ORFs were affected at least once ([Figure 1](#fig1){ref-type="fig"}A). Appropriate target sites were found by applying the web-based software TAL Effector Nucleotide Targeter.[@bib31] The target sites were specifically designed for the HBV genome used in the experiments (subtype, ayw3; genotype, D3), but the conservation profile of the binding sites among the different HBV genotypes was also analyzed using representative genotypes ([Figure S1](#mmc1){ref-type="supplementary-material"}). TALENs were cloned utilizing Voytas lab Golden Gate cloning method,[@bib31] and the respective expression vectors pCS2TAL3DD and pCS2TAL3RR[@bib32] were modified such as TALEN expression is driven from the hAAT liver-specific promoter. Besides TALENs, three CRISPR/Cas9 gRNA sequences were adopted from published studies by Kennedy et al.[@bib26] (HBV-RT) and Lin et al.[@bib27] (P1 and XCp), and then they were cloned into expression vectors following the Zhang lab cloning protocol.[@bib33] Furthermore, all three gRNA expression cassettes were combined into the expression vector pShV, also including the Cas9 expression cassette.[@bib34] The detailed localization of the TALEN, CRISPR/Cas9, and small interfering RNA (siRNA) target sites in the HBsAg-ORF is shown in [Figure 1](#fig1){ref-type="fig"}C. Because of the close proximity of these target sites, comparisons of efficacy in this study were mainly based on this location. The functionality of the different constructs ([Figures S2](#mmc1){ref-type="supplementary-material"} and [S3](#mmc1){ref-type="supplementary-material"}) was tested after transient co-transfection with the HBV replication-competent plasmid pTHBV2 into HEK293 cells. Induction of specific nuclease-mediated mutagenesis was detected by a mismatch-selective endonuclease assay ([Figure 2](#fig2){ref-type="fig"}A), and the estimated gene modification was calculated using the formula developed by Miller et al.[@bib35] ([Table 1](#tbl1){ref-type="table"}). The three different TALEN pairs induced mutations at a comparable rate, ranging from 5.7% induction through the TALEN pair targeting the HBcAg-ORF and 9.4% induction through the TALEN pair targeting the protein X-ORF. To conduct a comparative study to a previously published HCAdV-delivered small hairpin RNA (shRNA) coding sequence against HBV (HBVU6no.2, here shRNA-S) by Rauschhuber et al.,[@bib36] we continued the work in this study with the TALEN pair directed against the HBsAg-ORF, which achieved a targeted disruption of 7.3% of the corresponding ORF.Figure 2Proof of Concept of Anti-HBV Efficacy of TALENs or CRISPR/Cas9(A) HEK293 cells were co-transfected with an HBV expression plasmid pTHBV2 and either of the complete nuclease systems. DNA was isolated after 4 days and examined for mutagenesis at target sites by T7E1 assay. Undigested and digested products were separated on an agarose gel side by side for comparison. Expected cleavage product sizes are indicated by arrowheads. "M" indicates the molecular weight marker lane. (B) Huh7 cells were co-transfected with an HBV expression plasmid pTHBV2 and TALEN or CRISPR/Cas9 nuclease systems. HBsAg concentrations in the supernatant were measured after 4 days by ELISA. Data are represented as means of S/CO (sample to control ratio) values and error bars indicate SD of three replicates. Statistically significant differences to HBV only are indicated by an asterisk (\*p \< 0.01).Table 1Genome-Editing Efficacy Estimated by T7E1 Digest and Sequence Trace DecompositionS (%)X (%)C (%)HBV-RT (%)P1 (%)XCp (%)T7E1[@bib35]7.39.45.714.09.43.0TIDE[@bib37]37.49.210.1 Co-transfection of the CRISPR/Cas9 construct containing all three gRNA expression cassettes with pTHBV2 into HEK293 cells resulted in different target disruption efficiencies for the different target sites. The gRNA HBV-RT, which was directed to the coding sequence of the YYMD motif of the catalytic domain of HBV polymerase and at the same time also to the HBsAg-ORF, performed best with a targeted disruption of 14.0%. The gRNAs published by Lin et al. (P1 and XCp) were less efficient, with mutation induction percentages of 9.4% for P1 and 3.0% for XCp. The CRISPR/Cas9 genome-editing efficacy was additionally quantified by sequence trace decomposition[@bib37] ([Table 1](#tbl1){ref-type="table"}). Here, the targeted disruption percentage was confirmed for gRNA P1 with an efficiency of 9.2%. In contrast, the results for HBV-RT and XCp were about three times higher compared to the efficiencies determined by the mismatch-selective endonuclease assay. The gRNAs HBV-RT and XCp achieved 37.4% and 10.1% targeted disruption, respectively, as measured by sequence trace decomposition. In a next step, the expression cassettes of the left and right TALEN subunits directed against the HBsAg-ORF were combined in one plasmid under the control of the liver-specific hAAT promoter. After co-transfection of nuclease-expressing plasmids with pTHBV2 into the human liver cancer cell line Huh7, concentrations of HBsAg were measured with an enzyme immunoassay ([Figure 2](#fig2){ref-type="fig"}B). HBsAg concentrations were significantly reduced after treatment of the cells with the CRISPR/Cas9 system directed by the three gRNAs HBVRT, P1, and XCp in combination. In contrast, the TALEN pair directed against the HBsAg ORF caused no significant reduction of HBsAg concentration in the supernatant. Characterization of HCAdV Vector Constructs {#sec2.2} ------------------------------------------- Vectors containing the triple-guide RNA construct or the double HBsAg-TALEN construct were produced using a well-established protocol[@bib9], [@bib38] employing helper virus (HV) co-transductions for HCAdV amplification in the producer cell line 116.[@bib39] Production was finalized in purified large-scale batches of HCAdV, which were analyzed regarding concentration, purity, and vector integrity. After purification of the HCAdV, a basic characterization was performed based on the concentration of the HCAdV by optical density (viral particles \[vps\]) and the level of infectious particles of HCAdV versus HV in final vector preparations quantified by qPCR (infectious units \[IUs\]). For the vp titer, viral DNA was extracted from the vector preparation, and the OD~260~ was measured and converted to number of particles per milliliter by use of the extinction coefficient of wild-type adenovirus, as determined by Maizel et al.[@bib40] The IUs were identified by virus-selective qPCRs on genomic DNA isolated from cells 3 hr post-transduction. In [Table 2](#tbl2){ref-type="table"} the titers of different vector preparations are summarized, and parameters as particle-to-infectious unit ratio (vp:IU) for HCAdV, representing the infectivity of the HCAdV, and infectious unit-to-particle ratio (IU:vp) for HV, as an indication of HV contamination, are provided. The Food and Drug Administration has recommended that the infectivity of clinical grade Ad vector be \<30:1,[@bib41] and this objective was met by the vector preparations. Furthermore, low HV contamination levels (\<0.2%) could be achieved.Table 2Characterization of Virus PreparationsTotalHCAdVHelper Virusvp/mLIU/mLvp:IUIU/mLIU/vp (%)HCAdV-FTC/CBh-Cas9-VAI-RT-P1-XCp1.20E+115.19E+0923:11.44E+080.12HCAdV-FTC/hAAT-TALEN-S3.02E+111.07E+1028:12.50E+070.01 HCAdVs harboring TALEN sequences or the triple-gRNA construct were tested on genomic integrity by NotI and PspXI restriction enzyme co-digest of isolated viral DNA ([Figure 3](#fig3){ref-type="fig"}A). As controls, isolated HV DNA and isolated original empty and full vector plasmid DNA were digested and analyzed by agarose gel electrophoresis. The digested viral genomes revealed on the gel the band patterns that resembled their parental vector plasmids without the bacterial plasmid backbone. This was an indication that DNA rearrangements had been successfully avoided. In addition, the lanes containing the viral genomes clearly differed from the lane containing the digested HV genome, substantiating the titration outcomes. All vectors used in this study are summarized in [Figure 3](#fig3){ref-type="fig"}B.Figure 3Characterization of Vector Genome Structures in Final Vector Preparations(A) The results of NotI and PspXI restriction enzyme analyses of virion DNA of indicated vector preparations (HCAdV-Cas9-VAI-RT-P1-XCp and HCAdV-TALENs) and helper virus (HV) in comparison to parental plasmids (pAdFTC-Cas9-VAI-RT-P1-XCp and pAdFTC-TALENs) are shown. pAdFTC is the original plasmid containing the HCAdV genome without insert. Bands referenced with numbers on the left side of the gel represent the following: (1) the stuffer DNA, (2) the hAAT-TALEN-S insert, (3) the pAdFTC bacterial backbone, and (4) the Cas9-VAI-RT-P1-XCp insert. "M" indicates the molecular weight marker. (B) Schematic illustration of recombinant adenoviral vector genomes used in the study. HCAdV-Cas9-VAI-RT-P1-XCp, the CRISPR/Cas9 system was expressed from the CBh promoter; gRNAs RT, XCp, and P1 were expressed under the control of the U6 promoter; HCAdV-TALENs were expressed from the tissue-specific human alpha-1-antitrypsin (hAAT) promoter; HCAdV-HBVU6no.2, expresses a small hairpin RNA against HBsAg and the human coagulation factor IX (hFIX) under the control of the hAAT promoter; HCAdV-CRISPR-mock, instead of expressing gRNAs against HBV as shown in HCAdV-Cas9-VAI-RT-P1-XCp, this vector expresses an irrelevant gRNA (U6-HPV). Inhibition of HBV Infection *In Vitro* {#sec2.3} -------------------------------------- After producing HCAdVs that express either one of the nuclease systems, our vectors were challenged in infection models of HBV. Transduction of HepG2.2.15 cells with nuclease-expressing vectors for 9 days resulted in a reduction of HBsAg secretion of about 54% by the CRISPR/Cas9 system and about 14% by the previously published RNAi vector in relation to untreated HepG2.2.15 cells ([Figure 4](#fig4){ref-type="fig"}A). The TALEN vector did not induce any reduction of HBsAg secretion as well as the control CRISPR-mock vector. Measurement of secreted extracellular HBV DNA molecules by qPCR revealed a prominent decline at day 9 down to one-20th of the reference level in untreated cells ([Figure 4](#fig4){ref-type="fig"}B). In comparison, the HBV DNA levels in the medium of mock-treated cells ranged at three-quarters of the reference level. Intracellular HBV DNA levels were also analyzed by qPCR ([Figures 4](#fig4){ref-type="fig"}C and 4D). In untreated HepG2.2.15 cells, about 500 HBV DNA molecules were detected per cell. Interestingly, this number was raised about 4-fold in mock-treated cells. In contrast, the HBV-specific CRISPR/Cas9 system induced a decline to about 150 copies per cell. The HBV sequence amplified by qPCR was located outside of the nuclease-induced mutations, providing evidence for a mechanism of reduction of HBV DNA quantity. A corresponding outcome was found for cccDNA determination by cccDNA-selective qPCR from T5 exonuclease-digested samples. Interestingly, treatment with the CRISPR-HBV vector reduced the cccDNA content to 0.3 copies per cell, indicating a statistical clearance of 70% of the cells from cccDNA. To verify the required non-cccDNA elimination in the aforementioned assay, PCR products of T5 endonuclease-digested and undigested templates were separated on an agarose gel ([Figure 4](#fig4){ref-type="fig"}E). Reduction of PCR products after T5 endonuclease treatment proved the digestion of template DNA, which was not in a supercoiled circular state. In line with the premise that HBV-transgenic mouse serum does not contain cccDNA, there was no amplicon detected in T5 endonuclease-treated serum samples.Figure 4Inhibition of HBV in HepG2.2.15 CellsEither of the complete nuclease systems were delivered into cells by HCAdV and incubated for 9 days. (A) HBsAg concentrations in supernatant were measured at the indicated time points by ELISA. All measurements were performed with medium that has been on cells for 3 days. Data are represented as means of S/CO (sample to control ratio) values relative to HBV only, and error bars indicate SD of three replicates. Statistically significant differences to HBV only are indicated by an asterisk (\*p \< 0.01). (B) Extracellular HBV DNA was quantified by qPCR in extracted DNA from the same medium samples, which were examined by ELISA. HBV DNA was quantified relative to HBV-only samples using the ΔCt method. Data represent means of ΔCt ratios. Statistical differences in Ct values in comparison to HBV-only samples are indicated by an asterisk (\*p \< 0.01). (C) Intracellular HBV DNA was quantified by qPCR in extracted DNA from cells harvested on day 9 post-transduction. HBV DNA was quantified relative to the *B2M* reference gene using the 2^−ΔΔ^Ct method. Data represent means of HBV DNA copy numbers per cell, and error bars indicated SD of three replicates. Statistical differences in Ct values in comparison to HBV-only samples are indicated by an asterisk (\*p \< 0.01). (D) HBV cccDNA was quantified by qPCR in extracted and T5 exonuclease-digested DNA from cells harvested on day 9 post-transduction. cccDNA was quantified relative to the *B2M* reference gene in undigested but similarly treated templates using the 2^−ΔΔ^Ct method. Data represent means of cccDNA copy numbers per cell, and error bars indicate SD of three replicates. Statistical differences in Ct values in comparison to HBV-only samples are indicated by an asterisk (\*p \< 0.01). (E) PCR products from HBV templates of the transduction experiment or HBV-transgenic mouse serum with or without T5 exonuclease treatment prior to PCR to verify non-cccDNA elimination. To further evaluate the effect of the designer nuclease-bearing vectors, HepG2-NTCP cells were infected with HBV and transduced with the respective constructs. This HBV model mirrors in a more accurate way the HBV infection, as the viral DNA is derived from the natural HBV and expression of viral genes and replication of the genome depend on the newly formed cccDNA.[@bib42] After infection with HBV and subsequent transduction with the therapeutic or control vectors, HBeAg ([Figure 5](#fig5){ref-type="fig"}A) and HBsAg ([Figure 5](#fig5){ref-type="fig"}B) concentrations were measured 3 and 6 days post-transduction as parameters of viral gene expression. After 3 days, the therapeutic CRISPR-vector induced a 37% reduction, and, after 6 days, a 45% reduction in HBeAg concentration relative to HBV-only-treated cells. The mock-CRISPR vector also lowered the HBeAg secretion, but not to a statistically significant extent. The TALEN vector did not alter the expression of HBeAg at all in comparison to HBV-only-treated cells. The HBsAg levels after treatment were only measured on day 6 in therapeutic CRISPR-treated and HBV-only samples. The reduction of 76% of HBsAg concentration in the supernatant relative to HBV-only-treated cells was more prominent than the measured loss of HBeAg. The mock treatment induced a similar effect for HBsAg as for HBeAg. All significant differences to the reference antigen level in HBV-only-treated samples are indicated by an asterisk in [Figures 5](#fig5){ref-type="fig"}A and 5B. The results are in accordance with the change in total HBV transcripts measured in isolated RNA of treated cells ([Figure 5](#fig5){ref-type="fig"}C). CRISPR/Cas9-treated cells with therapeutic gRNAs showed a significant reduction of total HBV transcripts of 54% on day 3 and 64% on day 6, respectively, relative to HBV-only-treated cells. In line with the previous results was the loss of total HBV DNA content in therapeutic CRISPR-treated cells of 39% on day 3 and 78% on day 6, respectively, relative to HBV-only-treated cells. Of note, here too the mock treatment resulted in an elevated HBV DNA level on day 3 but without statistical significance ([Figure 5](#fig5){ref-type="fig"}D).Figure 5Inhibition of HBV in HepG2-NTCP CellsCells were infected with HBV 4 days prior to transduction of either of the complete nuclease systems by HCAdV and then incubated for 3 or 6 days. (A and B) HBeAg (A) and HBsAg (B) concentrations in supernatant were measured at the indicated time points by ELISA. All measurements were performed on medium that was on cells for 3 days. Data are represented as means of S/CO (sample to control ratio) values relative to HBV only, and error bars indicate SD of three replicates. Statistically significant differences to HBV only are indicated by an asterisk (\*p \< 0.01). (C) Total HBV RNA was measured by qRT-PCR from cells 3 and 6 days after transduction with the indicated vectors. RNA was quantified relative to the *GAPDH* reference gene using the 2^−ΔΔ^Ct method. Data represent means of HBV ratios relative to HBV-only treated samples, and error bars indicate SD of three replicates. Statistical differences in Ct values in comparison to HBV-only samples are indicated by an asterisk (\*p \< 0.01). (D) Intracellular HBV DNA was quantified by qPCR in extracted DNA from cells harvested on day 3 and 6 days after transduction. HBV DNA was quantified relative to the *B2M* reference gene using the 2^−ΔΔ^Ct method. Data represent means of HBV DNA copy numbers per cell, and error bars indicate SD of three replicates. Statistical differences in Ct values in comparison to HBV-only samples are indicated by an asterisk (\*p \< 0.01). Discussion {#sec3} ========== Gene-editing techniques provide a whole new repertoire of tools to correct certain maladies for which no option of treatment or cure at all exists. A critical issue in the development of new treatment strategies is the delivery of the therapeutic agent. Here we present a delivery strategy based on packaging expression units into adenoviral vectors. HCAdV vectors represent an advanced vehicle option for diverse and large genetic cargos. Their capacity and lower immunogenicity compared to earlier adenoviral vector generations belong to the main advantages.[@bib43], [@bib44] The lowered immunogenicity of these vectors counts only for the acquired immune system native for the applied serotype, because the viral gene expression is omitted. However, the immunogenicity of the incoming capsid is still present. Moreover, the vector used in this study is derived from human adenovirus type 5 (Ad5), which is the most commonly used type in gene therapy but also has high prevalence of pre-existing immunity in human populations. It is possible to bypass the immune response induced by the incoming viral proteins, for example, by shielding the viral particle with polyethylene glycol (PEG).[@bib45] In addition, PEG modification counteracts vector sequestration in the bloodstream.[@bib46] Other approaches include capsid modification to ablate blood coagulation factor X binding to the hexon[@bib47], [@bib48] or vector pseudotyping, in which Ad5 vectors are engineered with capsid parts from other serotypes with low pre-existing immunity to circumvent vector neutralization and, more importantly, acute toxicity.[@bib49], [@bib50] A comparable strategy for vector optimization is to completely switch the vector serotype to a rare type.[@bib51] A study from Wang et al. hypothesized that the cargo itself might pose another challenge by induction of Cas9-specific immune responses.[@bib52] By using adenoviral vectors, the transferred genetic material is delivered in an episomal and transient way. On the one hand this can be advantageous with respect to limitation of the cargo immunogenicity, but on the other hand the dosage has to be carefully evaluated to guarantee the desired effect. Translational targeting as it was implemented here by using a liver-specific promoter is a strategy to counteract undesired side effects in cells other than the hepatocytes.[@bib53], [@bib54] Another method for translational targeting is the post-transcriptional downregulation of transgenes.[@bib7] Such an approach would require the comparison of the microRNA (miRNA) expression profile in the optimal case from HCAdV- and HBV-infected primary hepatocytes against a panel of cells from various human cell types in order to find candidate miRNAs that downregulate the transgene expression in cells other than hepatocytes. Chronic HBV infection is still a worldwide threatening disease with no complete reliable cure. Although the lifespan of patients can be extended to a normal level with current therapeutics, the quality of life is impeded by regular medication administration and side effects. In addition, the elevated risks for liver damage and liver cancer accompanying the HBV infection are not restored to normal risk levels because of the sustained impact of the suppressed infection. A potential solution to this problem might be achieved by designer nuclease-based therapy, which promises the complete eradication of latent HBV genomes. The effective eradication of the HBV genome from infected cells can also decrease the risk of a superinfection with the hepatitis delta virus, which is dependent on HBsAg production. This is in contrast to nucleos(t)ide analog RT inhibitors, which repress reverse transcription of the HBV genome but leave viral protein translation unaffected. The approach applied in this study includes the delivery of expression units of three gRNAs along with one Cas9 in a one-vector system. In comparison to a recently published study, which utilizes ssAAVs to deliver one gRNA and one Cas9 expression unit from *Staphylococcus aureus*,[@bib23] this approach promises a more radical attack against the HBV genome. This study also confirmed the observation of others that the HBV cccDNA was rather degraded than mutated by CRISPR/Cas9 cleavage.[@bib55], [@bib56] In theory, the probability of complete degradation of HBV cccDNA increases with every gRNA, and additionally multi-species targeting is possibly independent from consensus sequences. The presented reductions of HBV markers of replication in this study never went below the detection limit, which can be achieved with current therapeutic treatments. Possible reasons for this are that either a relatively short time frame was examined or data were collected in a model with high HBV content and cell turnover, which may lead to higher thresholds to asses significant activity. Nevertheless, the reductions in these experimental settings give reason to initiate further studies in a clinically relevant HBV infection animal model *in vivo*. Comparison of TALENs with the CRISPR/Cas9 system in this study showed fewer efficacies of the TALENs in the context of the viral vector, although mutational activity was equal in transfection experiments. Of note, transfection experiments in a liver cell line also showed no effect from the TALEN system, but it was hypothesized that this was due to poor transfection efficiencies. Therefore, it was tried to enhance their potency by vectorization, which turned out unsuccessful in the later experiments. This suggests that there are difficulties with the vectorization of TALENs, which are not encountered with the CRISPR/Cas9 system, and we hypothesize that multiple factors may be responsible for the non-performance of HCAdV-TALEN vectors. It could be speculated that these issues are caused by the repeated sequences present in the repeat variable diresidues (RVDs). Although we did not observe major rearrangements in the analytic restriction enzyme digest, the occurrence of smaller deletions of, for instance, RVDs cannot be excluded. We performed Sanger sequencing only into the ends of the TALEN sequences, but not over the repetitive structures. Another important issue could be the used promoter driving expression of designer nucleases. In plasmid-based experiments, we used the strong CMV promoter to drive expression of TALENs, and in HCAdV-mediated TALEN delivery, we applied the tissue-specific hAAT promoter. In contrast, Cas9 in the context of the CRISPR/Cas9 system was expressed from the strong CBh promoter in both non-viral and HVAdV approaches. The designer nuclease systems were additionally compared to a previously vectorized shRNA expression cassette.[@bib36] Here we could find equal to or superior suppression of antigen expression by the CRISPR/Cas9 system. Furthermore, because of the mutational effect of the designer nucleases, we believe the designer nuclease-mediated reduction to be a more permanent effect than the one by inhibition by RNAi. Here we found a reduction of cccDNA copy numbers to less than one copy per three HepG2.2.15 cells. In concordance with previous studies,[@bib23] we speculate that this may be due to definite loss of cccDNA caused by degradation. It is of note that HepG2.2.15 cells are considered to contain about 10 copies of cccDNA per cell,[@bib57], [@bib58] but here only 1.3 copies per cell were measured in untreated samples, and, therefore, copy numbers are potentially underestimated. This may have a methodological reason but this phenomenon needs to be analyzed in further detail. Furthermore, we observed a difference for quantified total intracellular and extracellular HBV DNA and the amount of cccDNA ([Figure 4](#fig4){ref-type="fig"}). Especially the intracellular HBV DNA level in CRISPR-mock-treated HepG2.2.15 cells was significantly higher than those in the untreated control. That adenovirus can increase HBV infection parameters, such as HBsAg levels, in mice shortly after infection was also observed in another study in mice.[@bib36] To further shed light on this phenomenon, it is of note that previous studies suggested that adenovirus types binding to α~v~ integrins as co-receptor, such as Ad5 used in the present study, activate several signaling proteins, such as phosphoinositide-3-OH kinase (PI3K).[@bib59] PI3K plays a role in the autophagy pathway, which is also involved in the HBV DNA replication.[@bib60] Thus, circumventing activation of this signaling pathway by using alternative adenovirus types (e.g., serotypes 40 and 41[@bib61]) may, therefore, further increase CRISPR activity in adenovirus-based approaches. In summary, this study can be considered as a valuable improvement to the already known state of the art, which expands the possible use of the CRISPR/Cas9 system in the context of viral vectors by the inclusion of multiple gRNAs. Beyond that, the addition of further elements like immunomodulatory factors is imaginable to improve the system even more in the combat against chronic HBV infection. Future objectives are to test our vectors in animal models of HBV infection and eventually to optimize the vector for the needs of human application. Materials and Methods {#sec4} ===================== Cell Culture {#sec4.1} ------------ The cell line HepG2.2.15 (stably transfected with the HBV genome) was cultured in William's Medium E (PAN-Biotech, Aidenbach, Germany), supplemented with 10% fetal bovine serum (FBS, Standard Quality, EU approved, PAA, Pasching, Austria), 1× minimum essential medium (MEM) with non-essential amino acid (NEAA) solution (100×) without L-Glutamine (PAN-Biotech, Aidenbach, Germany), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Pen/Strep, PAN-Biotech, Aidenbach, Germany). Cell culture vessels were coated with collagen A (Biochrom, Berlin, Germany) prior to seeding. The human hepatoma cell line Huh7 was maintained in DMEM (PAN-Biotech, Aidenbach, Germany) supplemented with 10% FBS, 1× MEM NEAA, and Pen/Strep. HEK293 cells were grown in DMEM supplemented with L-glutamine, 10% FBS, and Pen/Strep. The HCAdV producer cell line 116, which is based on the HEK293 cell line stably expressing Cre recombinase,[@bib39] was cultured in MEM Eagle with Earle's balanced salt solution (PAN-Biotech, Aidenbach, Germany) supplemented with 10% FBS, 100 μg/mL hygromycin B (PAN-Biotech, Aidenbach, Germany), and Pen/Strep. The FBS concentration in the medium was reduced to 5% and antibiotics were omitted during virus production. This cell line is obtainable from Philip Ng (Baylor College of Medicine, Houston, TX). HepG2-NTCP-K7 cells, which stably overexpress the NTCP as an entry receptor for HBV, were cultured on collagen-coated plates in full DMEM (10% fetal calf serum \[FCS\], 1% Pen/Strep, 1% sodium pyruvate, 1% NEAA, and 1% Glutamin; Life Technologies, Darmstadt, Germany). Before HBV infection, the culture medium was replaced with differentiation medium (full medium plus 2.5% DMSO; Sigma-Aldrich, Taufkirchen, Germany). Cells were infected with HBV 100 genome equivalents/cell (GEq/cell) in differentiation medium plus 4% PEG6000. All cell lines were maintained in a humidified incubator at 37°C and 5% CO~2~. Plasmid Construction {#sec4.2} -------------------- TALEN pairs for HBsAg (S), HBcAg (C), and protein X (X) knockout were designed by the TALEN Targeter program (<https://tale-nt.cac.cornell.edu/>). The target sequences of the binding domains were for HBsAg 5′-T-CAGCCCGTTTCTCCTGGCTC and 5′-T-ACGAACCACTGAACAA with a 15-bp spacer, for HBcAg 5′-T-AGACGACGAGGCAGGTCCCC and 5′-T-GAGACCTTCGTCTGCGAGGC with a 17-bp spacer, and for protein X 5′-T-CTCTTTACGCGGACTCCCC and 5′-T-GCACACGGTCCGGCAGA with a 14-bp spacer sequence in between. TAL repeats were assembled by the Golden Gate TALEN and TAL Effector Kit 1.0 supplied by the Daniel Voytas laboratory (no longer available at Addgene, Cambridge, MA)[@bib31] and cloned into the TALEN expression cassette of pCS2TAL3DD or pCS2TAL3RR,[@bib32] which were before cloned into the multiple cloning site of a pBluescript-based vector.[@bib62] Promoters were exchanged with an hAAT liver-specific promoter by restriction enzyme cloning. Therefore, the hAAT promoter was amplified by PCR and ligated into the PstI restriction enzyme site of the respective vector. The resulting plasmids were named pBS-p/p-dNotI-hAAT-TALEN-DD-S1 and pBS-p/p-dNotI-hAAT-TALEN-RR-S2. Three gRNA sequences were adopted from published sources[@bib26], [@bib27] targeting either the ORF of HBV polymerase in the highly conserved reverse transcriptase domain (HBV-RT, TTCAGTTATATGGATGATG), in the RNase H domain (P1, GTTTTGCTCGCAGCAGGTCT, with one position adjusted to the HBV genome sequence used in this project), and the ORF of protein X (XCp, GGGGGAGGAGATTAGGTTAA). The HBV-specific gRNAs were cloned into pX330-U6-Chimeric_BB-CBh-hSpCas9 (42230, Addgene, Cambridge, MA), following the protocol from the Feng Zhang laboratory.[@bib33] The intermediate shuttle plasmid pShV[@bib34] was constructed by insertion of a synthetic DNA fragment into the pEX-K plasmid (Eurofins, Ebersberg, Germany). The synthetic DNA fragment is composed of a multiple cloning site (MCS), which is flanked by a 112-bp and a 116-bp non-coding random DNA sequence that do not share similarity with natural DNA sequences. They function as homology arms (HAs) for site-directed homologous recombination into the HCAdV genome contained in a bacterial artificial chromosome (BAC).[@bib63] The HA-flanked MCS is additionally flanked by the recognition sites for the homing endonucleases I-CeuI and PI-SceI, enabling the cloning into the HCAdV genome contained in the previously established plasmid pAdFTC.[@bib62] Outside of the I-CeuI and PI-SceI restriction sites, SwaI sites were added to release the synthetic DNA fragment from the pEX-K plasmid. The plasmid backbone was then digested with NotI to remove the original MCS of pEX-K, blunted, and ligated with the released synthetic DNA fragment. The resulting plasmid pShV served as the basis to create intermediate shuttle plasmids for cloning into the HCAdV genome. To clone a TALEN pair-containing construct, the blunted SacII fragment from pBS-p/p-dNotI-hAAT-TALEN-DD-S1 containing the left TALEN targeting the HBsAg ORF was cloned into the HincII restriction site of pShV. Next, the blunted SacII fragment from pBS-p/p-dNotI-hAAT-TALEN-RR-S2 containing the right TALEN targeting the HBsAg ORF was cloned into the EcoRV restriction site of pShV-hAAT-TALEN-S1, generating the intermediate plasmid pShV-hAAT-TALEN-S. To generate the intermediate plasmid containing the CRISPR/Cas9 system directed against HBV, the blunted XbaI/NotI fragment containing the hSpCas9 from pX330-U6-Chimeric_BB-CBh-hSpCas9 (Feng Zhang, plasmid 42230, Addgene, Cambridge, MA) was cloned into the EcoRV restriction site of pShV. The three gRNA expression cassettes were then subsequently cloned into pShV-CBh-hSpCas9. For this purpose, the expression units were amplified by PCR and restriction sites added by using primers with either NheI- (gRNA-NheI-fwd 5′-ATGCTAGCGAGGGCCTATTTCCCATGATTCC-3′; gRNA-NheI-rev 5′-ATGCTAGCCTGCAGAATTGGCGCACG-3′) or BamHI-restriction enzyme recognition sites (gRNA-BamHI-fwd 5′-ACTGGGATCCGAGGGCCTATTTCCCATGATTCC-3′; gRNA-BamHI-rev 5′-TGAGGATCCTGCAGAATTGGCGCACG-3′) at either end of the amplicon, respectively. First, the expression cassette for the HBV-RT gRNA was amplified using the NheI-primer set, digested with NheI, and cloned into the AvrII restriction enzyme site of pShV-CBh-hSpCas9, resulting in pShV-CBh-hSpCas9-RT. Second, the expression cassette for the P1 gRNA was amplified using the NheI-primer set, digested with NheI, and cloned into the NheI restriction enzyme site of pShV-CBh-hSpCas9-RT, resulting in pShV-CBh-hSpCas9-RT-P1. Third, the expression cassette for the XCp gRNA was amplified using the BamHI-primer set, digested with BamHI, and cloned into the BamHI restriction enzyme site of pShV-CBh-hSpCas9-RT-P1, resulting in pShV-CBh-hSpCas9-RT-P1-XCp. The I-CeuI/PI-SceI insert fragments of the different intermediate shuttle plasmids were cloned into the I-CeuI/PI-SceI of pAdFTC,[@bib62] resulting in the respective pAdV-FTC/insert plasmids pAdV-FTC/hAAT-TALEN-S and pAdV-FTC/CBh-hSpCas9-RT-P1-XCp. Adenovirus Production {#sec4.3} --------------------- The pAdFTC/insert plasmids were digested with NotI to linearize the pAdFTC/insert vector DNA and transfected with FuGENE 6 Transfection Reagent (Promega, Madison, WI) into 116 cells.[@bib39] At 18 hr post-transfection, cells were transduced with the first-generation HV AdNG163R-2[@bib39] containing a packaging signal flanked by loxP sites. To increase the titer of the HCAdV, five serial passages in 116 cells were performed. The virions containing the HCAdV-hAAT-TALEN-S or HCAdV-CBh-hSpCas9-RT-P1-XCp (in short HCAdV-CRISPR-HBV) vector genomes were purified by CsCl gradients. After one CsCl step gradient and one equilibrium gradient, the HCAdV vectors were purified at high titer (3E+09--1E+10 transducing particles per milliliter), with HV contamination lower than 0.2%. The genome integrity of the adenovirus vector preparations was tested on virion DNA, which was isolated with a proteinase K-SDS lysis solution (1×TE, 0.5% SDS, and 0.1 μg/μL proteinase K) for 2 hr at 56°C, precipitated with ethanol, and then resuspended in 20 μL ddH~2~O. Analytical restriction enzyme digests, PCRs amplifying junctions in the vector construct, and Sanger sequencing were performed on viral DNA. Transfection and Transduction Experiments to Test the Efficacy of Designed Plasmids and Vectors {#sec4.4} ----------------------------------------------------------------------------------------------- Plasmid constructs were co-transfected with an HBV replication-competent plasmid, pTHBV2[@bib28] with FuGENE 6 Transfection Reagent (Promega, Madison, WI), into HEK293 cells or Huh7 cells, according to the manufacturer's instructions. Briefly, cells were plated in a 24-well plate 1 day before transfection to reach approximately 50%--80% confluency on the day of transfection. In total, 900 ng plasmid DNA was transfected per well using 2.7 μL transfection reagent. A molar target plasmid-to-nuclease plasmid ratio of 1:5 was used and adjusted to 900 ng with unrelated plasmid DNA as stuffer DNA. Transfected cells were incubated for 4 days, harvested for genomic DNA isolation with a proteinase K-SDS lysis solution (10 mM Tris-Cl \[pH 8.0\], 100 mM EDTA \[pH 8.0\], 50 mM NaCl, 0.5% SDS, 20 μg/mL RNase, and 100 μg/mL Proteinase K),[@bib64] and HBsAg was measured in the supernatant. To analyze HBV knockdown by vector-delivered nucleases in the context of an established HBV infection, HepG2.2.15 cells were transduced for 9 days with HCAdV-FTC/CRISPR-mock (a CRISPR/Cas9 vector directed against HPV; E.E.-S., unpublished data), HCAdV-FTC/TALEN-S, HCAdV-FTC/HBVU6no.2 (an RNAi vector directed against HBV, which is here called shRNA-S[@bib36]), or HCAdV-FTC/CRISPR-HBV at an MOI of 200. Medium was collected every third day and renewed from the day of transduction (d0). Therefore, cells were washed before new medium was added. Total DNA was isolated on day 9 post-transduction. HBsAg was measured in the cell culture supernatant of Huh7 and HepG2.2.15 cells in a 1:10 dilution in PBS using the Monolisa HBs Ag ULTRA kit (Bio-Rad, CA). HBV-infected HepG2-NTCP-K7 cells were transduced with the same vectors at an MOI of 300. Medium and cells were harvested on days 3 and 6 post-transduction with HCAdVs. The medium was tested for HBeAg concentrations (Siemens Molecular Diagnostics, Marburg, Germany) and HBsAg concentrations (Abbott Laboratories, Chicago, IL). DNA and RNA were isolated from cells with the NucleoSpin Tissue Kit (Macherey-Nagel, Düren, Germany) and the NucleoSpin RNA Kit (Macherey-Nagel, Düren, Germany), respectively. Cell viability of NTCP-HepG2 cells was monitored before harvest with the CellTiter-Blue Reagent (Promega, Madison, WI), according to the manufacturer's instructions ([Figure S4](#mmc1){ref-type="supplementary-material"}). Mutation Detection {#sec4.5} ------------------ For the T7 endonuclease 1 (T7E1) (New England Biolabs, MA) mutation detection assay, DNA sequences that span the target sites were amplified from purified genomic DNA by PCR using standard conditions for the OneTaq 2X Master Mix with Standard Buffer (New England Biolabs, MA), with the following primer sets: S/HBV-RT, S-T7 for 5′-ttcctcttcatcctgctgct-3′ and S rev 5′-tgtaaaaggggcagcaaaac-3′; X, No. 5-forw 5′-actcctagccgcttgttttg-3′ and No.5-rev 5′-ataagggtcgatgtccatgc-3′; C, C-T7 for 5′-ctgggtgggtgttaatttgg-3′ and No.6-rev 5′-tacccgccttccatagagtg-3′; P1, P1_F 5′-gctttcactttctcgccaac-3′ and P1_R 5′-accttgggcaatatttggtg-3′; and XCp, XCp_F 5′-actctctcgtccccttctcc-3′ and XCp_R 5′-gcctgagtgcagtatggtga-3′. The PCR products were desalted by ethanol precipitation and resuspended in 1× NEB2 buffer (New England Biolabs, MA). Next, the PCR products were denatured and annealed to promote heteroduplex formation using the following program: 95°C-2 min, 95°C→85°C (Δ 2°C/s), 85°C→25°C (Δ 0.1°C/s), and 16°C. T7E1 was added to the reaction and incubated for 15 min at 37°C. The reaction was stopped using a gel loading dye containing SDS and then resolved by electrophoresis on a 2% agarose gel for 1 hr at 100 V. The gel was analyzed with the Gel Doc EZ System (Bio-Rad, CA) and Image Lab Software Version 5.2 (Bio-Rad, CA). The gene modification rates were estimated using the following approximation: fractional modification = (1- (1- (fraction of cleaved bands))^1/2^), as introduced by Miller et al.[@bib35] For quantification of the editing efficacy by a method named TIDE (tracking of indels by decomposition), the same PCR products were analyzed by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany) and the chromatograms analyzed using the TIDE online tool.[@bib37] Quantification of HBV Total DNA, cccDNA, and Total RNA {#sec4.6} ------------------------------------------------------ Total extracellular and intracellular DNA was quantified in duplicates from three separate samples by qPCR using My-Budget 5x EvaGreen QPCR-Mix II (Bio-Budget Technologies, Krefeld, Germany) in a 10-μL reaction volume, including 5 μL sample volume, according to the manufacturer's instructions, with the following primer set: C_qRT-for 5′-tagaagaagaactccctcgcctcg-3′ and C-T7 rev 5′-cccagtaaagttccccacct-3′. qPCR was performed on the CFX96 Touch System (Bio-Rad, CA) using the following program: 95°C-15 min; 40× (95°C-15 s, 59°C-20 s, 72°C-20 s, plate read); melt curve 65°C→98°C (Δ 0.5°C/5 s). Ct values were determined by use of the Bio-Rad CFX Manager 3.1 software using the single-threshold mode. Extracellular HBV DNA was isolated from 150 μL medium using an SDS-lysis buffer. Samples were normalized against same input volume and quantified relative to HBV-only samples using the ΔCt method. Intracellular HBV DNA was isolated from approximately 1E+05 cells using an SDS-lysis buffer. The same qPCR protocol and primer set were used as for extracellular HBV DNA quantification, and, additionally, HBV-Ct values were normalized by an *B2M* qPCR[@bib65] using the 2^−ΔΔCt^ method. For cccDNA detection, 8.5 μL total cellular DNA was subjected to 30 min of T5 exonuclease (New England Biolabs, MA) digestion, as described in Xia et al.,[@bib66] with the exception that the DNA was precipitated with ethanol and resuspended in 12 μL ddH~2~O before performing the qPCR protocol, as described for total HBV DNA with the following primer set: P1_F 5′-gctttcactttctcgccaac-3′ and P1_R 5′-accttgggcaatatttggtg-3′. Ct values were normalized against *B2M* from samples receiving the same treatment but without T5 exonuclease digestion. A control reaction was based on DNA isolated from HBV-transgenic mouse serum, as described previously,[@bib67] which is considered negative for cccDNA. Total cellular RNA was converted to HBV-specific cDNA and quantified with the Luna Universal One-Step RT-qPCR (New England Biolabs, MA). HBV total transcripts were amplified with primers binding on the common 3′ end, published by Yan et al.,[@bib42] and GAPDH qRT-PCR was taken as the reference gene.[@bib68] The qRT-PCR was performed on the CFX96 Touch System (Bio-Rad, CA, USA) using the following program: 55°C-10 min; 95°C-1 min; 45× (95°C-10 s, 60°C-30s, plate read); melt curve 60°C→95°C (Δ 0.2°C/5 s). Ct values were determined by use of the Bio-Rad CFX Manager 3.1 software using the single-threshold mode. Expression ratios were calculated in relation to HBV-only samples using the 2^−ΔΔCt^ method. Statistical Analysis {#sec4.7} -------------------- Statistical calculations were performed on technical replicates using an unpaired two-tailed Student's t test, and differences were considered statistically significant when p was less than 0.01. Author Contributions {#sec5} ==================== Conceptualization, M.S., E.E.-S., T.B., and A.E.; Methodology and Investigation, M.S.; Resources, A.O., T.M., U.P., and A.E.; Writing -- Original Draft, M.S.; Writing -- Review & Editing, M.S., E.E.-S., A.O., T.M., U.P., and A.E.; Funding Acquisition, M.S., U.P., and A.E.; Supervision, A.E. Conflicts of Interest {#sec6} ===================== The authors declare no conflict of interest. Supplemental Information {#appsec2} ======================== Document S1. Figures S1--S4Document S2. Article plus Supplemental Information We would like to thank Philip Ng (Baylor College of Medicine, Houston, TX, USA) for providing 116 cells and the helper virus for HCAdV production and Jan Postberg (HELIOS Medical Centre Wuppertal, Witten/Herdecke University, Germany) for providing HepG2.2.15 cells. This work was supported by the Else Kröner-Fresenius-Foundation (grant 2012_A303) and German Liver-Foundation (grant S163/10137/2017) to M.S. and the German Research Foundation (DFG) to U.P. via TRR 179 and TP18. Supplemental Information includes four figures and can be found with this article online at [https://doi.org/10.1016/j.omtn.2018.05.006](10.1016/j.omtn.2018.05.006){#intref0025}.

Introduction ============ Diffuse large B-cell lymphoma (DLBCL) is the most commonly diagnosed form of lymphoma in Western and developing countries, accounting for 40% of non-Hodgkin lymphoma cases among adults ([@b1-etm-0-0-4995]--[@b3-etm-0-0-4995]). Conventional chemotherapy and bone marrow transplantations have a curative effect and are able to effectively improve the survival rates of patients with DLBCL, with some patients being completely cured ([@b4-etm-0-0-4995]). Nevertheless, lymphoma relapse and drug resistance are a major challenge in clinical practice ([@b5-etm-0-0-4995]). In order to improve the therapeutic efficacy and reduce negative impacts of treatment, a combination of techniques are typically used to treat the disease ([@b6-etm-0-0-4995]). It has previously been reported that heat shock protein 90 (HSP90) has a notable role in tumor development; it has therefore become a novel target for tumor therapy ([@b7-etm-0-0-4995]). HSP90 regulates the stability, translation, degradation and function of its client proteins via an adenosine 5′-triphosphate (ATP)-regulated mechanism ([@b7-etm-0-0-4995]). Unlike other HSPs, HSP90 has \~200 cellular target proteins ([@b8-etm-0-0-4995]--[@b10-etm-0-0-4995]). HSP90 inhibitors, such as 17-dimethylaminoethylamino-17-demethoxy geldanamycin (17-DMAG), are a group of small molecules that interact and inhibit the activity of HSP90 dimers ([@b11-etm-0-0-4995],[@b12-etm-0-0-4995]). 17-DMAG is able to inhibit the chaperone activity of HSP90 via competing with ATP for its binding site, leading to the misfolding of cellular client proteins, including RAF proto-oncogene serine/threonine-protein kinase, mutant TP53 proteins, proto-oncogene tyrosine-protein kinase Src, receptor tyrosine-protein kinase-2 and the nuclear receptor subfamily 3 group C member 1, which results in protein degradation and destabilization via the proteasomal pathway ([@b13-etm-0-0-4995]). 17-DMAG is able to suppress tumor growth by inhibiting HSP90, which affects the degradation of key proteins involved in multiple signal transduction pathways, thus inhibiting cell proliferation and inducing cell apoptosis ([@b13-etm-0-0-4995]). The effect of 17-DMAG on DLBCL cells is not completely understood and so there is a requirement for further experimental and clinical studies to determine its value in clinical practice. The purpose of the present study was to determine the effect of 17-DMAG on DLBCL, whether 17-DMAG is able to induce apoptosis in DLBCL cells and the underlying mechanisms responsible. Materials and methods ===================== ### Cell lines and cell culture The SU-DHL-4 DLBCL cell line (Chinese Academy of Sciences, Shanghai Institute of Life Sciences, Cell Resource Center, Shanghai, China) was maintained in suspension at 37°C in an atmosphere containing 5% CO~2~. Cells were cultured in Dulbecco\'s modified Eagle medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA). Cells in the exponential growth phase were then seeded into 96-well plates at a density of 1×10^4^ cells/well. ### Antibodies and reagents The HSP90 inhibitor, 17-DMAG, was donated by Dr Hong Young-Soo (South Korean Living Engineering Research Institute, Seoul, South Korea). Rabbit anti-human B-cell lymphoma 2 (Bcl-2, SC-509), rabbit anti-human Bcl-2-associated X protein (Bax, SC-526) and rabbit anti-human HSP70 member 5 (HSPA5, ab21658) antibodies were purchased from Abcam (Cambridge, UK). Rat anti-human β-actin antibodies (sc-47778) were purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). ### Assessment of cell proliferation by MTT assays Cell proliferation was analyzed using an MTT assay (M2128, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). A total of 5×10^3^ SU-DHL-4 cells/100 µl/well were seeded into 96-well plates, treated with different concentrations of 17-DMAG (0, 25, 50, 100 and 200 µmol/l), and cultured for 24, 48 and 72 h in a 5% CO~2~ incubator at 37°C. Each experiment was performed in triplicate. The cells were subsequently incubated with 15 µl MTT reagent at 37°C for 4 h, and dimethyl sulfoxide was added to dissolve the formazan crystals. An ELI plate reader (Spectra Max M2; Molecular Devices LLC, Sunnyvale, CA, USA) was used to measure the optical density of each well at 570 nm. Finally, the cell survival rate was calculated using the following formula: Cell survival rate (%) = (experimental group A value - zero set A value) / (control group A value - zero set A value) × 100. ### Assessment of apoptosis using propidium iodide (PI) staining The apoptotic rate of the SU-DHL-4 cells was analyzed using PI. Cells in a 6-well plate at a density of 3×10^5^ cells/well were treated with different concentrations of 17-DMAG (0, 25, 50, 100 and 200 µmmol/l) for 24 h at 37°C. At the end of the treatment, cells were washed with cold PBS and fixed overnight with alcohol solution at 4°C. Then the fixed cells were washed twice with PBS, stained with PI solution (5 mg NaC~6~H~5~ PI, 0.1 g 100 triton-100 and 100 µl ddH~2~O) for 30 min at 37°C in the dark according to the manufacturer\'s protocol (BBI Life Science Corporation, Shanghai, China). Finally, ≥7,000 events per sample were acquired using a flow cytometer. CellQuest Pro software, version 3.0 (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the results of the flow cytometer analysis. ### Western blot analysis Western blot analysis was performed to detect the protein expression levels of Bax, Bcl-2 and HSPA5. Proteins were extracted from the DLBCL cells treated with 17-DMAG and from untreated control cells. The cells were plated in 6-well culture dishes at a density of 6×10^5^ cells/well. The harvested cells were then lysed on ice for 30 min in 100 ml of lysis buffer \[120 mmol/l NaCl, 40 mmol/l Tris (pH 8), 0.1% NP40\] and centrifuged at 8,600 × g for 30 min at 37°C. The bicinchoninic acid assay method (BCA1-1KT, Sigma Aldrich; Merck KGaA) was used to determine the protein concentration of the samples. Protein samples (20 µg/lane) were resolved using 10% SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The membranes were subsequently incubated with the appropriate primary antibodies against Bax (1:1,000), Bcl-2 (1:1,000) and HSPA5 (1: 1,000) to detect each the proteins of interest separately at 4°C overnight. β-actin (1:1,000) was used as a loading control. After being washed with PBS, the membranes were incubated with peroxidase-conjugated secondary antibodies (ab6728, Abcam, Cambridge, UK) for 2 h at room temperature. Finally, the detected protein bands were visualized using an Immobilon Western Chemiluminescent HRP Substrate kit (Merck KGaA) and the ImageQuant LAS 4000 software (GE Healthcare Life Sciences, Little Chalfont, UK). ### 2.5.5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolca-rbocyanine Iodide (JC-1) staining Changes in the mitochondrial membrane potential (MMP) were measured using JC-1 staining (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer\'s protocol. Following treatment with 17-DMAG for 24 h at 37°C, SU-DHL-4 cells from each group were incubated with 10 µM JC-1 dye at 37°C for 30 min. Subsequently, cells were washed twice with PBS and observed under a Zeiss 780 laser scanning confocal microscope (Carl Zeiss SAS, Jena, Germany; magnification, ×100), with excitation wavelength at 488 nm and emission wavelength at 527 and 590 nm. Image-Pro Plus 6.0 software was used to analyse the fluorescence intensity of JC-1 staining. ### Statistical analysis All statistical analyses were performed using one-way analysis of variance with least significant difference test on SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). All results were expressed as the mean ± standard deviation. P\<0.05 was considered to indicate a statistically significant difference. Results ======= ### 17-DMAG suppresses the proliferation of DLBCL cells The anti-proliferative activity of 17-DMAG was evaluated in the DLBCL cell line, SU-DHL-4. Cells were treated with different concentrations of 17-DMAG for 48 h, and then cell viability was analyzed using an MTT assay. The results revealed that 17-DMAG inhibited the proliferation of SU-DHL-4 in a dose-dependent manner ([Table I](#tI-etm-0-0-4995){ref-type="table"}; P\<0.05 vs. 0 µmol/l.). The proliferation of SU-DHL-4 cells was also decreased in a time-dependent manner by 17-DMAG treatment ([Fig. 1](#f1-etm-0-0-4995){ref-type="fig"} and [Table I](#tI-etm-0-0-4995){ref-type="table"}; P\<0.05 vs. 0 h). ### 17-DMAG treatment induces apoptosis in DLBCL cells The effect of 17-DMAG on apoptosis in SU-DHL-4 cells was evaluated to further characterize its antitumor activity. Cells were cultured with 17-DMAG at different concentrations for 24 h and stained with PI, following which the proportion of PI positive cells was measured using flow cytometry. The results revealed that the apoptosis of SU-DHL-4 cells was induced by 17-DMAG in a dose-dependent manner ([Fig. 2](#f2-etm-0-0-4995){ref-type="fig"}; P\<0.05 vs. 0 µmol/l). ### Cell apoptosis is induced by 17-DMAG via the mitochondrial pathway In order to determine whether 17-DMAG induces apoptosis through the activation of the mitochondrial apoptotic pathway, the expression levels of several proteins involved in the mitochondrial apoptotic pathway were measured using western blot analysis. The levels of Bcl-2 family members were detected following treatment with 17-DMAG for 24 h. The expression levels of anti-apoptotic proteins Bcl-2 were increased and HSPA5 were reduced following treatment with 17-DMAG, whereas levels of the proapoptotic protein Bax were constant ([Fig. 3](#f3-etm-0-0-4995){ref-type="fig"}). Taken together, these results suggest that 17-DMAG induces apoptosis in DLBCL cells via the mitochondrial apoptotic pathway. ### 17-DMAG alleviates changes in MMP MMP is an important determinant of early apoptosis ([@b14-etm-0-0-4995]). Accordingly, MMP was measured in DLBCL cells using JC-1 staining. In living DLBCL cells, JC-1 aggregates in the mitochondria and emits red fluorescence, whereas, in apoptotic DLBCL cells, JC-1 is a green fluorescence monomer and accumulates in the cytosol ([@b15-etm-0-0-4995]). The ratio of red to green fluorescence reflects the intensity of MMP. In the control group, cells appeared orange-red ([Fig. 4](#f4-etm-0-0-4995){ref-type="fig"}). In comparison, green fluorescence was more intense and red fluorescence was decreased in cells treated with 17-DMAG, indicating a decrease in the MMP. In the presence of 17-DMAG, the green fluorescent intensity was decreased, whereas the red fluorescence was increased. These results suggest that the 17-DMAG neuronal protective effect against cell apoptosis is achieved via the mitochondrial pathway. Discussion ========== In the search for a novel, effective and safe pharmacological agent of natural origin for the treatment of cancer, the effect of 17-DMAG extract on apoptosis in SU-DHL-4 DLBCL cells was assessed. The results indicated that 17-DMAG exerts antiproliferative and proapoptotic effects on SU-DHL-4 cells via decreasing cell viability in a dose-dependent manner. The intrinsic or mitochondrial pathway is recognized as one of the major apoptosis signaling pathways. Specifically, the intrinsic pathway comprises mitochondria-dependent processes that result in decreasing Bcl-2 levels and activation of Bax ([@b16-etm-0-0-4995]). The present study focused on the molecular changes that occur early in the intrinsic apoptotic pathway, and it was observed that 17-DMAG causes a rapid decrease in the MMP. Mitochondria serves an essential role in the intrinsic apoptotic pathway, which is mediated through amplification factors, including second mitochondria-derived activator of caspase, cytochrome *c* and apoptotic inducer factors, and the execution of this pathway is achieved hby regulating the Bcl-2 family of proteins ([@b17-etm-0-0-4995]). The proapoptotic members, including Bax and Bcl-2 homologous antagonist killer, and their antiapoptotic counterparts, including Bcl-2 and Bcl-2-like 1, typically maintain the balance ([@b18-etm-0-0-4995]). Bax translocates to the mitochondria and triggers the mitochondrial outer membrane potential, which facilitates the downstream apoptotic signaling assembly of pro-apoptotic factors in the apoptotic signal transduction pathways ([@b19-etm-0-0-4995]). In contrast, Bcl-2 overexpression blocks Bax from the mitochondria, thus inhibiting apoptosis ([@b20-etm-0-0-4995]). This is consistent with the results of the present study. 17-DMAG-induced apoptosis in SU-DHL-4 cells is associated with the downregulation of the *BCL2* gene, whereas the upregulation of *Bax* gene expression indicates the involvement of the MMP. The endoplasmic reticulum (ER) stress response has been demonstrated to be important for the growth and development of tumors under stressful growth conditions, including hypoxia and glucose deprivation, which are commonly encountered by the majority solid tumors ([@b21-etm-0-0-4995]). HSPA5 is a major ER stress-regulated protein that is involved in ER stress-induced apoptosis ([@b22-etm-0-0-4995]). HSPA5 is a major regulator of the unfolded protein response and an ER-resident chaperone. Increasing evidence has indicated that HSPA5 is highly expressed in many tumor cell lines, and contributes to the invasion and metastasis of various human tumors ([@b23-etm-0-0-4995]). In addition, HSPA5 downregulation can lead to the detection of the ER stress-inducing anticancer therapies ([@b21-etm-0-0-4995]). The results of the present study indicated that the downregulation of HSP5A in SU-DHL-4 cells affects cell viability, whereas the activation of Bax abolishes the expression of Bcl-2. Previous studies have revealed that HSPA5 directly regulates the expression of target genes that lead to cell death or apoptosis in a p53-dependent or independent manner in cancer cells under ER stress ([@b23-etm-0-0-4995],[@b24-etm-0-0-4995]). The present study demonstrated that 17-DMAG activates Bax and downregulates the expression of Bcl-2 and HSPA5 in SU-DHL-4 cells. The effect of 17-DMAG may be achieved via a reduction in the activity of Bcl-2 and the maintenance of activity of Bax. During the MMP, the suppression of HSPA5 expression may inhibit ER stress. The results of the present study indicated that 17-DMAG serves a role in SU-DHL-4 cell apoptosis. In conclusion, 17-DMAG induces apoptosis via the MMP pathway and blocks the ER stress pathway. Therefore, 17-DMAG may have potential as an effective therapeutic agent for DLBCL, although further study is required. {#f1-etm-0-0-4995} {#f2-etm-0-0-4995} {#f3-etm-0-0-4995} {#f4-etm-0-0-4995} ###### Cell survival rate of SU-DHL-4 cells. Cell survival rate, % ------- ----------------------------------------------------------- -----------------------------------------------------------     0 100.0000±0.6 100.0000±0.2   25 88.9913±0.5^[a](#tfn2-etm-0-0-4995){ref-type="table-fn"}^ 89.5851±1.1^[a](#tfn2-etm-0-0-4995){ref-type="table-fn"}^   50 71.5563±0.2^[a](#tfn2-etm-0-0-4995){ref-type="table-fn"}^ 58.1955±3.0^[a](#tfn2-etm-0-0-4995){ref-type="table-fn"}^ 100 62.2241±0.4^[a](#tfn2-etm-0-0-4995){ref-type="table-fn"}^ 55.1494±2.5^[a](#tfn2-etm-0-0-4995){ref-type="table-fn"}^ 200 52.4728±0.2^[a](#tfn2-etm-0-0-4995){ref-type="table-fn"}^ 39.4546±1.2^[a](#tfn2-etm-0-0-4995){ref-type="table-fn"}^ 17-DMAG, 17-dimethylaminoethylamino-17-demethoxy geldanamycin. P\<0.05 vs. 0 µmol/l 17-DMAG.

Invasive candidiasis (IC) comprises candidemia and deep-seated candidiasis with or without concomitant candidemia \[[@CIT0001]\]. The most important risk groups are patients in the intensive care unit (ICU) and those undergoing abdominal surgery, particularly if repeated or complicated \[[@CIT0002]\]. Candidemia is associated with significant morbidity, mortality, and cost \[[@CIT0001]\]. The overall mortality is around 40% \[[@CIT0007]\] but is higher among patients in the ICU \[[@CIT0008]\]. A positive blood culture (BC) or culture from a newly (\<24 hours) placed drain establishes the diagnosis of proven IC \[[@CIT0009]\]. However, BCs have a low sensitivity. Autopsy studies have suggested a sensitivity between 21% and 71%, which is highest if large-volume BCs (60 mL) are obtained daily and additional sets are obtained during febrile episodes \[[@CIT0010]\]. The use of mycosis culture bottles improves sensitivity for the detection of *Candida glabrata* when the BACTEC system is used and of polymicrobial infections when the BacT/ALERT system is used \[[@CIT0002], [@CIT0011]\]. However, such bottles are infrequently used. The time to BC positivity is around 2 days \[[@CIT0002], [@CIT0014]\]. A 2-day delay in antifungal therapy has been associated with more than a doubling of the mortality rate in several \[[@CIT0015], [@CIT0016]\] but not all studies \[[@CIT0002], [@CIT0017], [@CIT0018]\]. Hence, rapid diagnostic tests are warranted. β-D-glucan and the *Candida* mannan antigen (MAg) and antibody (MAb) tests are recommended biomarkers for the diagnosis and management of *Candida* diseases \[[@CIT0019]\]. The β-D-glucan is a panfungal test and hence cannot distinguish between candidiasis and other fungal infections. The sensitivity for IC is generally high (76.7%--100.0%), but the specificity is more variable (40.0%--91.8%) \[[@CIT0020]\]. Multiple sources for false positivity have been reported, many of which are particularly challenging in the ICU \[[@CIT0020], [@CIT0021], [@CIT0026]\]. This is an important caveat and may lead to inappropriate antifungal treatment of patients without candidiasis. *Candida* MAg and MAb detection has been found useful for the detection of IC \[[@CIT0027]\], although the performance of combined MAg and MAb detection was disappointing (sensitivity 55% and specificity 60%) in a recent study including ICU patients with severe abdominal conditions \[[@CIT0020]\]. The variation in reported performance for this test remains unexplained, but in a population mainly at risk of candidiasis, a *Candida*-specific rather than panfungal test may be preferable with respect to risk of false-positive results and interpretation. The T2Candida molecular test has recently been Conformité Européenne (CE) marked and Food and Drug Administration (FDA) cleared. The test detects (1) *C. albicans* and *C. tropicalis,* reported together as *C. albicans*/*C. tropicalis*; (2) *C. glabrata*, *C. krusei*, *S. cerevisiae,* and *C. bracarensis*, reported together as *C. glabrata*/*C. krusei*; and (3) *C. parapsilosis, C. orthopsilosis,* and *C. metapsilosis*, reported as *C. parapsilosis*. Together, these species formed the majority (93.5%) of the *Candida* bloodstream infections in Denmark in 2012--2015 \[[@CIT0011]\]. The combined sensitivity and specificity of the test for detecting candidemia were 91.1% and 99.4%, respectively, in the study that led to the FDA clearance \[[@CIT0032]\]. Subsequent studies have confirmed a high sensitivity using seeded blood samples \[[@CIT0033]\] and when comparing the results for follow-up BCs and T2Candida tests obtained from pediatric and adult patients with documented candidemia \[[@CIT0014], [@CIT0034]\]. Moreover, among patients already receiving antifungal therapy, the T2Candida assay yielded positive results in a number of cases with negative BC \[[@CIT0014]\]. Of note, studies performed so far included candidemic patients with or without enrichment of the study material with spiked blood samples. Hence the performance in cases with IC without concomitant candidemia is largely unknown. In this study, we compared the diagnostic performance of BC, T2Candida, and *Candida* MAg and MAb in patients at risk of IC in the 2 major university ICUs in a country with a high incidence of IC \[[@CIT0011], [@CIT0035]\]. To our knowledge, it is the largest and only non-single-center prospective study performed in a high-risk ICU setting for comparative diagnosis of IC. METHODS {#s1} ======= **Patients and Data Collection** In total, 126 ICU patients were included during the study period of October 1, 2014, to June 21, 2016. The characteristics at 2 major university ICU units are summarized in [Supplementary Table 1](#sup1){ref-type="supplementary-material"}. **Inclusion Criterion** The inclusion criterion was 1 of the following: (1) initiation of prophylactic, empiric, or preemptive antifungal treatment; (2) colonization index ≥0.5; or 3) temperature \>38°C or \<36°C despite 3 days of broad-spectrum antibiotic treatment *and* at least 2 of the following risk factors: abdominal surgery, secondary peritonitis, pancreatitis, central vein catheter (CVC) in place, total parenteral nutrition (TPN), dialysis, steroid treatment, immunosuppressive treatment, or liver transplantation. Patients who fulfilled the inclusion criterion but had candidemia before inclusion were not excluded. Presence or absence of each inclusion criterion was registered, along with each blood sampling. Upon completion of the study, patient records and clinical microbiology test databases were reviewed for each patient. The following data were extracted: underlying diseases, body temperature \>38°C or \<36°C, signs of sepsis (systolic blood pressure \<90 mmHg; mean arterial pressure \<65 mmHg; need of vasopressor infusion \[epi-/norepinephrine\] or blood pressure drop 40 mmHg from baseline), white blood cell count \>12 000 cells/µL, confirmation of ≥3 days of broad-spectrum antibiotics. The microbiological tests were reviewed and categorized into whether *Candida* were isolated from at least 2 nonsterile sites (±3 days) and whether there was an alternative microbiological diagnosis. From each patient, a 5.5-mL EDTA tube and a BC set were obtained simultaneously at the time of enrollment. The EDTA tube was used for T2Candida (T2 Biosystems, Lexington, MA) and for Platelia *Candida* MAg and Ab (Bio-Rad, Marnes-la-Coquette, France). BacT/ALERT (bioMérieux, Marcy l'Etoile, France) BC was used in Odense and BACTEC (Becton Dickinson, Franklin Lakes, NJ) at Rigshospitalet. Sampling was repeated twice weekly in Odense for as long as the patient fulfilled the enrollment criteria. At Rigshospitalet, a single follow-up sample set was obtained after 3 days. At both hospitals, routine urine and tracheal secretions were cultured at regular intervals. Additional microbiological tests were performed as indicated by the treating physician. Cases were classified based on vital signs and results of microbiological examinations within 3 days of enrollment (see below). 1. Proven IC:  either (1) growth of *Candida* in a BC or (2) fulfilling all the following: (i) growth of *Candida* in a tissue biopsy or sample from a drain placed within 24 hours, (ii) sampling from a normally sterile site using sterile procedures, and (iii) clinical or radiological signs of infection at that site (EORTC/MSG criteria \[[@CIT0009]\]). 2. Likely IC:  either (1) *Candida* isolated from at least 2 nonsterile sites (±3 days) and no alternative microbiological diagnosis (±3 days) and fulfilling ≥1 SIRS criterion despite 3 days of broad-spectrum antibacterial treatment or (2) *Candida* MAg \>250 mg/L and colonized with *Candida* at 2 sites (±3 days) (adopted from \[[@CIT0025], [@CIT0036]\]). 3. Possible IC: either (1) *Candida* MAg \>125 mg/L and colonized at 2 sites (±3 days) or (2) *Candida* MAg \>250 mg/L or 3) *Candida* isolated from at least 2 nonsterile sites (±3 days) and fulfilling SIRS criteria despite ≥3 days of broad-spectrum antibiotics. 4. No IC: remaining cases. Subsequently, we studied the medical files of all cases who were initially classified as either proven, likely, or possible IC or who had at least 1 positive marker for IC (T2 or mannan Ag) and revised the classification according to results of microbiological analyses within 21 days of enrollment and detailed information on clinical course, as presented in [Supplementary Table 2](#sup1){ref-type="supplementary-material"}. Statistical Analysis {#s2} -------------------- The sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values of the tests were assessed based on the sample drawn at inclusion and the subsequent sample drawn at day 2 or 3. If analyses of the 2 samples were discordant (ie, 1 positive and 1 negative), the test was considered positive. The Fisher exact test was used to compare the proportions of patients receiving antifungal therapy for BC-positive vs -negative patients. RESULTS {#s3} ======= In total, 334 sample sets from 126 ICU patients at risk for IC were included during the 21-month study period. The median age (range) was 65.5 (16--89) years, and 72 (57%) were men. The majority of patients met more than 1 inclusion criterion (82%) and received antifungal therapy at the time of inclusion (77%) ([Table 1](#T1){ref-type="table"}). Abdominal surgery was the most common risk factor (44%), followed by secondary peritonitis or immunosuppression. ###### Baseline Characteristics of the Study Population (126 Patients^a^) --------------------------------------------------------------------------------- --------------- Male, No. (%) 72 (57) Age, median (IQR), y 65.5 (53--75) Inclusion criterion, % Initiation of antifungal therapy 84 Colonization index ≥ 0.5 33 Fever despite antibiotics combined with the presence of at least 2 risk factors 80 Risk factors, No. (%) Central venous catheter 112/124 (90) Parenteral nutrition 24/121 (20) Dialysis 26/122 (21) Steroids 32/121 (26) Abdominal surgery 54/123 (44) Secondary peritonitis 20/120 (17) Pancreatitis 6/120 (5) Immunosuppressed 19/121 (16) Liver transplantation 1/121 (0.8) Antifungal Rx at time of inclusion, No. (%) None 27/119 (23) Fluconazole^b^ 76/119 (63) Other azole 3/119 (3) Echinocandin^b^ 14/119 (12) --------------------------------------------------------------------------------- --------------- Abbreviation: IQR, interquartile range. ^a^Data missing for some patients as indicated by varying denominators. ^b^One patient received caspofungin and fluconazole combination therapy. Microbiology {#s4} ------------ At the time of enrollment, 15 (11.9%) patients were positive by BC, T2Candida, and/or MAg testing ([Figure 1](#F1){ref-type="fig"}, [Table 2](#T2){ref-type="table"}). Overall, 7/11 T2Candida-positive samples and 7/10 MAg-positive samples were from patients without a concomitant positive BC at the time of inclusion. Significantly fewer patients with positive compared with negative BCs received antifungal therapy at the time of inclusion (2/5 \[40%\] vs 90/115 \[78%\], *P* = .008). For the T2Candida test and the MAg test, there were no statistically significant associations between test results and antifungal therapy at inclusion (7/11 \[64%\] vs 80/103 \[78%\], *P* = .287, and 9/10 \[90%\] vs 78/103 \[76%\], *P* = .449, respectively). ###### Comparison of Blood Culture, T2Candida, and *Candida* Mannan Antigen Results for the Initial Samples Taken at the Time of Inclusion Blood Culture T2Candida *Candida* MAg T2Candida and/or MAg ----------------------------------- ----------- --- ------- --- --------------- --- ----- ---------------------- --- --- ----- *C. albicans* (1) 1 1 1 *C. glabrata* (1) 1 1 1 *C. tropicalis* (1) 1 1 1 *C. tropicalis & C. glabrata* (1) 1 \-^a^ 1 1 *C. kefyr* (1) 1 1 1 Neg-BC (121) 5 2 107 7 7 7 107 4 3 3 111 In total (126) 8 3 108 7 10 7 109 6 5 4 111 Abbreviations: Int., intermediate; MAg, mannan antigen; Neg., negative; Neg-BC, blood culture without growth of *Candida*; Pos., positive. ^a^The *C. glabrata* found in the initial blood culture was reported on day 4 but not on day 0 by the T2Candida test. {#F1} T2Candida Test Results {#s5} ---------------------- The species distribution among patients with initial samples positive in T2Candida was *C. albicans*/*C. tropicalis* in 8/11 (72.3%) patients and *C. glabrata*/*C. krusei* in 3/11 (27.3%). The T2Candida test was positive for 4/5 patients with positive index BCs and for 2 additional patients with positive BCs 2 and 4 days before inclusion, whereas 5 patients had negative BCs ([Table 3](#T3){ref-type="table"}). The species group reported by the T2Candida test was confirmed by the identification of BC or colonizing isolates in 10/11 cases (*Candida* was not identified to the species level in 1 patient), although T2Candida failed to detect the second species at day 0 in a patient with polyfungal infection ([Table 3](#T3){ref-type="table"}). T2Candida was negative in the patient with *C. kefyr* candidemia, a species that is not included in the panel, and was invalid in 9/126 (7.1%) patients at enrollment. ###### Kinetics of the Blood Culture and Biomarker Results Classification at Inclusion, Pt No. Test Days From Study Enrollment --------------------------------------------- ------ ---------------------------- ---------- ----------- ------------------- ---------- ------- ------- ------- ------- ------ ------ Blood culture--pos. cases Proven, \#63 BC T & G Neg T2 A/T A/T & G/K MAg \>500 \>500 MAb Int Int Proven, \#103 BC T T Neg T Neg Neg Neg Neg Neg T2 A/T A/T A/T Neg A/T Neg Neg Neg Neg MAg \>500 \>500 \>500 \>500 \>500 \>500 \>500 \>500 \>500 MAb Neg Neg Neg 15.9 37.2 38.2 37.2 35.7 38.6 Proven, \#4 BC G Neg T2 G/K Neg MAg Neg Neg MAb Int Int Proven, \#98 BC A Neg T2 A/T A/T MAg Neg Neg MAb Neg Neg Proven, \#33 BC *C. kefyr* T2 Neg MAg \>500 MAb Int T2Candida+, BC-neg. cases Proven (*C. albicans*), \#89 T2 A/T A/T MAg Neg Int MAb 14.2 11.7 Likely (*C. krusei*), \#60 BC Neg Neg *C. dubliniensis* Neg T2 G/K Neg P Neg MAg \>500 \>500 \>500 \>500 MAb 15.1 13.2 19.8 32.6 Likely (*C. tropicalis*), \#22 T2 A/T A/T MAg \>500 \>500 MAb Int 35.3 Likely (*C. albicans*), \#28 T2 A/T A/T MAg \>500 \>500 MAb Neg Neg Proven (*Candida* no ID), \#90 T2 G/K Neg Neg MAg \>500 \>500 250--500 MAb Neg Neg Neg Likely (prov d-4, *C. albicans*), \#54 T2 A/T MAg Neg MAb Int Possible *C. albicans*, \#114 T2 A/T Invalid A/T A/T A/T A/T Neg MAg Neg Neg Neg Neg Neg Neg Neg MAb Int Int Int 37.2 58.6 58.6 58.6 Possible *C. albicans*, \#74 T2 Neg Neg G/K MAg Neg Neg 125--250 MAb Neg Neg 10.4 Unlikely, \#36 T2 Neg Neg Neg Neg Neg Neg Neg P Neg MAg Neg Int 125.2 Neg Neg Neg Neg Neg Int MAb 20.9 24.7 45.2 38.9 37.8 41.1 39.8 35.2 40.8 Unlikely, \#83 T2 Neg P Neg Neg Neg MAg Neg Neg Neg Neg Neg MAb Neg Neg Neg Neg Neg Mannan AG--pos., T2C- and BC-neg. cases Unlikely, \#109 T2 Neg MAg \>500 MAb Neg Unlikely (prov d-5,-7, *C. albicans*), \#20 T2 Neg MAg \>500 MAb 71.1 Unlikely, \#24 T2 Neg Neg Neg Neg Neg MAg 125--250 125--250 125--250 125--250 \>250 MAb 71.7 65.9 75 54.7 75.4 Possible (Prov d+4), \#121 T2 Neg Neg Neg Neg Neg MAg Neg Neg Neg Neg 125--250 MAb Int 11.2 14.6 19.2 34.7 Unlikely, \#70 T2 Neg Neg Neg MAg Neg 125--250 125--250 MAb Neg Neg Neg Empty space indicates that no samples were obtained in that time period. For patients with no BC row included, the accompanying blood cultures were negative. Patient 20 was blood culture positive with *C. albicans* 5 and 7 days before enrollment in the study. Mannan antigen interpretative cutoffs: Neg.: \<62.5 pg/mL; Int.: ≥62.5--125 pg/mL; and Pos.: ≥125 pg/mL. Antimannan antibody interpretative cutoffs: Neg.: \<5 AU/mL; Int.: 5--\<10 AU/mL; and Pos.: ≥10 AU/mL. Abbreviations: BC, blood culture; Int, intermediate; MAb, mannan antibody (AU/mL); MAg, mannan antigen (pg/mL); Neg, negative; T2, T2Candida. Fungal species abbreviations: A, *Candida albicans*; G, *Candida glabrata*; K, *Candida krusei*; P, *Candida parapsilosis*; T, *Candida tropicalis.* In 4 BC-negative patients, a single follow-up sample was positive by T2Candida, 3 of these with *C. parapsilosis* in the absence of concomitant *C. parapsilosis* colonization or other indication of IC ([Table 3](#T3){ref-type="table"}). These 3 cases were likely false positives. The fourth patient became positive with *C. glabrata*/*C. krusei*, MAg, and MAb at day 4. Including these, the overall species distribution as determined by T2Candida was *C. albicans*/*C. tropicalis* 8/15 (53%), *C. glabrata*/*C. krusei* 4/15 (27%), and *C. parapsilosis* 3/15 (20%). *Candida* Mannan Ag and Ab {#s6} -------------------------- The MAg test was positive in 3/5 patients with positive BC at enrollment, including the patient with *C. kefyr* candidemia ([Table 3](#T3){ref-type="table"}). Seven additional patients were MAg positive at enrollment, 4 of whom were T2Candida positive and 1 of whom had been BC positive 5 and 7 days earlier. During the follow-up period, 4 additional BC-negative patients changed to MAg positive (2 of whom also developed MAb), whereas 5 patients became MAb positive during follow-up (1 of whom was BC positive at inclusion, 4 of whom were T2 positive, and 4 of whom were or subsequently became MAg positive). Kinetics of BC and Biomarker Results {#s7} ------------------------------------ T2Candida stayed positive longer (mean \[range\], 3.2 \[0--5\] days) than BC. Eight of 9 patients with positive MAg and follow-up samples remained MAg positive during the entire observation period (up to 20 days) ([Table 3](#T3){ref-type="table"}). Clinical Classification and Performance of Diagnostic Tests {#s8} ----------------------------------------------------------- The numbers of patients classified as proven, likely, or possible IC were 11 (8.7%), 6 (4.8%), and 11 (8.7%), respectively ([Table 4](#T4){ref-type="table"}; [Supplementary Table 2](#sup1){ref-type="supplementary-material"}). Abdominal IC was the most common manifestation (16/22 \[72.7%\]) among BC-negative patients with proven (2/5), likely (4/6), or possible IC (10/11), respectively. Significantly fewer patients with proven IC received antifungal treatment (AF) before enrollment (5/11) than among likely and unlikely cases (6/6, *P* = .004, and 82/95, *P* = .047) ([Table 4](#T4){ref-type="table"}). ###### Performance of Blood Culture, T2Candida, and Mannan Antigen Based Upon the 2 Initial Blood Sample Sets for Patients Classified With Proven (11), Likely (6), Possible (11), or Unlikely (98) Invasive Candidiasis Final Classification No. of Patients No. w/ Prior AF BC Pos T2 Pos MAg Pos -------------------------------------------------------------- ----------------- ------------------ -------- -------------- --------- Proven 11 5/11 5/11 6/11 4/11 Candidemia within ±3 d of inclusion 6 2^a,a^ 5/6 5/6 3/6 Abdominal candidiasis incl. aortaprothesis 1 0 0 0 0 Abdominal candidiasis 1 1^e^ 0 1/1 1/1 Mediastinal or pleural candidiasis 2 2^b,e^ 0 0 0 Polymicrobial necrotizing fasciitis 1 0 0 0 0 Likely 6 6/6 0/6 4/6 3/6 Abdominal candidiasis & prior proven abdominal candidiasis 3 3^a,d,e^ 0 1/3 0 Abdominal candidiasis 1 1^c^ 0 1/1 1/1 Pulmonary candidiasis in hematological Pt 1 1^e^ 0 1/1 1/1 Tissue candidiasis 1 1^a^ 0 1/1 1/1 Possible 11 10/11 0/11 1 (&1 d4)/11 2/11 Abdominal candidiasis 8 7^a,a,b,b,b,e,e^ 0 1 (&1 d4)/8 1/8 Abdominal candidiasis & prior proven candidemia, d --5 & --7 1 1^c^ 0 0 1/8 Abdominal candidiasis & proven, d +4 1 1^e^ 0 0 0 Renal candidiasis & prior proven 1 1^e^ 0 0 0 Unlikely 98 82/95^f^ 0/98 1^g^ /98 2/98 Information on prior systemic antifungal therapy at inclusion is indicated (No. w/ Prior AF). Abbreviations: AF, antifungal treatment; BC, blood culture; MAg, mannan antigen (pg/mL); T2, T2Candida. ^a^Duration of AF before inclusion: \<1 day. ^b^Duration of AF before inclusion: 1--3 days. ^c^Duration of AF before inclusion: 4--7 days. ^d^Duration of AF before inclusion: \>7 days. ^e^No information on duration of AF before inclusion. ^f^Information on prior antifungal therapy was missing for 3/98 patients. ^g^ *Candida parapsilosis*. The sensitivity was higher for T2Candida compared with BC and MAg for proven IC (55% vs 45% and 36%), for proven or likely IC (59% vs 29% and 41%), and for proven, likely, or possible IC (39% vs 8% and 32%), respectively, compared with others ([Table 5](#T5){ref-type="table"}); however, as the number of cases of IC was limited, these differences did not reach statistical significance. When combining the diagnostic tests, the sensitivity increased to 64%--65% for test combinations including T2Candida compared with 53%--55% for BC+MAg for proven vs others and proven/likely vs others. The specificity was high and above 90% for all tests and test combinations except T2Candida-BC-MAg triple testing for proven vs others. The PPV was higher for T2Candida than for MAg (50% vs 36% for proven and 83% vs 64% for proven/likely vs other, respectively). The negative predictive value was similar across the tests (94%--96% for proven and 90%--95% for proven/likely, respectively) but somewhat lower if including possible cases of IC (81%--88%). Overall, T2Candida combined with BC seemed to have the best diagnostic performance for proven/likely IC compared with other single tests or test combinations. ###### Performance Characteristics for the Diagnostic Tests Using the Clinical Classification of Proven (11), Likely (6), Possible (11), or Unlikely Invasive Candidiasis (98) as the Gold Standard Candidiasis Classification and Diagnostic Test Sensitivity, % Specificity, % PPV, % NPV, % ------------------------------------------------ ---------------- ---------------- -------- -------- Proven vs others BC 45 100 100 95 T2 55 93 50 96 MAg 36 94 36 94 T2+BC 64 93 54 96 T2+MAg 64 89 41 96 BC+MAg 55 94 38 96 T2+BC+MAg 64 89 41 96 Proven or likely vs others BC 29 100 100 90 T2 59 96 83 94 MAg 41 96 64 91 T2+BC 65 96 85 95 T2+MAg 65 92 65 94 BC+MAg 53 96 54 93 T2+BC+MAg 65 92 65 94 Proven, likely, or possible vs unlikely BC 18 100 100 81 T2 39 97 92 85 MAg 32 98 82 83 T2+BC 43 97 92 86 T2+MAg 50 95 82 88 BC+MAg 39 98 85 85 T2+BC+MAg 50 98 82 88 Abbreviations: BC, blood culture; Mag, mannan antigen; NPV, negative predictive value; PPV, positive predictive value; T2, T2Candida. Discussion {#s9} ========== In this study comparing the performance of diagnostic tests among high-risk ICU patients, the highest sensitivity and NPV for IC were found for T2Candida and for test combinations including T2Candida. No single test or test combination resulted in a sensitivity above 65% for IC, but the combination of T2Candida and BC seemed to have the best diagnostic performance for proven/likely IC. So far, the only other study prospectively enrolling patients before the establishment of an IC diagnosis found the T2Candida test positive in 4/6 patients with positive BCs, in line with the 4/5 found in our study \[[@CIT0032]\]. In that study, a 91.1% sensitivity and 99.4% specificity for T2Candida compared with BC were estimated, enriching the sample size with 250 samples spiked with *Candida* cells at concentrations similar to what is expected during (untreated) candidemia \[[@CIT0032]\]. We estimated test performance based on the clinical classification of high-risk patients, the majority of whom were suffering from deep-seated IC. We speculate that this difference in study design and the fact that many patients had received several days of antifungal therapy before enrollment may in part explain the lower performance of the T2Candida test in our setting. The expected IC rate is around 10% among severely ill high-risk ICU patients \[[@CIT0037]\]. In this study, 8.7% were classified with proven IC and additional 4.8% with likely IC. In this context, the rate of BC-proven candidiasis was notably low (4%). Most of our patients received antifungal therapy at the time of enrollment, with fluconazole being the predominating compound. Notably, none of the patients with candidemia had received AF for ≥24 hours at the time of inclusion. Fluconazole (20 mg/L) reduced overall BC sensitivity by 7.5%--12.5% in a recent laboratory experiment \[[@CIT0038]\]. In line with these findings, significantly fewer patients with proven infection received prior antifungal therapy in our study, a diagnosis that requires a positive culture from blood or a sterile site. In contrast, fluconazole was observed not to impact the sensitivity of the T2Candida assay in the above-mentioned laboratory study \[[@CIT0038]\]. We observed a trend toward a lower T2Candida-positive rate for patients receiving antifungal therapy before sampling, suggesting that T2Candida sensitivity may also be reduced during antifungal therapy. Fluconazole therapy may lower the fungal load or clear the bloodstream despite not clearing the foci of patients with deep-seated candidiasis and hence result in a lower T2Candida sensitivity that is not observed in studies of spiked blood. It may therefore be advisable to initiate diagnostic testing before initiation of antifungals whenever feasible. The species group provided for positive T2Candida test results was in agreement with the *Candida* species diagnosis established by culture from blood or other sites for patients with IC. The overall T2Candida species distribution including positives during follow-up was *C. albicans*/*C. tropicalis* 53%, *C. glabrata*/*C. krusei* 27%, and *C. parapsilosis* 20%. In comparison, these species accounted for 52%, 36%, and 3%, respectively, in the national surveillance program in 2012--2015 \[[@CIT0011]\]. *C. parapsilosis* is a skin and biofilm--associated organism. None of the 3 patients with a positive *C. parapsilosis* T2Candida result were diagnosed as IC or colonized with *C. parapsilosis* by culture. We therefore regard these findings as contaminations, illustrating the importance of thoroughly decontaminating the skin or catheter before blood draws for T2Candida. In settings with a higher true prevalence of *C. parapsilosis* infections like in neonates and in endemic areas such as Latin America, Southern Europe, and Asia, it may be difficult to differentiate clinically relevant *C. parapsilosis* findings from contaminations, potentially leading to unnecessary use of antifungal therapy \[[@CIT0039]\]. T2Candida is an expensive diagnostic tool. At the time of writing, the price in our country for a test kit is US\$300 per sample, and an annual fee for a service contract of US\$15 500. Although cost analyses have suggested lower candidemia-related inpatient costs and mortality with implementation of the T2Candida test \[[@CIT0040]\], cost may be prohibitive in some settings, particularly at a time of declining cost for the echinocandins. The combination of MAg with BC was superior to BC alone. However, in addition to being less sensitive compared with T2Candida alone and when combined with BC, a drawback is that species identification is not provided for MAg-positive samples to inform whether the species is a likely target for de-escalation to fluconazole once the patient is stable. The time to negativity was longer for the T2Candida test than for BC, supporting recent findings \[[@CIT0041]\]. This may suggest that whereas the appropriate duration of therapy after resolution of symptoms and BC negativity is 14 days, this may be shorter after resolution of symptoms and T2Candida negativity \[[@CIT0042]\]. In contrast, the mannan antigen remained positive throughout the observation period and up to 14 days after BC documented clearance of the bloodstream and thus should not alone prompt continued antifungal therapy. This study has limitations. First and foremost, the number of proven and likely cases was limited, and more than half of the proven IC cases were BC negative at the time of inclusion, suggesting a low amount of circulating *Candida* cells. Therefore, numbers were insufficient for statistical comparative analysis of the diagnostic tests. In the absence of a reliable gold standard for the diagnosis of IC, classification of patients was done adopting the EORTC/MSG definitions for proven infection and criteria adopted from 2 recent studies for likely and possible IC \[[@CIT0009], [@CIT0025], [@CIT0036]\]. A positive blood culture defined proven infection, and a highly positive MAg in a colonized patient with clinical signs and symptoms defined likely IC, whereas a positive T2Candida did not. Consequently, the relative diagnostic performance of T2Candida compared with BC and MAg may have been underestimated. Most of the patients with deep-seated IC were patients with complicated infections receiving broad-spectrum antibiotics in whom it is difficult to dissect the clinical significance of the findings of *Candida* in microbiological samples. Finally, colonization was difficult to compare, as neither sampling nor mycologic examination was standardized. In conclusion, our study underscores the complexity associated with diagnosing IC. Most patients were BC negative, probably in part because the majority were already on fluconazole prophylaxis, as recommended in a multimorbid high-risk population. T2Candida was the biomarker that contributed the most to improving the diagnostic sensitivity while retaining a good specificity and NPV. This makes it a promising addition to the diagnostic armamentarium. However, our study also suggests that maximal benefit of diagnostic efforts may require that sampling is initiated as early as possible, preferably before initiation of antifungal therapy, when the sensitivity and NPV of T2Candida and BC testing are maximal and probably sufficient for excluding IC. Supplementary Data {#s10} ================== Supplementary materials are available at *Open Forum Infectious Diseases* online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. ###### Click here for additional data file. ***Author contributions.*** The study was designed by M.C.A., J.S., and J.S.A. N.R., J.S.A., K.K., and M.K.H. were responsible for enrollment and data collection. M.C.A. was responsible for laboratory tests. M.H. reviewed patient records and analyzed the data. The clinical classification was done by M.H. and M.C.A. The manuscript was drafted by M.C.A. and M.H. and reviewed and approved by all authors. ***Financial support.*** This work was supported in part (test kits provided, and part of the hourly fee for the ICU doctors spent on patient record review was reimbursed to the ICU departments) by T2Biosystems. MCA has received research grants from Savværksejer Jeppe Juhls og hustru Ovita Juhls Mindelegat. 295 MH received support from the Danish National Research Foundation, grant \#126. ***Availability of data.*** Data are not publicly available. ***Potential conflicts of interest.*** M.C.A. has received personal speaker honoraria in the past 5 years from Astellas, Basilea, Gilead, MSD, Pfizer, T2Biosystems, and Novartis. She has received research grants and contract work payment, paid to the Statens Serum Institute, from Astellas, Basilea, Gilead, T2Biosystems, F2G, Cidara, and Amplyx. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. [^1]: Equal contribution

Due to restrictions set by the BC Centre for Excellence (BCCfE) IRB, the data are available upon request. Any requests to redistribute these data should be forwarded to the BCCfE laboratory director (<prharrigan@cfenet.ubc.ca>) and must pass through the standard BCCfE data sharing protocol. Introduction {#sec001} ============ The HIV-1 Nef gene has a single exon of about 620 nucleotides, which partially overlaps a 3'-long terminal repeat. It encodes an immunogenic accessory protein with multiple activities during HIV infection. Although Nef is not essential for viral replication *in vitro*, it is an important virulence factor *in vivo* \[[@pone.0163688.ref001], [@pone.0163688.ref002]\], and it is highly expressed from the early stages of infection \[[@pone.0163688.ref003]\]. Recent findings have elucidated the poorly understood and complex mechanisms by which Nef enhances viral infectivity \[[@pone.0163688.ref004]--[@pone.0163688.ref006]\]. Among other mechanisms, it downregulates CD4 cell surface expression and major histocompatibility complex I antigens, and it also modulates T-cell signaling pathways \[[@pone.0163688.ref007]\]. Although most studies focus on the Nef protein, the Nef gene also encodes an RNA structure, whose contribution to HIV virulence is largely unexplored. Experimental evidence shows that from the early stages of viral replication, Nef sequences appear within HIV mRNAs \[[@pone.0163688.ref008]\] that may facilitate viral rebound during antiretroviral therapy \[[@pone.0163688.ref009]\]. Based on computational approaches \[[@pone.0163688.ref010]\], Peleg et al. \[[@pone.0163688.ref011]\] noted that Nef RNA contains extensive information beyond the information required merely to encode the Nef protein, and they proposed that the information corresponds to conserved RNA structures. The reverse transcriptase in HIV lacks double-stranded proofreading, so its high mutation rate generates viral variants, some of which eventually escape selective pressures from the host immune system. The viral variability includes RNA motifs corresponding to "local" RNA secondary structures \[[@pone.0163688.ref012]\], which are in turn informative of 3D conformations \[[@pone.0163688.ref013]\]. Viral RNA conformations may in fact be evolving at a much faster rate than the underlying sequence itself \[[@pone.0163688.ref014]\]. Regardless of evolutionary rates, the prediction and identification of RNA motifs help to construct and to explore important biological hypotheses about the viral life cycle \[[@pone.0163688.ref015]--[@pone.0163688.ref018]\]. The inherent flexibility of RNA can yield alternative conformations ("folds") with distinct biological functions. Riboswitches typically have two alternative folds, e.g., for regulating bacterial genes \[[@pone.0163688.ref019]--[@pone.0163688.ref021]\]. Although viral RNAs are much shorter than bacterial RNAs, with correspondingly less sequence to encode information, their conformational changes can still indicate transitions between competing processes, or even RNA-protein interactions such as RNA processing, among other possibilities \[[@pone.0163688.ref022]\]. RNA's ability to assume different low-energy conformations, its "plasticity," can also be indicative of an ability to evolve \[[@pone.0163688.ref023]\], since only a few sequence mutations are then required to trigger dramatic conformational rearrangements. HIV-1 RNA may in fact be particularly flexible \[[@pone.0163688.ref024]\], and several alternative folds may be critical in the HIV-1 life cycle. As examples, the HIV-1 untranslated leader RNA influences RNA dimerization \[[@pone.0163688.ref025], [@pone.0163688.ref026]\]; nascent transcripts of TAR RNA influence transcription and gene expression \[[@pone.0163688.ref027]\]; and the Rev Response Element modulates nucleocytoplasmic export to control HIV replication rates \[[@pone.0163688.ref028], [@pone.0163688.ref029]\]. RNA interference (RNAi) is a cellular process expressing RNA molecules to inhibit gene expression, typically by accelerating RNA degradation. As such, RNAi provides an evolutionarily conserved defense against viruses \[[@pone.0163688.ref030], [@pone.0163688.ref031]\]. *In vitro*, Nef RNA can evade RNAi with alternative folds \[[@pone.0163688.ref032]--[@pone.0163688.ref034]\]. *In vivo*, HIV-1 RNA is under selective pressure to evade host-induced RNAi \[[@pone.0163688.ref035]\] to avoid the downregulated viral replication occurring when certain cellular and Nef RNAs interact \[[@pone.0163688.ref036], [@pone.0163688.ref037]\]. HIV also encodes RNAs, including a Nef-derived miRNA, miR-N367, to regulate its own transcription \[[@pone.0163688.ref038], [@pone.0163688.ref039]\]. Although induced RNAi can inhibit viral replication \[[@pone.0163688.ref040], [@pone.0163688.ref041]\], HIV is resistant to many RNAi-based antiviral therapies \[[@pone.0163688.ref042]\]. These considerations motivate the present investigation of the evolution of Nef RNA structures and their ability to evade the host immune system via alternative folds. This article measures RNA plasticity (i.e., the ability to take alternative folds) primarily with a computed quantity "Capacity for Alternative Folds" (CAF). CAF can contribute insights into RNA kinetics, because it incorporates more than just the minimum free energy structure into its measurement of plasticity. To relate changes in RNA plasticity to the evolution of the HIV epidemic, we collected HIV-1 sequences from the Los Alamos Database \[[@pone.0163688.ref043]\] and from a study on the North American HIV-1 Epidemic \[[@pone.0163688.ref044]\]. Based on sampling year, we partitioned each of the two datasets into Historic and Modern subsets. If the human immune system places detectable selective pressure on viral plasticity, each Modern dataset should be statistically different from the corresponding Historic dataset. Because the local RNA dynamics observed *in vitro* correspond to local changes to the RNA secondary structure \[[@pone.0163688.ref034]\], and because local predictions are generally more dependable than global predictions, our study primarily computed local secondary structures from subsequences, rather than global secondary structures from the full-length Nef sequence. Because small changes in a sequence can propagate dramatically to surrounding RNA structures \[[@pone.0163688.ref045]\], we examined overlapping subsequences. Many computational approaches to the *ab initio* prediction of alternative folds from RNA sequence exist \[[@pone.0163688.ref046]--[@pone.0163688.ref052]\]. Although these approaches do not have established success in discriminating riboswitches from genomic background, they have proved useful for investigating the structural features that accompany alternative folding. Ensemble diversity, based on sampling the energy landscape of RNA secondary structures, e.g., can characterize alternative structures in riboswitches \[[@pone.0163688.ref053]--[@pone.0163688.ref055]\]. Given an RNA sequence, ensemble diversity provides a competitive method for investigating thermodynamically stable alternative folds, and in addition, it has the merits of simplicity and generality. CAF, our measure of RNA plasticity, in fact partially characterizes the diversity in an ensemble of RNA conformations. Our other measures also quantify evolutionary changes in the full-length Nef RNA secondary structure, and in tertiary structural motifs such as pseudoknots and G-quadruplexes (G4). Materials and Methods {#sec002} ===================== Ethics statement: The sequences in the Test Datasets were obtained from subjects enrolled under REB-approved protocols who gave written, informed consent in the original studies collecting the specimens. The Institutional Review Boards at Providence Health Care/University of British Columbia and Simon Fraser University granted ethical approval for the study that provided the sequences in the Test Datasets \[[@pone.0163688.ref044]\]. The Office of Human Subjects Research Protections at NIH determined the present study was excluded from IRB review per 45 CFR 46 and NIH policy. HIV-1 Nef datasets: Historic and Modern Test Datasets: Cotton et al. \[[@pone.0163688.ref044]\] classified their cohorts into two datasets according to specimen collection date: a Historic Dataset (1979--1989) and a Modern Dataset (2000--2011). We used the same classification and coordinates (1--621) as their HXB2-alignments. Cotton et al. \[[@pone.0163688.ref044]\] collected sequences from untreated patients from various locations in North America and verified that the sequences belonged to HIV-1 subtype B. We discarded sequences whose alignments covered \< 41% of the HXB2 Nef gene, resulting in Historic and Modern Datasets of 335 sequences each. [Table 1](#pone.0163688.t001){ref-type="table"} displays additional details about the datasets. 10.1371/journal.pone.0163688.t001 ###### Features of the Test and Training Datasets of Nef sequences. {#pone.0163688.t001g} Dataset Name No. of patients (= Nef sequences) Collection date Sampling region Reference ---------- ---------- ----------------------------------- ----------------- ----------------- ------------------------------------------ Test Historic 335 1979--1989 North America Cotton et al. \[[@pone.0163688.ref044]\] Modern 335 2000--2011 Training Historic 9 1979--1989 Worldwide Los Alamos Database as of Feb 12, 2015 Modern 125 2000+ All Nef sequences are of HIV-1 subtype B and have \>95% coverage. No dataset shares a sequence with any other. Sequences of Historic and Modern Datasets are derived from drug-naïve patients. Historic and Modern Datasets for each category do not share a common patient. Historic and Modern Training Datasets: For the Training Datasets, we used the intra-patient Nef sequence data available in Los Alamos HIV Database as of Feb 12, 2015 \[[@pone.0163688.ref043]\]. We selected drug naïve patients infected with HIV-1 Subtype B worldwide (a total of 66 studies and 179 patients), with \> 95% Nef coverage, from the latest available sampling point (Fiebig stages 4--6). Our statistical tests require independent sampling, so each patient contributed only one sequence. If more than one sequence satisfied our criteria, we selected a single sequence at random. To parallel protocols for the Test Dataset, we split the Training Data into Historic (1979--1989) and Modern (2000+) Training Datasets. No patient contributed to both the Historic and Modern Training Datasets. The Historic Training Dataset contained 9 Nef sequences; the Modern Training Dataset, 125 Nef sequences. HIVAlign \[[@pone.0163688.ref043]\] aligned the Training Dataset sequences to the HXB2 reference sequence (Genbank accession: K03455). In every procedure described below, we optimized any adjustable parameters with the Training Datasets. Some optimizations were informal (by eye), whereas others had a formal figure of merit. In either case, the corresponding procedure with the Test Dataset always fixed its adjustable parameters and used the values optimized on the Training Dataset. For example, the Modern Training Dataset originally had 136 sequences but we reduced it to 125 to permit a useful but informal optimization from the resulting alignment. The Training and Test Datasets had no sequences in common. Measuring distance: Hamming distances quantified the dissimilarity between aligned pairs of sequences by the number of differing nucleotides (*d*~*nt*~) or amino acids (*d*~*aa*~). Dissimilarity between two RNA secondary structures can be measured by the number of differing base pairs (*d*~*bp*~) or the tree-editing distance (*d*~*te*~) \[[@pone.0163688.ref056]\], the minimum number of base-pair changes required to edit one RNA secondary structure into another. Symbols with over-bars represent average pairwise values, e.g., $\overline{d_{bp}}$ represents an average *d*~*bp*~ (see, e.g., the definition of CAF below). Extracting windowed Nef subsequences: Two parameters, the window size (Win, an even integer) and the skip size (Skip), were adjustable. Positions of the form *Window*/2 + *k* × *Skip*, (*k* = 0,1,...) within Nef alignment positions 1 to 621 provided coordinates for centering subsequences of length *Window* within the aligned Nef. We extended the Nef subsequences on both sides from their center so that each side included *Window*/2 letters of the original Nef sequence on either side. We optimized *Window* and *Skip* on the Training Dataset, minimizing by eye the overall *p*-values from our statistical tests. To facilitate the interpretation of biological features, the center of each Nef subsequence was mapped back into the reference HXB2 sequence. Capacity for alternative folds (CAF): For each windowed Nef subsequence *s* described above, according to standard thermodynamic models of RNA secondary structure \[[@pone.0163688.ref010], [@pone.0163688.ref057]--[@pone.0163688.ref060]\], the ViennaRNA (version 2.1.9) \[[@pone.0163688.ref056], [@pone.0163688.ref061]\] `command RNAsubopt -s -p 500` sampled *M* = 500 secondary structures *s*~*m*~ from the Boltzmann distribution. The command `RNAdistance -DF–Xm` calculated the base-pair Hamming distance *d*~*bp*~{*s*~*m*~,*s*~*n*~} between distinct sampled secondary structures *s*~*m*~ and *s*~*n*~, (*m*,*n* = 1,2,...,*M*). The CAF for the subsequence *s* is the average base-pair Hamming distance: $$CAF \equiv \overline{d_{bp}} = \frac{2}{M\left( {M - 1} \right)}{\sum\limits_{1 \leq m < n \leq M}{d_{bp}\left\{ {s_{m},s_{n}} \right\}}}$$ Thus, if a subsequence *s* has a large CAF, then on average, its folds contain very diverse base pairs. Test of statistical significance: The `R` function `wilcox.test` calculates two-sided *p*-values for the Mann-Whitney U-tests \[[@pone.0163688.ref062]\]. The U-test, which corrects for different sample sizes if necessary, evaluated the statistical significance of CAF changes between Historic and Modern Datasets of Nef subsequences. Adjusted *p*-values \[[@pone.0163688.ref063]\] based on a variant of the Benjamini Hochberg (BH) procedure \[[@pone.0163688.ref064]\] corrected for multiple tests by providing an upper bound on False Discovery Rates (FDRs). Selecting regions (R1, R2, and R3) with significant changes in their CAF: Fifty-three (⌈(*NefLength* − *Window*)/*Skip*⌉ = 53) different locations contributed to the comparison of Historic and Modern Datasets for *Window* = 100 nt and *Skip* = 10 nt, the parameters optimized on the Training Dataset. Of the locations, 6/53 showed a statistically significant CAF change between Historic and Modern Datasets, with *p*-values corresponding to a FDR \< 0.001 (shown in red in [S1 Fig](#pone.0163688.s001){ref-type="supplementary-material"}). The six regions overlapped, so we selected the three regions with the smallest *p*-values for further investigation, naming them R1 (112--211), R2 (362--461), and R3 (492--591). R1, R2, and R3 do not overlap, and all other regions overlap with one of them. Calculating RNAshapes Entropy: RNAshapes (version 2.1.6) \[[@pone.0163688.ref046]\], command `RNAshapes–p` calculated the probabilities for abstract RNA shapes. RNAshapes Entropy is the Shannon entropy of the resulting probability distribution (for details, see Section B.1 in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"}). Like CAF, RNAshapes Entropy is a measure of RNA plasticity. Computing RNA secondary structural stability and the most stable conformation: For each RNA subsequence *s*, the ViennaRNA command `RNAfold–T 37C` calculated the minimum free energy (MFE), which corresponds to the most stable secondary structure. Similarly, using default parameters, the ViennaRNA command `centroid_fold` calculated the consensus secondary structure, as follows. The ViennaRNA command `centroid_fold` randomly sampled *M* = 1000 RNA structures *s*~*m*~ from the Boltzmann distribution, listed all base pairs {*bp*~1~...*bp*~*N*~} occurring in at least half (*M*/2 = 500) of the structures *s*~*m*~, and then combined the base pairs {*bp*~1~...*bp*~*N*~} into a single, consensus structure (for details, see Ding et al. \[[@pone.0163688.ref065]\]). The free energy of the consensus structure is abbreviated as CFE (consensus free energy). To summarize, therefore, the free energy of the most stable structure is the MFE; of the consensus structure, the CFE. Typically, either a lower MFE or a lower CFE can suggest greater stability of the dominant RNA conformation. In passing, we note that according to the gold standard of RNA structural alignments, the CFE typically predicts RNA structure more accurately than the MFE \[[@pone.0163688.ref065]\]. Clustering RNA secondary structures for a single sequence *s* or a set *S* of sequences: Consider an arbitrary set of RNA secondary structures. The `R` function `pam` implements the Partitioning Around Medoids (PAM) algorithm to partition a set of structures into two clusters. Calculating dominant and alternative structures for a single R2 subsequence: For each R2 subsequence *s*, the ViennaRNA command `RNAsubopt -e 3 –s` sampled the Boltzmann distribution of secondary structures. The sampled structures were partitioned into two clusters, as described above. The "dominant structure" of the subsequence *s* was its MFE structure (which was the most stable structure in one of the clusters); and its "alternative structure" was the most stable structure in the other cluster (see Section B.2 in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"} for details). Calculating the Dominant-Alternative Hamming Distance (DAHD) for a single R2 subsequence *s*: The DAHD is the base-pair distance (*d*~*bp*~) between the dominant and alternative structures (see Section B.2 in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"} for details). Calculating dominant and alternative R2 structures from a set *S* of R2 subsequences: Each R2 subsequence *s* has a most stable structure. The PAM algorithm partitioned the R2 subsequences into two clusters according to their most stable structures, as described above. The larger cluster is the "dominant cluster" *C*~1~; the smaller, the "alternative cluster" *C*~2~. Each cluster has a medioid, an R2 subsequence within it whose structure has the minimum average base-pair distance *d*~*bp*~ to the other structures. Each medioid therefore provides a convenient representative of its cluster. The dominant cluster has medioid $\overline{C_{1}}$, "the dominant R2 subsequence" (of the set *S* of R2 subsequences), whose most stable structure is "the dominant R2 structure". Similarly, the medioid $\overline{C_{2}}$ is "the alternative R2 subsequence", whose most stable structure is "the alternative R2 structure". (A warning for all that follows: the adjective "dominant" always refers to the dominant cluster of structures.) Regressing CAF on MFE: Nested linear regression models of CAF on MFE, with and without a variable indicating whether a sequence was Modern, assessed the statistical significance of CAF changes (see Section B.3 in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"} for details). Mutation test: Among the set of R2 subsequences in the Modern Dataset, the dominant (most stable) structure $\overline{C_{1}\left( {Modern\ R2} \right)}$ corresponds to a particular R2 subsequence, which we call the "wildtype". The R2 subsequences corresponding to the dominant and alternative structures differed in two consecutive nucleotides M1M2 (398,399) and M5M6 (453,454), and two single nucleotides M3 (405) and M4 (447), corresponding to four hypothetical mutants M1M2, M3, M4, and M5M6 of the wildtype sequence. ViennaRNA compared the MFEs and the most stable structures of the mutants to the wildtype. Back-Translated (BT) Nef sequences: The EMBOSS (version 6.3.1) \[[@pone.0163688.ref066]\] command `backtranseq` generated random sets of hypothetical encoding full-length Nef sequences \[[@pone.0163688.ref011]\], based on back-translation with the HIV-1 codon-usage file at <http://www.kazusa.or.jp/codon/>. If a gap caused the amino acid corresponding to a real codon to become ambiguous, the randomization replaced the real codon triplet with three gaps. Thus, BT sequences typically had more gaps than real sequences. RNA-enforced Back-Translated (REBT) subsequence set: To investigate the significance of Modern R2 plasticity, we generated random RNA sequences such that both their secondary structure (hence, RNA-Enforced) and translated residues (hence, "Back-Translated") would resemble Modern R2 subsequences as much as possible. The random REBT sequences had the same length (100 nt) as the R2 subsequences, and each of 335 Modern R2 subsequences and its most stable structure served as a template for generating a REBT sequence, as follows: If a pair of nucleotide positions corresponded to a base pair in the most stable structure of the Modern sequence and both positions corresponded to the third codon, then the corresponding random bases in the REBT sequence were also a potential base pair. The empirical distribution of the base pair was p(A,U) = p(U,A) = 0.169, p(C,G) = p(G,C) = 0.273, and p(G,U) = p(U,G) = 0.058, the probabilities being the base pair frequencies in predictions of most stable structures for all 335 Modern R2 sequences. The frequencies are similar to Rfam base pair frequencies, so our results are likely robust for any reasonable choice of empirical base-pair frequencies. The Infernal command `esl-alistat` listed base pairs in the most stable structure \[[@pone.0163688.ref067]\]. If a nucleotide position corresponded to an unpaired nucleotide on the most stable structure of the Modern sequence, then the REBT procedure drew the corresponding base at random from a uniform distribution. All other nucleotide positions in the REBT sequence remained unchanged. The most stable structure generally paired very few third codon positions. Hence, the REBT procedure prefers to randomize base pairs, even if it changes an amino acid. Pseudoknot prediction: For a given sequence s, PknotsRG (version 1.3) \[[@pone.0163688.ref068]\] predicted pseudoknots within the RNA structure. For the R2 region only, the option `–m` predicted pseudoknots within the MFE structure. Calculating (Doubly) Differenced Relative Entropy: Relative Entropy calculations followed Peleg et al. \[[@pone.0163688.ref011]\], which uses MFE predictions from full-length Nef RNA sequence. The EMBOSS (version 6.3.1) \[[@pone.0163688.ref066]\] command `backtranseq` generated BT random sequences. Section C in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"} contains the details of calculating (Doubly) Differenced Relative Entropy ΔΔ*I* from Historic, Modern, and Back-Translated (BT) random sequences. Predicting RNA G-quadruplexes (locally and globally stable G4): The ViennaRNA command `RNAfold -g` predicted thermodynamically stable RNA G4 for both the 100-nt long windowed Nef subsequences (locally stable G4) and the full-length Nef RNA sequence (globally stable G4). See Section D in [S1 Text](#pone.0163688.s007){ref-type="supplementary-material"} and [S3 Table](#pone.0163688.s009){ref-type="supplementary-material"} for detailed results. Visualizing structures: The VARNA software \[[@pone.0163688.ref069]\] provided RNA secondary structure diagrams. Results {#sec003} ======= Three regions of Nef show noticeable change in RNA plasticity {#sec004} ------------------------------------------------------------- CAF quantified the RNA plasticity of windowed Nef subsequences in the Test Dataset. For each Nef subsequence of length 100 nt (the Window parameter) at 10 nt intervals along the Nef RNA sequence (the Skip parameter), the corresponding two-sided Mann-Whitney *p*-value compared the CAFs within the Historic and Modern Datasets (an exploratory optimization on the Training Dataset fixed the Window and Skip parameters used on the Test Dataset). All results presented correspond to the Test Dataset from Cotton et al. \[[@pone.0163688.ref044]\]. [S1 Fig](#pone.0163688.s001){ref-type="supplementary-material"} gives the complete set of *p*-values comparing the CAFs of all Historic and Modern Nef subsequences. Our focus here is on the three Nef regions with the most statistically significant changes in CAF, namely, the regions R1, R2, and R3 appearing in [Table 2](#pone.0163688.t002){ref-type="table"}. 10.1371/journal.pone.0163688.t002 ###### Summary of Nef regions with the most significant change in CAF. {#pone.0163688.t002g} Location on Nef (1--621) Datasets and *p*-values *d*~*aa*~ CFE GC CAF -------------------------- ------------------------- ---------------------- ---------------------- ---------------------- -------------- R1 (112--211) Historic 4.69 ± 4.70 -5.08 ± 4.10 0.47 ± 0.02 23.21 ± 7.00 Modern 5.69 ± 4.48 -6.26 ± 4.66 0.48 ± 0.02 20.77 ± 7.48 *p*-value 8.69x10^‒4^ 5.48x10^-2^ **3.26x10**^**‒5**^ (*q*-value) (5.76x10^-3^) (1.12x10^-1^) (5.75x10^-4^) R2 (362--461) Historic 2.73 ± 1.51 -11.90 ± 4.60 0.52 ± 0.02 13.15 ± 7.25 Modern 3.46 ± 1.59 -10.88 ± 5.07 0.52 ± 0.02 18.88 ± 8.73 *p*-value 6.01x10^-3^ 4.54x10^-1^ **5.44x10**^**‒19**^ (*q*-value) (2.22x10^-2^) (5.32x10^-1^) (2.89x10^-17^) R3 (492--591) Historic 4.45 ± 2.78 -8.07 ± 4.83 0.54 ± 0.02 16.84 ± 6.35 Modern 5.23 ± 2.52 -5.85 ± 4.39 0.52 ± 0.03 19.20 ± 6.71 *p*-value **5.67x10**^**‒10**^ **5.36x10**^**‒13**^ **1.13x10**^**‒6**^ (*q*-value) (3.01x10^-8^) (9.48x10^-12^) (3.00x10^-5^) Values with symbol ± denote mean ± standard deviation. The *p*-values in bold are significant at FDR levels 0.001. FDR calculations were performed separately for each measure of CFE, GC, and CAF. [Table 2](#pone.0163688.t002){ref-type="table"} quantifies some important sequence and structure features of the R1, R2, and R3 regions in the Historic and Modern Datasets. The Methods section describes our windowed Nef subsequences. Within each window (i.e., within each set of alignment columns), the average Hamming amino acid distance $\overline{d_{aa}}$ between translated pairs of subsequences quantified the amino acid diversity (the opposite of its conservation). The average of $\overline{d_{aa}}$ over all windows was $\hat{d_{aa}}\left( {Historic} \right) = 3.33$ in the Historic Dataset and $\hat{d_{aa}}\left( Modern \right) = 4.54$ in the Modern Dataset (see Section A in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"}). [Table 2](#pone.0163688.t002){ref-type="table"} shows that in both datasets, the amino acid diversity of R2 was lower than in its average over windowed subsequences (i.e., $2.73 = \overline{d_{aa}}\left( Historic\ R2 \right) < \hat{d_{aa}}\left( Historic \right) = 3.33$ and $3.46 = \overline{d_{aa}}\left( Modern\ R2 \right) < \hat{d_{aa}}\left( Modern \right) = 4.54$); the amino acid diversity of R1 and R3, higher. [Table 2](#pone.0163688.t002){ref-type="table"} also shows that R2 had a lower CFE (i.e., a more stable consensus RNA structure) than R1 and R3. In fact, when we varied the Window parameter (subsequence length) from 100 nt to values between 75 nt to 150 nt, both Historic and Modern Nef sequences in the Training Dataset consistently had the lowest CFE in regions overlapping R2, indicating that R2 contains the most stable local secondary structures within Nef. In [Table 2](#pone.0163688.t002){ref-type="table"}, the GC compositions of R1 and R2 in particular do not change much from Historic to Modern Datasets, so their GC composition is unlikely to influence directly the other structural features in [Table 2](#pone.0163688.t002){ref-type="table"}. RNA plasticity in region R2 increased significantly from Historic to Modern Datasets {#sec005} ------------------------------------------------------------------------------------ ### Modern R2 sequences have gained RNA plasticity as measured by CAF {#sec006} Overall, the CAF increase from Historic to Modern Datasets was most significant in R2 (*q*-value = 2.89x10^-17^); in contrast to other features such as GC composition and CFE, which changed less in R2 than in R1 or R3. The GC composition of R2 was similar in both Historic and Modern Datasets (0.52 ± 0.02, [Table 2](#pone.0163688.t002){ref-type="table"}). Moreover, at the FDR threshold of 0.01, R2 displayed no significant differences in either GC composition or CFE between the Historic and Modern Datasets. Notably, therefore, the R2 CAF increase from Historic to Modern Datasets lacks a co-association with changes in CFE or GC composition. ### Other measures confirm increased RNA plasticity in Modern R2 {#sec007} Additional procedures on R2 confirmed its increased RNA plasticity (see the [Materials and Methods](#sec002){ref-type="sec"}, and Section B in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"} for details). First, the Dominant-Alternative Hamming Distance (DAHD) counted the base-pair differences between structures (cluster medioids) representative of dominant and alternative RNA conformations of each R2 subsequence \[[@pone.0163688.ref053], [@pone.0163688.ref054]\]. Second, the software RNAshapes (version 2.1.6) \[[@pone.0163688.ref046]\] helped calculate RNAshapes Entropy, yet another, but very different measure of RNA plasticity and alternative folding. Third, an analysis regressing CAF on the MFE quantified the dependency of the increased CAF (as a measure of RNA plasticity) on changes in the MFE (as a measure of RNA stability). The analysis also compared the regression to a similar regression using Simian Immunodeficiency Virus homologs taken from *Pan troglodytes troglodytes* (SIVcpz.ptt), miRNAs, and TPP riboswitches. Genbank \[[@pone.0163688.ref070], [@pone.0163688.ref071]\] provided 5 SIVcpz.ptt sequences \[[@pone.0163688.ref072]\]. The NCBI/BLAST/TBLASTN tool \[[@pone.0163688.ref073]\] then extracted regions similar to HIV-1 Nef R2. Rfam \[[@pone.0163688.ref074]\] provided the miRNA and TPP riboswitch sequences. Like the CAF analysis, the DAHD and RNAshapes Entropy of Modern R2 were higher than those of the Historic R2 (comparing corresponding values in rows "Modern" and "Historic" in [Table 3](#pone.0163688.t003){ref-type="table"} and also RNAshapes Entropy bar plots in [S2 Fig](#pone.0163688.s002){ref-type="supplementary-material"}). Regression analyses also supported an increased RNA plasticity in Modern R2 where the F-statistic derived from ANOVA was 82.87 (p = 2.2 x10^‒16^) (see [S3 Fig](#pone.0163688.s003){ref-type="supplementary-material"} for regression plots). Section B in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"} provides extensive detail on the above tests. 10.1371/journal.pone.0163688.t003 ###### Measures of diversity and plasticity for Modern, REBT (random) Modern, and Historic R2 sets. {#pone.0163688.t003g} R2 $\overline{\mathbf{d}_{\mathbf{n}\mathbf{t}}}$ $\overline{\mathbf{d}_{\mathbf{a}\mathbf{a}}}$ $\overline{\mathbf{d}_{\mathbf{b}\mathbf{p}}}$ MFE CAF DAHD RNAshapes Entropy ------------- ------------------------------------------------ ------------------------------------------------ ------------------------------------------------ --------------- -------------- --------------- ------------------- Modern 8.30 3.46 38.55 -28.67 ± 4.04 18.88 ± 8.73 30.05 ± 18.11 0.51 ± 0.57 REBT Modern 20.51 4.09 41.43 -32.81 ± 4.07 13.93 ± 6.94 19.55 ± 16.35 0.36 ± 0.51 Historic 4.59 2.73 26.14 -29.49 ± 4.17 13.15 ± 7.25 19.32 ± 16.27 0.20 ± 0.41 ### Modern R2 has more plasticity than random RNA {#sec008} Column $\overline{d_{aa}}$ of [Table 3](#pone.0163688.t003){ref-type="table"} shows that although the amino acid diversity ($\overline{d_{aa}}$) of R2 was lower than that of R1 or R3, it was greater in Modern R2 than in Historic R2. We examined possible relationships between amino acid diversity and RNA plasticity with a set of RNA-Enforced-Back-Translated (REBT) random sequences, as follows. For each Modern R2 subsequence, the REBT Modern Dataset contained a random subsequence matched to it by length (100 nt), amino acid sequence, and RNA structural features (see the [Materials and Methods](#sec002){ref-type="sec"}). In [Table 3](#pone.0163688.t003){ref-type="table"}, the rows correspond to Modern, REBT Modern, and Historic Datasets of R2 subsequences. The columns correspond to various quantities associated with the sets of R2 subsequences, given as mean ± standard deviation where possible. Although the REBT Modern R2 subsequences had higher nucleotide ($\overline{d_{nt}}$), amino acid ($\overline{d_{aa}}$), and structure ($\overline{d_{bp}}$) diversity than real Modern R2 subsequences, they had lower RNA plasticity (as measured by CAF, DAHD, or RNAshapes Entropy) than real Modern sequences (e.g., 13.93 \< 18.88 for CAF). Most stable structures of R2 fall naturally into two clusters, the dominant cluster and the alternative cluster {#sec009} --------------------------------------------------------------------------------------------------------------- Consider each of the Historic and Modern Datasets in turn. Each R2 subsequence in the Dataset has a most stable structure (its predicted MFE structure). For each Dataset, the structures partitioned naturally into two clusters (i.e., the optimal clustering index under *d*~*bp*~ equaled 2). The larger cluster (which we examine first) we called the dominant cluster; the smaller, the alternative cluster. We chose a "dominant R2 structure" to represent the dominant cluster (the cluster medioid: see the [Materials and Methods](#sec002){ref-type="sec"}; also, [S1 Table](#pone.0163688.s007){ref-type="supplementary-material"}). The dominant R2 structure corresponds to an R2 subsequence, which for brevity and consistency with our other terminology, we call the "dominant R2 subsequence" (but see the warning about terminology in Methods subsection "Calculating dominant and alternative R2 structures from a set *S* of R2 subsequences"). Substitute "alternative" for "dominant" throughout to obtain analogous definitions of "alternative R2 subsequence" and "alternative R2 structure". The dominant R2 structure was the same in both Historic and Modern Datasets, whereas the alternative R2 structure in the Historic Dataset differed by a single base pair from its Modern counterpart. The size of the alternative cluster increased significantly from the Historic to the Modern Dataset, from 40/335 to 101/335 (two-tailed Fisher Exact p = 8.5x10^‒9^). ### In the Modern Dataset, the RNA structures predicted from the dominant R2 subsequence and from its full-length Nef sequence share features (and similarly, for the alternative R2 subsequence) {#sec010} Until further notice, consider only the Modern Dataset. If an RNA secondary structure predicted from a full-length sequence shares features with the structure predicted from a subsequence, the agreement increases confidence in the features. With this agreement in mind, define "the dominant Nef sequence" as the full-length Nef sequence (Nef coordinates 1--621) containing the dominant R2 subsequence. The dominant Nef sequence has a most stable RNA structure, called "the dominant Nef structure". Define similarly "the alternative Nef sequence" and "the alternative Nef structure". [S4 Fig](#pone.0163688.s004){ref-type="supplementary-material"} compares the dominant and alternative Nef structures to each other and to the dominant and alternative R2 structures. In [S4 Fig](#pone.0163688.s004){ref-type="supplementary-material"}, the dominant Nef structure contains the hairpin-like structure called P0 (in blue), as do both the dominant and alternative R2 structures. [S4(A) and S4(C) Fig](#pone.0163688.s004){ref-type="supplementary-material"} show that the dominant R2 structure shares features with the dominant Nef structure, with both containing both P0 and P1 hairpins. Furthermore, [S4(B) and S4(D) Fig](#pone.0163688.s004){ref-type="supplementary-material"} show that the alternative R2 structure shared similarities with the alternative Nef structure, with both containing the P2 hairpin. The agreements confirm that the hairpins probably have biological functions. ### Phylogenetic trees argue against an oversampling bias within our datasets {#sec011} The phylogenetic trees in [Fig 1](#pone.0163688.g001){ref-type="fig"} applied Neighbor-Joining \[[@pone.0163688.ref075]\] and the *p*-distance method \[[@pone.0163688.ref076]\] to amino acid distances (*d*~*aa*~) between: (A) the 335 full-length Nef sequences of the Historic Dataset; (B) the 335 Historic R2 subsequences; (C) the 335 Modern R2 subsequences; All analyses were conducted in MEGA6 \[[@pone.0163688.ref077]\]. Each blue triangle indicates an amino acid sequence whose R2 subsequence had a most stable RNA structure in the dominant cluster; each red circle, in the alternative cluster. In the Historic Dataset, a few R2 subsequences in the dominant cluster generated similar amino acid sequences (see [Fig 1(B)](#pone.0163688.g001){ref-type="fig"}, occasional concentrations of blue triangles). The similar amino acid sequences dispersed, however, in a phylogenetic tree based on full-length Nef sequences (see [Fig 1(A)](#pone.0163688.g001){ref-type="fig"}). The dispersion argues against biases from oversampling any clade in the Historic Dataset. In the Modern Dataset, R2 subsequences displayed no noticeable concentrations of either blue triangles or red circles (see [Fig 1(C)](#pone.0163688.g001){ref-type="fig"}). Thus, oversampling biases within our datasets appear unlikely. {#pone.0163688.g001} ### Five or six nucleotide mutations in R2 can cause a subsequence's most stable structure to switch between the dominant and alternative structures {#sec012} The tree-editing distance (*d*~*te*~) between dominant and alternative R2 structures for the Modern Dataset was 82; the base pair distance (*d*~*bp*~), 51 (see [S1 Table](#pone.0163688.s007){ref-type="supplementary-material"}); the nucleotide distance (*d*~*nt*~) between the dominant and alternative R2 subsequences, 6. (The corresponding figures for the Historical Dataset were *d*~*te*~, 82; *d*~*bp*~, 50; *d*~*nt*~, 5.) For random RNA sequences of length 100 nt, [Fig 3](#pone.0163688.g003){ref-type="fig"} of Schuster et al. \[[@pone.0163688.ref045]\] shows that the average tree-editing distance (*d*~*te*~) between pairs was about 35, whereas the nucleotide distance (*d*~*nt*~) usually exceeded 20. Thus, the distances between the dominant and alternative R2 structures differ quantitatively from distances between random sequences. ### The switch between dominant and alternative R2 structures depends mostly on synonymous nucleotide changes {#sec013} [Fig 2](#pone.0163688.g002){ref-type="fig"} displays the six point mutations converting the dominant R2 subsequence into the alternative R2 subsequence. (See *d*~*nt*~ in the previous paragraph.) As described in the Materials and Methods, the alternative R2 subsequence corresponded to four hypothetical mutants of the dominant "wildtype" sequence: consecutive-base mutants (M1M2, M5M6) and two single nucleotide mutants (M3, M4). Of these, only M3 and M4 are synonymous mutations. The codon containing M1M2 may reflect HLA restrictions \[[@pone.0163688.ref044]\]. {#pone.0163688.g002} Let ΔMFE denote a mutant MFE minus the wildtype MFE. On one hand, mutations M1M2 in codon (397,398,399) and M5M6 in codons (451,452,453), and (454,455,456) stabilized the dominant structure (ΔMFE = ‒2.2 kcal/mol for M1M2; ΔMFE = ‒0.9 kcal/mol for M5M6). M1M2 also stabilized the important hairpin P1. On the other hand, the synonymous mutations M3 and M4 destabilized it (ΔMFE = +1.4 kcal/mol and + 4.7 kcal/mol, respectively), apparently by unwinding stems. ### The Alternative conformation for R2 may contain pseudoknots {#sec014} Pseudoknots are a frequent type of base pairing in RNA structures. Because the secondary structure models in ViennaRNA do not predict pseudoknots, we used PknotsRG (version 1.3) \[[@pone.0163688.ref068]\] to assess pseudoknot formation in all Historic and Modern R2 subsequences (see [S2 Table](#pone.0163688.s008){ref-type="supplementary-material"}). PknotsRG predicted no pseudoknots in the dominant R2 structure, but it did predict three pseudoknots in the alternative R2 structure (see [S5 Fig](#pone.0163688.s005){ref-type="supplementary-material"}). Apart from the pseudoknots, the alternative R2 structure prediction was consistent with that of ViennaRNA. The synonymous change at M3 (mentioned above, at Nef coordinate 405) contributed to pseudoknots (see the black square in [S5 Fig](#pone.0163688.s005){ref-type="supplementary-material"}). The region between R2 and R3 has increased base-pair variability {#sec015} ---------------------------------------------------------------- Following Peleg et al. \[[@pone.0163688.ref011]\], each full-length Nef RNA sequence yielded a most stable secondary structure in a dot-bracket representation (dots, and left and right parentheses). For four datasets (Historic and Modern, and the corresponding random RNA back-translations preserving protein sequences), background frequencies of dots and brackets were noted (see Section C in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"}). Then, we aligned all full-length Nef RNA sequences in the Historic and Modern Datasets together. For each of the four datasets, RNA structural diversity within each alignment column was quantified by the Relative Entropy (***I***) of the frequencies of its dot-bracket characters relative to the background frequencies (with all gap characters ignored). In each column, subtracting the Relative Entropy for the random sequences from that for the real sequences yielded the Differenced Relative Entropy at each aligned position, which accounts for the effect of random sequence variations on structure. Then, subtracting the difference for the Historic Dataset from the difference for the Modern Dataset yielded the (Doubly) Differenced Relative Entropy, denoted by ΔΔ*I*. At each position, ΔΔ*I* represents the change in RNA structural diversity from Historic to Modern Dataset (see Section C in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"} for details). The diversity peaked at Positions 476, 482, and 485, i.e., 15 nt downstream of R2 and 15 nt upstream of R3 (see [S6 Fig](#pone.0163688.s006){ref-type="supplementary-material"}). Although the position of the ΔΔ*I* peaks do not match the previous study exactly, our analyses showed that the known (historic) structural diversity near R2 \[[@pone.0163688.ref011]\] increased even further from the Historic Dataset to the Modern Dataset. Modern R1 contains significantly fewer stable RNA G-quadruplexes than Historic R1 {#sec016} --------------------------------------------------------------------------------- G4s are abundant structural elements in both RNA and DNA, and simple energy models permit RNA secondary structure programs to predict them \[[@pone.0163688.ref078]\] (see Section D in [S1 Text](#pone.0163688.s007){ref-type="supplementary-material"} and [S3 Table](#pone.0163688.s009){ref-type="supplementary-material"} for details). Both local prediction (on 100-nt subsequences) and global prediction (on the full-length Nef RNA sequence) suggested that the positions of both locally and globally stable G4s are highly conserved within and between Historic and Modern sequences, particularly at two locations: 27--39 and 186--200. Locally stable G4s at location 186--200 decreased significantly from the Historic to the Modern Dataset (257/335 to 137/335, p = 3.0x10^‒21^, 76.7% to 40.9%) as did their corresponding globally stable G4 predictions (187/335 to 89/335, p = 1.6x10^‒14^, 55.8% to 26.6%). G4s at location 27--39 displayed a similar (but non-significant) trend (see [S3 Table](#pone.0163688.s009){ref-type="supplementary-material"}). Discussion and Conclusions {#sec017} ========================== To discover biologically important structural changes in Nef RNA during the evolution of the North American HIV epidemic, we compared two sets of HIV-1 sequences from untreated patients (Historic and Modern) and identified RNA changes in plasticity, quantified here as the capacity for alternative folds (CAF). In addition to plasticity and local RNA structure, our statistical analysis of RNA changes examined pseudoknots and G-quadruplexes, as well as the relative entropies relevant to structural diversity. [Fig 3](#pone.0163688.g003){ref-type="fig"} summarizes relevant results about the Nef gene by others and by the present work. {ref-type="table"} for exact coordinates.) The green region (sites 186--200) displays significantly reduced stable G4s. The region in black (sites 476--485) showed high diversity (quantified by the Doubly Differenced Relative Entropy ΔΔ*I*). The grey segment labeled **A** (segment 123--190) refers to the region coinciding with R1 identified by Westerhout et al. \[[@pone.0163688.ref034]\]; the gray label **B** points to segment 416--446, referred to by Peleg et al. \[[@pone.0163688.ref011]\]. In Fig 3, the term "Sequence and Structural Variations Increasing" refers to the observed simultaneous changes in the GC composition, CFE, and CAF of Modern R3.](pone.0163688.g003){#pone.0163688.g003} Many studies focus on the Nef protein, with its subtle and complex activities in viral infectivity. The function of the Nef protein complicates and therefore must temper any interpretation of evolutionary changes in RNA structure. The Nef gene does contain occasional conserved protein regions, however, where protein function is unlikely to drive systematic changes in the Nef RNA. Caveats aside, in moving from the Historic to the Modern Dataset of HIV sequences, significant differences in the 621 nt of Nef RNA included fewer G4s, more pseudoknots, and changes in RNA plasticity depending on the Nef region (less plasticity in R1 (112--211); more plasticity in R2 (362--461) and R3 (492--591). In R3, the GC composition, stability (CFE), and plasticity (CAF) all changed significantly, so correlations complicate any inference about specific selective pressures on R3. Accordingly, our inferences focus on R1 and R2. R1 secondary structure became more rigid over time {#sec018} -------------------------------------------------- Modern R1 (region 123--190) displays less nucleotide conservation than most of Nef, and its RNA plasticity and number of thermodynamically stable RNA G4s decrease from the Historic to the Modern Dataset. In fact, RNA base pairs mostly replace the G4s near a conserved region (164--182) targeted by RNAi. An experimental mutation increasing plasticity within R1 permitted an alternative fold that stopped RNAi from binding to the target, enabling Nef RNA to escape RNAi \[[@pone.0163688.ref034]\]. Here, the emergence of a more stable and rigid RNA secondary structure (decreased CFE and CAF) in R1, where Historic subsequences had an unusually high RNA plasticity (average CAF = 23.21), suggests a recent selective pressure for RNA secondary structural rigidity. To summarize, although R1 plasticity correlated positively with HIV replication in an experiment, it diminished during the North American HIV epidemic, in seeming contradiction with probable selective pressures on HIV. The biological implications are therefore unclear. Although RNAi-based antiviral therapies might be able to target R1 because of its decreased plasticity, they might also reverse the evolutionary trend to decreased R1plasticity by provoking unwanted escape mutants. Unusually strong evidence supports selective pressures on RNA plasticity in R2 {#sec019} ------------------------------------------------------------------------------ RNA in R2 encodes for thirty-two Nef amino acids within the anti-parallel β sheet on β3/β4. In Historic and Modern Datasets, the R2 amino acids are highly conserved (i.e., R2 subsequence pairs have a small *d*~*aa*~). Although the region upstream of R2 sometimes varies in length (see [Fig 2](#pone.0163688.g002){ref-type="fig"} of Reference \[[@pone.0163688.ref072]\]), an alignment of HIV-1 and SIV~cpz~ Nef proteins displays moderate amino acid conservation in R2. R2 has a low structural free energy (low CFE and MFE), strongly suggesting a locally stable RNA secondary structure, including a highly stable hairpin P0, confirming previous predictions \[[@pone.0163688.ref011]\]. Despite the amino acid conservation and the RNA structural stability, the most significant change in Nef RNA plasticity occurs in R2. (CAF, DAHD, and RNAshapes Entropy all increased, and regression analysis (Section B.3 in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"}) showed that CAF increased, even after accounting for confounding effects from the MFE.) A randomization (REBT) of the R2 subsequence had less plasticity (CAF, DAHD, and RNAshapes Entropy) than real Modern R2 subsequence, suggesting that the high plasticity in R2 may have biological functionality. Moreover, five or six nucleotide mutations can switch the most stable structure for R2 between the dominant and alternative structures, far fewer than the switch requires for random 100 nt sequences. Finally, the Training Dataset from the Los Alamos HIV database showed that the increase in plasticity (CAF) was robust against changes in the Window size (from 50 nt to 150 nt), even for HIV-1 B Nef sequences drawn worldwide. The relative stability of the dominant and alternative R2 structures does not correlate obviously with Nef protein sequence (see [Fig 1](#pone.0163688.g001){ref-type="fig"}). In fact, nonsynonymous nucleotide mutations in R2 (M1M2 and M5M6) usually stabilized one RNA conformation, as opposed to increasing plasticity to accommodate both conformations (see [Fig 2](#pone.0163688.g002){ref-type="fig"}). Although sparse, the evidence therefore tends to contradict selection at the amino acid level as a cause of increasing R2 plasticity. ### Apparent diversity of most stable (MFE) structures in the set of R2 subsequences is likely due to the high RNA plasticity at an individual sequence level {#sec020} Peleg et al. \[[@pone.0163688.ref011]\] noted (despite the severe limitations of the computations and data available to them) that the most stable structures for full-length Nef sequences fell into two groups, with the highly conserved hairpin P0 within R2 having length either 5 nt or 7 nt. Although not focused on P0 loop-length, our results showed increased abundance of the alternative most stable structure in Modern R2, in agreement with their conclusions about two different structures near R2. Tests of base-pair variability (ΔΔ*I*, Section C in [S1 Text](#pone.0163688.s010){ref-type="supplementary-material"}) also supported increased variability of the most stable structure near R2. Finally, tests of plasticity (CAF) show a noticeable increase in thermodynamically stable and RNA conformations available to each individual Modern R2 subsequence. To summarize, both the RNA plasticity of individual R2 subsequences and the diversity of the most stable structures of R2 subsequences increased over time. In agreement with Peleg et al. \[[@pone.0163688.ref011]\], our results show that there are two possible most stable structures within R2, a dominant structure and a less common alternative structure, and that the most stable structure of each individual R2 subsequence resembles either the dominant structure or the alternative structure. Our computations show in addition, however, that kinetics take each individual R2 subsequence through equilibrium conformations resembling in turn the dominant and alternative structures. The two findings, about stability and kinetics, relate naturally to each other. On one hand, in a set of R2 subsequences with high RNA plasticity, a few nucleotide mutations in each sequence can toggle its most stable structure between two conformations corresponding to the dominant and alternative structures. On the other hand, R2 is in fact highly conserved, with only minor sequence variability. R2 sequence conservation suggests heavy selective pressure, possibly to preserve RNA plasticity, so every individual Nef sequence has alternative folds available to it. R2 plasticity can help evade host responses and might even function in viral RNA switching {#sec021} ------------------------------------------------------------------------------------------ The increasing plasticity and potential for alternative folds in and near R2 may therefore be an HIV response to selective pressures from its human host. Such pressures may include cellular and viral RNA molecules in HIV-1-host interactions. As a specific example, Nef-derived miR-N367 targets the 3′-UTR of Nef, which includes R2, to regulate HIV-1 transcription and replication \[[@pone.0163688.ref038]\], and an alternative fold might evade miR-N367. Alternative folds could also operate like bacterial riboswitches. In some bacterial riboswitches, ligand binding causes allosteric conformational changes propagating along RNA like a domino effect. Among other attractive possibilities, HIV might exploit changes in Nef RNA (either directly or allosterically) to regulate reverse transcription, intra-subtype recombination, or even frameshifting during translation. Evidence in support of Nef RNA-protein coevolution {#sec022} -------------------------------------------------- The alternative most stable structure for R2 is more frequent in the Modern than Historic Dataset. The transition from the dominant to alternative most stable structure in R2 requires the P1 hairpin to destabilize, which synonymous changes (M3 and M4) facilitate, and nonsynonymous changes (M1M2 and M5M6) impede. Cotton et al. \[[@pone.0163688.ref044]\] suggest (see their Fig 6) that mutations at codon (397,398,399) preadapt Nef to its interactions with a restricted Human Leukocyte Antigen (HLA). According to our computations, however, the nonsynonymous mutation M1M2 actually stabilizes P1, favoring the dominant most stable structure and reducing plasticity. Our results suggest that in R2, Nef RNA is under selective pressure to accommodate alternative folds (e.g., the alternative most stable structure). The mutation M3 critical to increasing RNA plasticity is synonymous, ruling out selective pressures on the corresponding amino acid and confining adaptation to RNA (not protein). Possibly, the synonymous changes maintain or increase RNA plasticity and the frequency of alternative folds in R2, while permitting compensatory responses to selection pressures from HLA-polymorphisms at the protein level. The hairpin P1 within R2 may be an attractive target for antiviral therapeutics {#sec023} ------------------------------------------------------------------------------- Targeting stable RNA motifs may be a particularly fruitful therapeutic strategy \[[@pone.0163688.ref079]\]. R2 is the most stable region of Nef RNA. Within R2, the P1 hairpin appears in predictions of the most stable structure for both R2 and full-length Nef RNA, and its sequence may have to accommodate evolutionary pressures on both protein function and RNA plasticity. Furthermore, experiments suggest that certain HIV-1 viruses can evade RNAi \[[@pone.0163688.ref080], [@pone.0163688.ref081]\]. Hence, if increased R2 plasticity helps HIV evade RNAi, then computational predictions about alternative R2 folds may eventually contribute usefully to clinical decisions about RNAi therapies, by identifying evolutionary bottlenecks for HIV sequences that evade RNAi. Moreover, computations suggest that mutation M3 at position 405 in loop of P1 hairpin forms a pseudoknot only in the alternative R2 structure, which would increase RNA plasticity near P1 by stabilizing the alternative over the dominant R2 structure. Many considerations therefore point to the loop of P1 hairpin in R2 as a particularly attractive target for RNAi-based anti-viral therapeutics. Supporting Information {#sec024} ====================== ###### Two-sided Mann--Whitney U-test *p*-values at 10-nt intervals, comparing average Hamming distance between 335 Historic and 335 Modern Nef subsequences of length 100 nt. The blue points correspond to FDR not exceeding 0.01; the red, to an FDR not exceeding 0.001. The three bold red line segments correspond to the regions designated R1, R2, and R3. (PNG) ###### Click here for additional data file. ###### RNAshapes Entropy histogram of Historic and Modern Nef R2 subsequences. Colors blue, red, green, and black roughly correspond to number of structures 1, 2, 3, and 4, respectively (see [Materials and Methods](#sec002){ref-type="sec"}). (PNG) ###### Click here for additional data file. ###### MFE vs. CAF linear regression of R2. \(A\) CAF values of Historic R2 subsequences (335 red data points), Modern R2 subsequences (335 green data points), and SIVcpz.ptt (5 blue data points). Colored lines represent the corresponding linear regression models. Adjusted R-squared value of the linear model CAF\~MFE by combining Historic and Modern Datasets (670 data points) was 0.2455. Adjusted R-squared value of the model CAF\~MFE+R2 distinguishing Historic and Modern Datasets in the model, was 0.3279. ANOVA test between the two models including and excluding variable R2 gives F-value of 82.87 (p = 2.2 x10^‒16^). (B) CAF values from sequence datasets corresponding to miRNA (19 red data points) and TPP-riboswitch (42 blue data points). Colored lines represent the corresponding linear regression models. Adjusted R-squared value of the model CAF\~MFE by combining miRNA and TPP-riboswitch Sets (61 data points) was 0.3112. Adjusted R-squared value of the linear model CAF\~MFE+ncRNA distinguishing miRNA and TPP-riboswitch sets, was 0.3199. ANOVA test between the models including and excluding variable ncRNA gives F-value of 1.7588 (p = 0.19). The gray area in each plot shows a 95% confidence band for the linear regression model. (PNG) ###### Click here for additional data file. ###### Most stable RNA secondary structures of two representative Modern R2 structures. \(A\) The dominant R2 structure. (B) The alternative R2 structure. (C) The dominant Nef structure (i.e., the most stable structure of the full-length Nef sequence containing the R2 subsequence whose most stable structure is the dominant R2 structure). (D) The alternative Nef structure (i.e., the most stable structure of the full-length Nef sequence containing the R2 subsequence whose most stable structure is the alternative R2 structure). In A and B, red nucleotides mark the six mutations that switch the most stable structure between the dominant and alternative R2 structures. The conserved hairpin P0 is shown in blue. In C and D, R2 is marked in red, to contrast the structural predictions for the R2 subsequence and the full-length Nef sequence. In C and D, the green circles indicate notable ΔΔ*I* peaks. (PNG) ###### Click here for additional data file. ###### Pseudoknot prediction in the alternative R2 structure. The triple pseudoknot is shown with blue arrows. Hairpins P0 (shown in red stripe) and P2 are identical to prediction of the alternative structure. Nucleotide differences from those of the dominant structure are shown in red. Black square shows location of the synonymous nucleotide difference that corresponds to position of mutation M3. PKnotsRG \[[@pone.0163688.ref068]\] program was used for prediction. Visualization was done using VARNA \[[@pone.0163688.ref069]\]. (PNG) ###### Click here for additional data file. ###### (Doubly) Differenced Relative Entropy ΔΔ*I*. Top five values are shown in black dots. Positions 145, 476, 482, 485, and 490. Standard deviation resulting from sampling bias shown in red. Standard deviation value of each position was equal to the square root of sum of the individual variance measures corresponding to the four datasets. See [Materials and Methods](#sec002){ref-type="sec"} for more details. (PNG) ###### Click here for additional data file. ###### Clustering statistics of region R2 structures. MFE predictions of R2 sequences were used for clustering (See [Materials and Methods](#sec002){ref-type="sec"}). Base-pair Hamming distances between the two medoids of clusters are shown in column $d_{bp}\left\{ {\overline{C_{1}\left( {R2} \right)},\overline{C_{2}\left( {R2} \right)}} \right\}$. (DOCX) ###### Click here for additional data file. ###### Prediction of locally stable pseudoknots in R2. Total No. of predicted pseudoknots shows the number of pseudoknot pairs observed in a total of 335 sequences in each set. Locations of pseudoknots varied. The location of predicted pseudoknots in the alternative structure was conserved between the Historic and Modern counterparts. PKnotsRG \[[@pone.0163688.ref068]\] program was used for prediction. (DOCX) ###### Click here for additional data file. ###### Location and frequency of locally and globally stable RNA G-quadruplexes (G4) in the Historic and Modern Nef RNA datasets. Predictions made using RNAfold--g command from ViennaRNA Software (version 2.1.9) \[[@pone.0163688.ref056]\]. Rows "Global Historic" and "Global Modern" represent results corresponding to the full-length Nef sequence (globally stable RNA G4s). Rows "Local Historic" and "Local Modern" represent results corresponding to 100 nt windowed Nef subsequences (locally stable RNA G4s) that best surround locations derived from the global predictions. The blue column shows the location of the RNA G4 that had a noticeable decrease in frequency in Modern sequences. (DOCX) ###### Click here for additional data file. ###### Supplementary Materials. Section A: The average of Hamming amino acid distance over all 100-nt windows of Nef sequences. Section B: Additional Tests of RNA plasticity in Historic and Modern R2. Section C: Base-pair variability using full-length Nef RNA secondary structures. Section D: Globally and locally thermodynamically stable RNA G-quadruplexes within region R1. (DOCX) ###### Click here for additional data file. We thank Dr. Zabrina Brumme and Dr. Richard Harrigan at the British Columbia Centre for Excellence in HIV/AIDS for providing the sequence data from patients in North America. We are grateful for the help of Dr. Alexander Bolshoy at the Institute of Evolution in providing coordinates mapping our results to his findings. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: **Conceptualization:** JS MG AM.**Methodology:** JS MG AM.**Project administration:** JS.**Supervision:** JS MG.**Writing -- original draft:** AM.**Writing -- review & editing:** JS MG.

Introduction {#section1-1534735418816824} ============ Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer death in females worldwide, accounting for 23% (1.38 million) of the total new cancer cases and 14% (458 400) of the total cancer deaths in 2008.^[@bibr1-1534735418816824][@bibr2-1534735418816824]-[@bibr3-1534735418816824]^ As in most other countries, breast cancer is by far the most common cancer in women in China and has posed a formidable potential threat to public health owing to its high morbidity and mortality.^[@bibr4-1534735418816824][@bibr5-1534735418816824]-[@bibr6-1534735418816824]^ Important risk factors associated with breast cancer included estrogen receptor, progesterone receptor, growth factors, and others.^[@bibr7-1534735418816824]^ Despite the remarkable advances achieved in the fields of surgery, endocrine therapy, chemotherapy, radiotherapy, and targeted therapy against breast cancer over the past several decades, most cases are still suffering from the metastasis, recurrence, and adverse drug reactions (ADRs).^[@bibr8-1534735418816824]^ As an important part of complementary and alternative medicine, traditional Chinese medicine (TCM) has become one of the main methods for comprehensive anticancer treatment owing to its advantages in treating complications, preventing drug resistance, and so on.^[@bibr9-1534735418816824]^ According to TCM theory, the basic pathogenesis of breast cancer is the meridian barrier, phlegm retention, qi stagnation, and blood stasis.^[@bibr10-1534735418816824]^ Therefore, the therapeutic principle is to nourish liver and kidney, strengthen body resistance, and eliminate pathogen.^[@bibr11-1534735418816824]^ As a new formulation of TCM, Chinese herbal injections (CHIs) own the features of notable curative efficiency and high bioavailability.^[@bibr12-1534735418816824]^ Among the variety of CHIs, SQFZI has long been extensively used in the clinical setting. It is composed of *Codonopsis pilosula* (Franch) Nannf and *Astragalus membranaceus* (Fisch) Bunge and was approved by the State Food and Drug Administration of the People's Republic of China (CFDA) in 1999.^[@bibr13-1534735418816824]^ It possesses the effects of nourishing the spleen and stomach, promoting blood circulation, and removing blood stasis. Modern research has revealed that SQFZI has the characteristics of enhancing efficacy and reducing toxicity.^[@bibr14-1534735418816824]^ SQFZI combined with chemotherapy is currently widely applied for treating breast cancer in China. Considering that a relevant systematic review remains lacking, we intended to investigate the efficacy and safety of SQFZI for breast cancer using meta-analysis to provide valuable evidence for clinical decision making. Material and Methods {#section2-1534735418816824} ==================== Inclusion and Exclusion Criteria {#section3-1534735418816824} -------------------------------- The inclusion and exclusion criteria were prespecified according to the PICOS (patients, intervention, comparator, outcomes, study design) criteria through discussion by the authors. Only randomized controlled trials (RCTs) meeting the following criteria were included in this meta-analysis: (1) Types of studies: RCTs focused on the effect of SQFZI combined with chemotherapy for the treatment of breast cancer. (2) Participants: all the involved participants were diagnosed as breast cancer according to the pathological, cytological, and histological features. (3) Interventions: the interventions of the control group included conventional chemotherapy agents such as cyclophosphamide, doxorubicin, epirubicin, pirarubicin, 5-fluorouracil, paclitaxel, docetaxel, methotrexate, Changchun ruisabine, gemcitabine, capecitabine, cisplatin, mitomycin, and so forth. The experimental group included studies of SQFZI combined with the same chemotherapeutic drugs as the control group. (4) Outcomes: The primary outcomes of the research included the clinical total effective rate and the performance status. According to the therapeutic effect criterion of the World Health Organization for solid tumors,^[@bibr15-1534735418816824]^ the clinical total effective rate was calculated by the following formula: (number of complete response patients + number of partial response patients)/total number of patients × 100%. Karnofsky performance status (KPS) was used to assess the performance status of patients. An increase of more than 10 points after treatment was deemed as significant improvement. Additionally, the incidence of immune functions changes (T lymphocyte subsets such as CD3+, CD4+, CD8+, CD4+/CD8+, NK cell, and peripheral hemogram) and ADRs (leukopenia, nausea and vomiting, hepatorenal dysfunction, and so on) were evaluated as secondary outcomes. The criterion of the ADRs met the World Health Organization criteria for common toxicity of chemotherapy drugs released in 1981.^[@bibr16-1534735418816824]^ Exclusion criteria were as follows: (1) Types of studies: RCTs for which full-text versions were unavailable, case reports, animal experiments, editorials, letters, and review articles; as for any publications shared overlapping information, the more recent and comprehensive article was included. (2) Interventions: The chemotherapeutic drugs, dose, and duration of treatment was incomplete or incorrect. (3) Outcomes: RCTs did not report the data of clinical total effective rate, performance status, and ADRs. Literature Search {#section4-1534735418816824} ----------------- A systematic literature search was conducted to identify the published RCTs with SQFZI for the treatment of breast cancer. The retrieval was performed in the following databases from their inception to October 29, 2017: PubMed, the Cochrane library, Embase, China National Knowledge Infrastructure Database (CNKI), Wan-Fang Database, China Science and Technology Journal Database (VIP), and the Chinese Biomedical Literature Database (SinoMed). "Breast Neoplasm" was regarded as MeSH term. All the searching strategies were developed and adapted for each database. The search strategies of PubMed are listed as follows: 1. \#1 Breast Neoplasm\[MeSH Terms\] 2. \#2 Breast Cancer\[Title/Abstract\] OR Mammary Cancer\[Title/Abstract\] OR Breast Malignant Neoplasm\[Title/Abstract\] OR Breast Carcinoma\[Title/Abstract\] OR Breast Malignant Tumor\[Title/Abstract\] OR Human Mammary Carcinoma\[Title/Abstract\] OR Human Mammary Neoplasm\[Title/Abstract\] 3. \#3 \#1 OR \#2 4. \#4 Shenqi Fuzheng 5. \#5 Randomized Controlled Trial\[Publication Type\] 6. \#6 Controlled Clinical Trial\[Publication Type\] 7. \#7 random\*\[All Fields\] 8. \#8 \#5 OR \#6 OR \#7 9. \#9 \#3 AND \#4 AND \#8 Data Extraction and Quality Assessment {#section5-1534735418816824} -------------------------------------- Two independent reviewers performed the data extraction and in case of discrepancies, a third reviewer would be consulted. The following contents were considered in data extraction: (1) baseline characteristics of included RCTs---the first author, publication date; (2) characteristics of patients---the number of patients in the experimental group and the control group, age, TNM stage; (3) details of intervention---the names, dosages, and treatment cycles of SQFZI; and (4) outcomes---the measured data about clinical total effective rate, performance status, ADRs and immune function. All literature was managed by NoteExpress (Wuhan University Library, Wuhan, China). Methodological quality assessment of each RCT was conducted by the Cochrane risk of bias tool (*Cochrane Handbook for Systematic Reviews of Interventions*, version 5.1.0).^[@bibr17-1534735418816824]^ Disagreements were resolved by methodological experts to reach consensus. Besides, 5 domains of bias that are relevant to the quality of RCTs, namely random sequence generation (selection bias), allocation concealment (selection bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), selective outcome reporting (reporting bias), and other bias were considered. If any RCTs described a correct random generation, or implemented blinding or reported complete measure outcomes, this RCT belonged to "low risk." Otherwise, trials were judged as "high risk." The evaluation of "Unclear" meant that the literature did not provide enough information for judgments. Statistical Analysis {#section6-1534735418816824} -------------------- This current meta-analysis pooled data from clinical trials via Review Manager 5.3 (Cochrane Collaboration, Oxford, UK).^[@bibr18-1534735418816824]^ Dichotomous outcomes were presented as relative risk (RR), whereas continuous variable was evaluated by mean difference, and 95% confidence intervals (95% CIs) of outcomes were calculated to indicate the range of results. The chi-square test was applied to evaluate heterogeneity among studies, and *I*^2^ was used to show the magnitude of this heterogeneity. Results of *P* ≥ .1 and *I*^2^ ≤ 50% suggested a lack of significant heterogeneity; the fixed-effect model was used accordingly.^[@bibr19-1534735418816824]^ For cases with *P* \< .1 and *I*^2^ \> 50%, we adopted a random-effect model, and subgroup analysis was presented to explore the sources of heterogeneity.^[@bibr20-1534735418816824]^ Meanwhile, the visual inspection of publication bias was demonstrated by funnel plot. Egger's test and Begg's test were also adopted, the result of *P* \> .05 showed that there was no obvious publication bias among included studies.^[@bibr21-1534735418816824]^ In addition, sensitivity analysis was conducted in clinical total effective rate so as to test the stability of results, by excluding the RCT seriatim to resynthesize the data. Egger's test, Begg's test, and sensitivity analysis were estimated and processed using STATA 13.0 software (Stata Corporation, College Station, TX, USA).^[@bibr22-1534735418816824]^ Results {#section7-1534735418816824} ======= Literature Search and the Characteristics of Included RCTs {#section8-1534735418816824} ---------------------------------------------------------- Our research yielded a total of 209 RCTs by a primary search of the aforementioned literature databases. After reading the titles and abstracts by 2 reviewers respectively, 63 RCTs were retrieved in full text, of which 32 were excluded due to the following reasons: not complying with the intervention of inclusion criteria (12 RCTs); not referring to the diagnostic standard or therapeutic criteria (15 RCTs); improper randomization method (3 RCTs); could not obtain the full text (2 RCTs). Ultimately, 31 related RCTs were deemed eligible in this meta-analysis. The PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flow diagram in [Figure 1](#fig1-1534735418816824){ref-type="fig"} illustrates the selection process. {#fig1-1534735418816824} There were 1292 and 1251 patients in the experimental group and control group, respectively. Among included RCTs, the sample size ranged from 40 to 185. All the patients qualified were women, who received treatment once a day intravenously, and the most frequent period of treatment was 21 days in 9 trials (29%). More details form the included studies are presented in [Table 1](#table1-1534735418816824){ref-type="table"}. ###### The Basic Characteristics of the Included Studies.  Study ID N (E/C) Age, Years (E/C) TNM Stage Therapy of Experiment Therapy of Control Course (Days) Outcome^[a](#table-fn2-1534735418816824){ref-type="table-fn"}^ ------------------------------------------- --------- ----------------------------- ----------- ------------------------------- -------------------- --------------- ---------------------------------------------------------------- Qi QG 2013^[@bibr23-1534735418816824]^ 20/26 52 (median age) II-IV SFI 250 mL + CAF CAF 21 ② Ma FL 2015^[@bibr24-1534735418816824]^ 36/36 --- --- SFI 250 mL + CAF CAF 63 ①②③④ Jia CF 2016^[@bibr25-1534735418816824]^ 50/50 32-65 / 31-63 --- SFI 250 mL + CAF CAF 21 ①④ Wang W 2015^[@bibr26-1534735418816824]^ 65/65 31-72 / 34-71 --- SFI 250 mL + CAF CAF 63d ①④ Wang DJ 2013^[@bibr27-1534735418816824]^ 38/38 31-62 / 31-63 II-III SFI 250 mL + CAF CAF 63 ① Xie F 2014^[@bibr28-1534735418816824]^ 45/45 37-66 / 35-68 --- SFI 250 mL + CAF CAF 63 ①④ Yuan JW 2008^[@bibr29-1534735418816824]^ 38/35 \>19 II-III SFI 250 mL + CAF CAF 20 ①④ Lu MY 2010^[@bibr30-1534735418816824]^ 58/52 32-69 --- SFI 250 mL + CAF CAF 21 ① Chen XC 2016^[@bibr31-1534735418816824]^ 42/42 42.65 ± 8.27 / 42.63 ± 8.24 --- SFI 250 mL + CAF CAF 42 ①③④ Liu Y 2017^[@bibr32-1534735418816824]^ 52/52 30-58 / 31-58 I-III SFI 250 mL + CAF CAF 21 ① Fu YJ 2014^[@bibr33-1534735418816824]^ 45/45 32-52 II-III SFI 250 mL + CAF CAF 63 ②③ Liang F 2014^[@bibr34-1534735418816824]^ 27/27 29-57 --- SFI 250 mL + CTF CTF 42 ①② Huang ZF 2008^[@bibr35-1534735418816824]^ 30/30 24-68 / 26-66 II-IV SFI 250 mL + CTF CTF 42 ①②③④ Sun SH 2005^[@bibr36-1534735418816824]^ 43/39 35-70 / 30-73 I-III SFI 250 mL + CTF CTF 42 ① Zou TL 2006^[@bibr37-1534735418816824]^ 32/32 29-65 --- SFI 250 mL + CTF CTF 14 ① Chen F 2007^[@bibr38-1534735418816824]^ 34/34 38-64 --- SFI 250 mL + CEF CEF 21 ① Yang F 2016^[@bibr39-1534735418816824]^ 40/40 25-67 / 26-65 I-III SFI 250 mL + CEF CEF 28 ①④ Dai ZJ 2007^[@bibr40-1534735418816824]^ 65/61 27-69 / 26-70 II-III SFI 250 mL + CEF CEF 28 ①③④ Wang SM 2006^[@bibr41-1534735418816824]^ 40/32 45.2 ± 9.8 / 46.7 ± 10.5 --- SFI 250 mL + CEF CEF 21 ① Xiao HW 2005^[@bibr42-1534735418816824]^ 55/53 43-67 --- SFI 250 mL + CEF CEF --- ②③④ Zhao BB 2012^[@bibr43-1534735418816824]^ 63/47 30-74 / 29-75 --- SFI 250 mL + ATC ATC --- ④ Li XL 2004^[@bibr44-1534735418816824]^ 40/35 56.4 / 54.2 --- SFI 250 mL + NE NE 28 ①②③④ Wu M 2012^[@bibr45-1534735418816824]^ 36/36 35-69 / 36-68 --- SFI 250 mL + CMF CMF 28 ① Song ZJ 2011^[@bibr46-1534735418816824]^ 21/25 32-65 / 35-61 II-III SFI 250 mL + CMF CMF 14 ① Qiao YC 2013^[@bibr47-1534735418816824]^ 20/20 40-65 --- SFI 250 mL + ECX ECX 14 ① A TK 2011^[@bibr48-1534735418816824]^ 40/40 28-65 --- SFI 250 mL + TA TA 21 ①② Chen JM 2010^[@bibr49-1534735418816824]^ 90/95 42/45 III-IV SFI 250 mL + TD TD --- ①④ Wang L R2016^[@bibr50-1534735418816824]^ 30/30 35-60 --- SFI 250 mL + TC TC 21 ①④ Nie JY 2005^[@bibr51-1534735418816824]^ 30/30 37-65 / 36-70 --- SFI 250 mL + 5-Fu + NVB NVB + 5-Fu 21 ②③ Zhang Q2013^[@bibr53-1534735418816824]^ 32/32 32-67 --- SFI 250 mL + GEM + DDP GEM + DDP 21 ① Li YQ 2002^[@bibr54-1534735418816824]^ 35/27 47.2 ± 10.8 / 46.7 ± 10.5 --- SFI 250 mL + 5-Fu + DDP + MMC 5-Fu + DDP + MMC 21 Abbreviations: E, experimental group; C, control group; CAF, cyclophosphamide+ doxorubicin+ 5-fluorouracil; CTF, cyclophosphamide+ pirarubicin+ 5-fluorouracil; CEF, cyclophosphamide+ epirubicin + 5-fluorouracil; ATC, anthracyclines; NE, navelbine+ epirubicin; CMF, cyclophosphamide + methotrexate + 5-fluorouracil; ECX, capecitabine; TA, paclitaxel + doxorubicin; TD, pirarubicin + docetaxel; TC, docetaxel + Epirubicin; NVB, navelbine; GEM, gemcitabine; DDP, cisplatin; MMC, mitomycin. ①, the clinical total effective rate; ②, the performance status; ③, adverse drug reactions (ADRs); ④ immune function. Quality Assessment {#section9-1534735418816824} ------------------ After performing quality assessment, only 3 RCTs adopted a random number table to generate the group, 1 RCT grouped patients by the toss of a coin.^[@bibr28-1534735418816824],[@bibr29-1534735418816824],[@bibr37-1534735418816824],[@bibr52-1534735418816824]^ Therefore, their selection bias was evaluated as "low risk." Five RCTs grouped in congruence with the therapeutic methods or admission time, so the selection bias was remarked as "high risk."^[@bibr36-1534735418816824],[@bibr42-1534735418816824],[@bibr48-1534735418816824],[@bibr54-1534735418816824]^ The other 22 RCTs did not illustrate the specific method of random sequence generation, therefore their selection bias was "unclear risk." Information on allocation concealment and blinding was not observed in the trials. Hence this study evaluated the selection bias of allocation concealment, performance bias, and detection bias as "unclear." Moreover, none of the included RCTs assessed had incomplete data, thus the attrition bias and reporting bias were assessed as "low risk." Additionally, none of the RCTs offered any details contributing to high risk for other bias, so this item was appraised as "unclear risk." Graphical description about quality assessment is shown in [Figure 2](#fig2-1534735418816824){ref-type="fig"}. {#fig2-1534735418816824} Outcomes {#section10-1534735418816824} -------- ### Clinical Total Effective Rate {#section11-1534735418816824} Depending on use of anthracycline, all chemotherapy regimens were divided into anthracycline-based subgroup and other chemotherapeutic drugs subgroup. The former contained 3 main chemotherapies: cyclophosphamide + doxorubicin + 5-fluorouracil (CAF), cyclophosphamide + pirarubicin + 5-fluorouracil (CTF), or cyclophosphamide + epirubicin + 5-fluorouracil (CEF).We assessed the clinical total effective rate in 2 subgroups respectively. The results of subgroup analysis are as follows. Anthracycline-based subgroup: 9 RCTs were available in this subgroup and displayed no heterogeneity (*P* = .90 \> .1, *I*^2^ = 0% \< 50%); a fixed-effect model was used.^[@bibr24-1534735418816824],[@bibr27-1534735418816824],[@bibr28-1534735418816824],[@bibr31-1534735418816824],[@bibr32-1534735418816824],[@bibr34-1534735418816824],[@bibr35-1534735418816824],[@bibr39-1534735418816824],[@bibr40-1534735418816824]^ The results indicated a statistically significant difference between SQFZI group and control group; thus, in terms of clinical total effective rate, SQFZI combined with anthracycline-based chemotherapy was superior to the anthracycline-based chemotherapy alone (RR = 1.36, 95% CI 1.21-1.53, *P* \< .00001). Other chemotherapeutic drugs subgroup: 4 RCTs demonstrated no evidence of heterogeneity in this subgroup, so the fixed-effect model was adopted (*P* = .12 \> .1, *I*^2^ = 48% \< 50%).^[@bibr46-1534735418816824],[@bibr48-1534735418816824],[@bibr49-1534735418816824],[@bibr52-1534735418816824]^ The results of meta-analysis presented a better impact when adding SQFZI than using other chemotherapeutic drugs alone. The difference between two groups was statistically significant (RR = 1.22, 95% CI 1.03-1.44, *P* = .02). There was no obvious interstudy heterogeneity reported among the subgroups (*P* = .31 \> .1, *I*^2^ = 4.3% \< 50%), hence it is acceptable that the results derived from 2 subgroups could be amalgamated. The pooled analysis demonstrated that SQFZI group performed even better on improving clinical total effective rate than control group, which received chemotherapeutic alone. The difference between SQFZI group and control group was considered as statistically significant (RR = 1.31, 95% CI 1.19-1.44, *P* \< .00001; [Figure 3](#fig3-1534735418816824){ref-type="fig"}). {#fig3-1534735418816824} Sensitivity Analysis and Publication Bias {#section12-1534735418816824} ----------------------------------------- For the outcome of clinical total effective rate, a sensitivity analysis was carried out to verify the stability of result. As shown in [Figure 4](#fig4-1534735418816824){ref-type="fig"}, since the clinical total effective rate did not show a qualitative transform, the result of sensitive analysis was robust. {#fig4-1534735418816824} Although the results of Egger's test (*t* = 2.20, *P* = .05) and Begg's test (*z* = .92, *P* = .360 \>.05) indicated no significant publication bias, the funnel plot on publication bias for clinical total effective rate presented modest asymmetry ([Figure 5](#fig5-1534735418816824){ref-type="fig"}), which suggested that there might be potential publication bias among included RCTs. {#fig5-1534735418816824} Performance Status {#section13-1534735418816824} ------------------ Anthracycline-based subgroup: No significant heterogeneity was detected among 10 RCTs of this subgroup (*P* = .24 \> .1, *I*^2^ = 23% \< 50%), thus we applied the fixed-effect model.^[@bibr23-1534735418816824][@bibr24-1534735418816824][@bibr25-1534735418816824]-[@bibr26-1534735418816824],[@bibr30-1534735418816824],[@bibr34-1534735418816824][@bibr35-1534735418816824]-[@bibr36-1534735418816824],[@bibr41-1534735418816824],[@bibr42-1534735418816824]^ Compared with anthracycline-based therapy alone, the SQFZI group was more effective in raising performance status (RR = 2.29, 95% CI 1.79-2.93, *P* \< .00001); the difference between 2 groups was considered to be statistically significant. Other chemotherapeutic drugs subgroup: There were 5 RCTs in this subgroup.^[@bibr44-1534735418816824],[@bibr46-1534735418816824][@bibr47-1534735418816824]-[@bibr48-1534735418816824],[@bibr53-1534735418816824]^ After examination, no heterogeneity was found, so we adopted fixed-effects model (*P* = .43 \> .1, *I*^2^ = 0% \< 50%). It turned out that SQFZI combined with other chemotherapeutic drugs was superior to chemotherapy based on other drugs alone for improving performance status of patients. The difference between the groups was statistically significant (RR = 2.04, 95% CI 1.55-2.68, *P* \< .00001). No visible heterogeneity was obtained between the two subgroups (*P* = .55 \> .1, *I*^2^ = 0% \< 50%), so we merged these subgroups into one group. The result demonstrated that SQFZI group achieved better effects than the control group, which received chemotherapeutic drugs alone; the difference between the above 2 groups had statistical significance (RR = 2.18, 95% CI 1.82-2.62, *P* \< .00001; [Figure 6](#fig6-1534735418816824){ref-type="fig"}). {#fig6-1534735418816824} Other Outcomes {#section14-1534735418816824} -------------- This study made a qualitative description for immune function of patients, which was considered as secondary outcome. The pooled analysis demonstrated that SQFZI group performed better in preventing the loss of peripheral T-lymphocyte subsets (CD4+, CD4+/CD8+), NK cell, leukopenia, and platelets. However, the combination of SQFZI and chemotherapy failed to achieve a better effect on CD3+, CD8+, and hemoglobin. More details regarding immune function were presented in [Table 2](#table2-1534735418816824){ref-type="table"}. ###### Results of Other Outcomes.  Outcomes N *I* ^2^ Effect Model Mean Difference \[95% CI\] *P* ------------ ---- --------- -------------- ---------------------------- ---------- CD3+ 10 98% Random 4.90 \[−1.34, 11.13\] .12 CD4+ 12 98% Random 7.51 \[3.31, 11.70\] .0005 CD8+ 12 97% Random 0.96 \[−2.56, 4.49\] .59 CD4+/CD8+ 12 70% Random 0.30 \[0.21, 0.39\] \<.00001 NK 10 91% Random 9.06 \[5.62, 12.50\] \<.00001 Leukopenia 5 0% Fixed 1.31 \[0.99, 1.63\] \<.00001 Hemoglobin 5 94% Random 3.76 \[−6.24, 13.76\] .46 Platelet 5 0% Fixed 12.53 \[1.96, 23.10\] .02 Adverse Drug Reactions {#section15-1534735418816824} ---------------------- In this meta-analysis, we mainly discussed three representative ADRs (leukopenia, nausea, and vomiting and hepatorenal dysfunction) and other ADRs to assess the curative effect of experimental group: 1. Twelve RCTs reported leukopenia.^[@bibr24-1534735418816824],[@bibr27-1534735418816824],[@bibr32-1534735418816824][@bibr33-1534735418816824]-[@bibr34-1534735418816824],[@bibr36-1534735418816824],[@bibr39-1534735418816824],[@bibr44-1534735418816824],[@bibr45-1534735418816824],[@bibr47-1534735418816824],[@bibr48-1534735418816824],[@bibr52-1534735418816824]^ The overall results demonstrated that the combination of SQFZI and chemotherapy was more efficient in relieving leukopenia than the control group which only received the chemotherapy; these between-group differences were statistically significant (RR = 0.51, 95% CI 0.41-0.64, *P* \< .00001; [Figure 7a](#fig7-1534735418816824){ref-type="fig"}). 2. Eight RCTs covered data on nausea and vomiting: pooled results showed that SQFZI plus chemotherapy decreased nausea and vomiting compared with chemotherapy alone.^[@bibr27-1534735418816824],[@bibr32-1534735418816824][@bibr33-1534735418816824]-[@bibr34-1534735418816824],[@bibr38-1534735418816824],[@bibr45-1534735418816824],[@bibr48-1534735418816824],[@bibr52-1534735418816824]^ There was no statistically significant between-group difference (RR = 0.51, 95% CI 0.40-0.66, *P* \< .00001; [Figure 7b](#fig7-1534735418816824){ref-type="fig"}). 3. Seven RCTs investigated hepatorenal dysfunction.^[@bibr24-1534735418816824],[@bibr27-1534735418816824],[@bibr28-1534735418816824],[@bibr31-1534735418816824][@bibr32-1534735418816824]-[@bibr33-1534735418816824],[@bibr48-1534735418816824]^ The results demonstrated that the conjunctive use of SQFZI and chemotherapy can decrease the incidence of hepatorenal dysfunction observably, and no significantly statistical difference was found (RR = 0.38, 95% CI 0.25-0.59, *P* \< .0001; [Figure 7c](#fig7-1534735418816824){ref-type="fig"}). {#fig7-1534735418816824} Additionally, this meta-analysis demonstrated that the SQFZI group had better efficacy in relieving other ADRs caused by chemotherapeutic drugs, including electrocardiogram changes, alopecia, intestinal reaction, thrombocytopenia, hemoglobin reduction, myelosuppression, and fatigue ([Table 3](#table3-1534735418816824){ref-type="table"}). ###### Results of Other Adverse Drug Reactions (ADRs).  ADRs N Effect model Relative Risk \[95% CI\] *P* --------------------------- --- -------------- -------------------------- --------- Electrocardiogram changes 5 Fixed 0.27 \[0.13, 0.56\] .0004 Alopecia 4 Fixed 0.43 \[0.26, 0.73\] \<.002 Intestinal reaction 3 Fixed 0.49 \[0.35, 0.68\] \<.0001 Thrombocytopenia 3 Fixed 0.61 \[0.40, 0.92\] .02 Hemoglobin reduction 3 Fixed 0.48 \[0.29, 0.78\] .003 Myelosuppression 2 Fixed 0.56 \[0.37, 0.87\] .009 Fatigue 2 Fixed 0.51 \[0.36, 0.72\] .0001 Discussion {#section16-1534735418816824} ========== This meta-analysis assessed the available evidences derived from 31 RCTs to detect the efficacy and safety of SQFZI combined with chemotherapy in treating breast cancer. According to the foregoing results, SQFZI plus chemotherapy can make a nonnegligible influence versus chemotherapy alone in terms of improving clinical total effective rate and performance status. Subgroup analysis revealed that especially in improving performance status of patients, SQFZI combined with chemotherapy showed a more impressive effect. Simultaneously, this combined chemotherapy approach also enhanced immune function of breast cancer patients and relieved ADRs. Breast cancer poses threats to the health and safety of human life. Although the treatment of this disease has been constantly improved, ADRs of patients have still increased year by year, which becomes a significant handicap to enhancing the curative effect of breast cancer.^[@bibr3-1534735418816824]^ In TCM theories, the occurrence of breast cancer is mostly associated with invasion of exopathogens and the deficiency of Qi-blood.^[@bibr54-1534735418816824]^ TCM possesses unique advantages of overall regulation and syndrome differentiation treatment as well as addressing both the symptoms and the root causes of the disease.^[@bibr55-1534735418816824]^ Related pharmacological studies have shown that SQFZI promotes the proliferation of macrophages in vitro, thereby improving the immunosuppression caused by chemotherapeutic drugs.^[@bibr56-1534735418816824]-[@bibr57-1534735418816824]^ Furthermore, research suggested that astragaloside could inhibit the proliferation of tumor cells in S phase and G2/M phase, suppress the expression of p21 and reduce the activity of cyclin-dependent kinase.^[@bibr58-1534735418816824]^ The authors concluded that astragaloside could be used as an effective adjuvant chemotherapeutic drug in cancer treatment.^[@bibr59-1534735418816824]^ As one of the effective components of *Codonopsis pilosula, Codonopsis pilosula* polysaccharides (CPP) could cause the inhibition of SMMC-7721 cells of hepatoma cells without obvious toxicity to viscera of Kunming mice.^[@bibr60-1534735418816824]^ To summarize, these 2 kinds of Chinese herbs synergistically nourish Qi-blood, reinforce kidney and spleen, and eliminate stagnation. Currently, there is a lack of systematic reviews comparing SQFZI combined with chemotherapy in the treatment of breast cancer. In this regard, our meta-analysis provides relevant medical evidence in this field and has the following advantages: First, to our knowledge, the present study is the first meta-analysis which delved into the efficacy and safety of SQFZI combined with chemotherapy in treating breast cancer. Second, we conducted a comprehensive literature search to identify the published studies through the combination of MeSH terms and text words. Clearer inclusion and exclusion criteria have been introduced, and the patients were diagnosed with breast cancer by a definite diagnostic standard, with a relatively consistent baseline. Third, we carried out subgroup analysis in the light of whether anthracyclines were used or not, which demonstrated that regardless of use of anthracyclines, chemotherapy drugs combined with SQFZI had superior curative effect. Arguably, no conspicuous intergroup heterogeneity was found between 2 subgroups. However, because of the limited data available for this population, the present meta-analysis had some limitations. First, only 5 RCTs specifically described randomization method, whereas they did not make a detailed description of random sequence connation, allocation concealment, or blinding methods. Besides, most items were assessed as unclear risk, which may have therefore affected the reliability of the results. Second, though the Egger's test and Begg's test manifested that there was no potential publication bias in present study, the deficiency of the funnel plot's bottom also indicated a lack of RCTs with large sample. Third, our results might have limited generalizability because all of the included RCTs were performed in China among Chinese populations; therefore, it is unclear whether the effect may change when SQFZIs are used in populations of other ethnicities and in different geographical locations. Fourth, because of the original research limitation, we failed to evaluate the long-term effect of SQFZI. On account of the limitations mentioned above, we raise several suggestions: first, RCTs are supposed to be registered in advance and implemented according to CONSORT standard so as to ensure the transparency of trial process.^[@bibr61-1534735418816824]^ Meanwhile, the clinical trials should pay more attention to randomization, concealment, blinding methods, and long-term follow-up to provide high-quality evidence-based medical evidence for clinical decision making. In addition, clinicians ought to strengthen monitoring of ADRs while remaining concerned on the measurement of effectiveness. It is the responsibility of the medical staff to use SQFZI as per the instruction guidelines and monitor the occurrence of ADRs. Conclusions {#section17-1534735418816824} =========== In summary, this study revealed that the combination of SQFZI and chemotherapy had a better effect on treating breast cancer. However, due to the limitations of the current meta-analysis, the strength of evidence needs to be promoted by rigorously designed, multicentered, large-sample randomized double-blind controlled trials. **Declaration of Conflicting Interests:** The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. **Funding:** The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the National Nature Science Foundation of China (No. 81473547; No. 81673829).

Correction to: *Nature Communications* 10.1038/s41467-019-13542-2, published online 11 December 2019. The original version of this article omitted references to previous work in 'Bondar, T. & Medzhitov, R. p53-mediated hematopoietic stem and progenitor cell competition. *Cell Stem Cell* **6**, 309--322 (2010)' and 'Marusyk, A., Porter, C. C., Zaberezhnyy, V. & DeGregori, J. Irradiation selects for p53-deficient hematopoietic progenitors. *PLoS Biol.* **8**, e1000324 (2010)'. These have been added as references 23 and 24 in the Introduction, the original sentence 'We discovered that mutant p53 enhances the repopulating potential of HSPCs^22^' has been amended to 'We discovered that mutant p53 enhances the repopulating potential of HSPCs^22^, similar to what has been reported previously^23,24^.' This has been corrected in the PDF and HTML versions of the article.

Treatments of type 2 diabetes must balance the prevention of microvascular complications with the minimization of clinically significant hypoglycemia. The difficulty in safely achieving these goals, combined with epidemic increases in diabetes worldwide, has spurred the search for novel therapeutic strategies. Among these, inhibition of the Na^+^-glucose cotransporter type 2 (SGLT2) has emerged as a promising therapy ([@B1],[@B2]). SGLT2 is a member of the *SLC5* gene family and transports glucose across cells using the Na^+^ gradient established by Na^+^-K^+^-ATPases ([@B3]). SGLT2 is a low-affinity, high-capacity transporter expressed predominantly in the early proximal tubule of the kidney and accounts for about 90% of renal glucose reabsorption ([@B4]--[@B6]). Given that the kidney filters approximately 180 g of glucose daily, SGLT2 inhibition may not just reduce hyperglycemia but may also promote negative energy balance and weight loss. Type 2 diabetes is characterized by fasting hyperglycemia as a result of insulin resistance, but is often preceded by hyperinsulinemia and normal blood glucose levels, a state that is maintained by compensatory insulin secretion by the pancreatic β-cell ([@B7]). The ability of the β-cell to counteract an increased glucose load is short-lived, however, and eventually pancreatic islets fail, giving rise to hyperglycemia. Rodent and human studies have both shown that glucose toxicity is implicated in β-cell failure by increasing the rate of β-cell death by the induction of proapoptotic genes ([@B8]--[@B10]). Inhibition of SGLT2 therefore has the potential to not only acutely lower hyperglycemia but to also improve glucose homeostasis by reducing glucose toxicity and preventing islet failure. Despite recent interest in SGLT2 as a potential target for diabetes treatment, relatively few long-term models of SGLT2 deficiency have been characterized. Previously, nonselective inhibition of both SGLT1 and SGLT2 for 4 weeks in partially pancreatomized diabetic rats by injection of phlorizin led to increases in insulin sensitivity and insulin secretion ([@B11],[@B12]). More recently, improvements in glucose homeostasis were demonstrated in diabetic rodent models after treatment with SGLT2-specific inhibitors for periods of 2 to 9 weeks ([@B13]--[@B16]). As many as seven different SGLT2 inhibitors designed for use in humans have been characterized in cell culture and animal studies, and many of these have moved on to clinical trials ([@B2],[@B17]--[@B22]). Here, we describe the first in vivo characterization of glucose homeostasis in a SGLT2 knockout mouse model. We investigated the behavioral and metabolic consequences of SGLT2 deletion, and furthermore, we determined the effect of renal glucose excretion on glucose homeostasis, insulin sensitivity, and β-cell function in the context of both high-fat feeding and genetically determined obesity (*db/db*) and diabetes. RESEARCH DESIGN AND METHODS {#s5} =========================== Animals and diets. {#s6} ------------------ The SGLT2 (*Slc5a2*) deficient mice were generated by Lexicon Pharmaceuticals, Inc. (The Woodlands, TX) as previously described ([@B6]). SGLT2 and *db/db* backcrosses were performed at The Jackson Laboratory (Bar Harbor, ME) and shipped to Yale for studies. Mice were housed at Yale University School of Medicine and maintained in accordance with the Institutional Animal Care and Use Committee guidelines. Mice were housed at 22 ± 2°C on a 12-h light/dark cycle with free access to food and water. Mice were fed regular chow (RC; 18% fat, 58% carbohydrate, 24% protein by calories; TD2018; Harlan Teklad, Madison, WI) or 4 weeks of high-fat diet (HFD; 55% fat, 24% carbohydrate, 21% protein by calories; TD93075; Harlan Teklad). Body composition was determined by ^1^H magnetic resonance spectroscopy (Bruker Minispec). The Comprehensive Laboratory Animal Monitoring System (Columbus Instruments, Columbus, OH) was used to evaluate activity, energy expenditure, feeding, drinking, and respiratory quotient over the course of 48 h. Data are the 24-h average normalized to body weight. For the urine collection studies, mice were housed for 24 h with free access to food and water in wire-bottomed cages designed to separate and collect urine and feces. In vivo glucose homeostasis. {#s7} ---------------------------- Glucose tolerance tests were performed after an overnight fast. Mice were injected intraperitoneally with 1 mg/kg glucose, and blood was collected by tail bleed at set times for plasma insulin and glucose measurements. Hyperinsulinemic euglycemic clamps were performed as previously described ([@B23]), with minor modifications. Specifically, clamp duration was extended to 180 min and the insulin dose was increased to 20 mU · kg^−1^ · min^−1^. Hyperglycemic clamps were conducted after an overnight fast. Mice were given a primed/variable infusion of glucose to reach and maintain hyperglycemia during the 120-min experiment. Blood was collected at set intervals by tail bleeds for determination of plasma glucose and insulin. Because of differences in fasting plasma glucose and insulin levels between genotypes, infusion rates were chosen to achieve matched changes in plasma glucose (Δ glucose), and the change in plasma insulin from baseline (Δ insulin) was used to assess the pancreatic β-cell response. Pancreas and islet studies. {#s8} --------------------------- Isolated islet studies were conducted as previously described ([@B24]). In brief, pancreata were removed from anesthetized mice and minced in CMRL media with type P collagenase to disperse cells. Islets were hand picked under a light microscope and allowed to recover overnight in media. Eighty islets were picked and loaded into a perifusion chamber with acrylamide gel column beads (Bio-Gel P4G; Bio-Rad, Hercules, CA) and perfusion buffer (Krebs-Ringer buffer with 3 mmol/L glucose and 0.2% fatty acid-free BSA). Perifusions were conducted with a Bio-Rep (Miami, FL) perifusion instrument that allows for precise temperature and flow control and collection of samples into 96-well format. After a 1-h equilibration period, islets were perifused with the indicated agonists, and samples were collected at 1-min intervals for 40 min. At the end of the perifusion, islet DNA was isolated and quantified for normalization of insulin and glucagon data using a Picogreen dsDNA quantitation kit (Invitrogen) according to the manufacturer's instructions. Preparation and staining of all histologic samples were conducted at the Department of Pathology Histology Core at Yale. Samples were fixed in 4% formalin overnight and set in paraffin wax. Sections were cut at 5 μm, dried, deparafinized, and rehydrated with distilled water. For kidney studies, hematoxylin and eosin (H&E) stains were analyzed in a blinded fashion by a pathologist. For pancreas studies, transferase-mediated dUTP nick-end labeling (TUNEL) reaction was performed according to the manufacturer's instructions (Millipore S7100; Millipore, Billerica, MA), after which slides were incubated with polyclonal anti-insulin (Dako 0564) and visualized by an alkaline-phosphatase--based method (Dako K5355). Frequency of cell death was calculated as the number of insulin/TUNEL-positive cells corrected by relative islet area (islet area/pancreas area) for 10 to 15 fields per section and three sections per mouse. Islet number was determined by counting all islets within a given field, corrected to pancreas area. Approximately 30 fields were taken at random, and three sections per mouse were analyzed. Sections were staggered to reduce the possibility of analyzing the same islet twice. For proliferation studies, slides were incubated with anti-Ki-67 (Biocare Medical CRM325) and anti-insulin (Dako A0564) and visualized with peroxidase and fluorescent secondary antibodies (Dako K4003, Invitrogen A11073). Frequency of cellular proliferation was calculated in a similar fashion as frequency of cell death. Pancreas weights were not available, so relative β-cell volume was calculated as the ratio of islet area/pancreas area. Cell size measurements were made using ImageJ software. Biochemical analyses. {#s9} --------------------- Plasma and urine glucose concentrations were measured by a glucose oxidase method using a Beckman Glucose Analyzer II. Plasma insulin was measured by radioimmunoassay kit (Linco Research, St. Louis, MO), and fatty acids were measured by a spectrophotometric technique (Wako NEFA Kit, Osaka, Japan). Hepatic glycogen content was measured as previously described, except that a 10:1 volume/weight dilution was used for homogenization ([@B25]). Insulin and glucagon from isolated islets were measured with the LINCOplex platform (Millipore). Plasma chemistries were measured with a Roche COBAS Mira automated chemistry analyzer at the Yale Mouse Metabolic Phenotyping Center Analytical Core. Plasma creatinine levels were determined by high-performance liquid chromatography/mass spectrometry/mass spectrometry. Statistical analysis. {#s10} --------------------- Data are reported as the mean ± SEM. Comparisons between groups were made using unpaired, two-tailed Student *t* tests. For time course data, two-way and one-way ANOVA were used where appropriate. A value of *P* \< 0.05 was considered significant. Statistical analyses were performed using GraphPad Prism 5 software. RESULTS {#s11} ======= Phenotype of SGLT2^−/−^ RC- and HFD-fed mice. {#s12} --------------------------------------------- The RC-fed SGLT2^−/−^ mice weighed approximately 10% less than wild-type (WT) mice at age 16 weeks ([Table 1](#T1){ref-type="table"}). To determine the effect of SGLT2 deletion in the presence of increased body weight and adiposity, a second group of SGLT2^−/−^ and WT mice were fed an HFD for 4 weeks. HFD mice had an increased body weight compared with controls for both genotypes, but unlike RC-fed mice, HFD/SGLT2^−/−^ mice only trended toward a lower body weight (*P* = 0.12; [Table 1](#T1){ref-type="table"}). There were no differences in fat mass between RC/WT and RC/SGLT2^−/−^ mice, and HFD/SGLT2^−/−^ animals trended toward reduced adiposity (*P* = 0.08; [Table 1](#T1){ref-type="table"}). ###### Physiologic overview of SGLT2^−/−^ mice Body weight Body fat Kidney weight Plasma BUN Plasma creatinine Urine output Glucosuria ---------------- -------------------------------------------- ------------ --------------- ------------ ------------------- ------------------------------------------ ----------------------------------------- WT 29.4 ± 0.7 6.9 ± 0.7 6.55 ± 0.42 23.2 ± 0.3 0.074 ± 0.001 1.5 ± 0.3 0.9 ± 0.3 SGLT2^−/−^ 26.7 ± 0.6[\*](#t1n1){ref-type="table-fn"} 7.4 ± 0.3 6.49 ± 0.14 23.8 ± 1.1 0.077 ± 0.007 6.7 ± 0.4[†](#t1n2){ref-type="table-fn"} 502 ± 30[†](#t1n2){ref-type="table-fn"} HFD/WT 35.0 ± 0.7 26.4 ± 1.2 ND 20.8 ± 0.7 0.103 ± 0.005 1.7 ± 0.2 1.0 ± 0.3 HFD/SGLT2^−/−^ 33.0 ± 1.0 22.3 ± 1.9 ND 18.8 ± 0.7 0.119 ± 0.007 5.4 ± 0.5[†](#t1n2){ref-type="table-fn"} 431 ± 31[†](#t1n2){ref-type="table-fn"} Data were collected from 16-week-old, ad libitum fed mice. Kidney weight represents the weight of one kidney corrected by body weight. Mice were housed in custom cages for 24 h for urine output and glucosuria measurements. HFD was fed for 4 weeks. *n* = 12--16 per genotype for body weight and fat data; *n* = 6--8 for remaining data. \**P* \< 0.05; [†](#t1n2){ref-type="table-fn"}*P* \< 0.001 for comparison of WT with SGLT2^−/−^ for each diet. Data analyzed by Student *t* test. ND, not done. Daily urine volume was 4.5-fold greater in SGLT2^−/−^ than in WT mice, which translated to a 500-fold increase in glucosuria, with similar findings in the HFD mice ([Table 1](#T1){ref-type="table"}). Despite increased urine volume and glucosuria, plasma chemistries were unchanged in SGLT2^−/−^ mice ([Supplementary Table 1](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1328/-/DC1)), in agreement with a recently published description of the SGLT2 knockout model ([@B6]). There was no difference in the blood urea nitrogen (BUN)/creatinine ratio between WT or SGLT2^−/−^ mice, suggesting that volume depletion could not account for the lower weight ([Supplementary Table 1](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1328/-/DC1)). Histologic analysis of kidney sections revealed normal glomerular structures and tubulointerstitial compartments, without significant histopathologic changes in the proximal or distal nephron of SGLT2^−/−^ mice ([Supplementary Fig. 1](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1328/-/DC1)), and there was no difference in kidney weight between genotypes ([Table 1](#T1){ref-type="table"}). To determine the effect of SGLT2 deletion on whole-body metabolism and behavior, SGLT2^−/−^ and WT mice were housed in metabolic cages. During resting hours (light cycle), there was no difference in activity, energy expenditure, or food intake between genotypes ([Supplementary Fig. 2*A*--*C*](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1328/-/DC1)), although water intake was elevated during this time for SGLT2^−/−^ mice ([Supplementary Fig. 2*D*](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1328/-/DC1)). In contrast, there were marked differences in each parameter between mice during active hours (dark cycle). Activity, energy expenditure, and food and water intake were all significantly increased in SGLT2^−/−^ mice during the dark cycle ([Fig. 1*A*](#F1){ref-type="fig"}). Notably, there was a 20% increase in feeding and a 190% increase in drinking compared with controls. Despite the increase in caloric intake, SGLT2^−/−^ mice maintained a lower body weight through a combination of 7% higher energy expenditure during the dark cycle and 13% (1.9 kcal/day) caloric loss of carbohydrate in the urine. {#F1} Under HFD conditions, mice consume significantly less carbohydrate. Interestingly, unlike RC-fed mice, there were no differences in activity, energy expenditure, or food intake in the light or dark cycle during HFD ([Supplementary Fig. 3](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1328/-/DC1)), consistent with the lower carbohydrate composition of the chow. Drinking, however, was clearly increased relative to HFD controls throughout the day, reflecting continuous glucose loss. In comparison with RC/SGLT2^−/−^ mice, HFD/SGLT2^−/−^ mice had lower water consumption (36.9 ± 2.5 vs. 45.6 ± 2.9 μL/h; *P* \< 0.05) and urine output (5.2 ± 0.5 vs. 6.5 ± 0.4 mL/day; *P* \< 0.05), again suggesting that carbohydrate content of the chow had a large influence on glucosuria. The lower respiratory quotient in SGLT2^−/−^ mice ([Fig. 1*A*](#F1){ref-type="fig"}) reflects a higher rate of lipid relative to carbohydrate oxidation, as might be expected for mice that are carbohydrate-depleted by profound glucosuria. Indeed, there was a 2.5-fold decrease in hepatic glycogen content in SGLT2^−/−^ compared with WT mice (5.1 ± 1.1 vs. 12.7 ± 2.0 μg/mg tissue; *P* \< 0.05). This was even more dramatic on the HFD, where the respiratory quotient was 0.78 and approached that of nearly a complete dependence on lipid oxidation ([Supplementary Fig. 3*E*](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1328/-/DC1)). Glucose homeostasis is improved in RC- and HFD-fed SGLT2^−/−^ mice. {#s13} ------------------------------------------------------------------- Next, in vivo glucose homeostasis was assessed. Compared with RC/WT mice, RC/SGLT2^−/−^ mice had significantly lower fasting plasma glucose concentrations and a nonsignificant reduction in plasma insulin concentrations (9.0 ± 0.6 and 6.4 ± 1.8 μU/mL insulin; *P* = 0.17; [Fig. 1*B* and *C*](#F1){ref-type="fig"}). After 4 weeks of the HFD, fasting glucose was drastically reduced in SGLT^−/−^ compared with WT mice and was indistinguishable from RC/WT controls ([Fig. 1*B*](#F1){ref-type="fig"}). Plasma insulin concentrations were 50% lower in the HFD/SGLT2^−/−^ compared with WT mice, as would be expected for the lower plasma glucose levels. HFD/SGLT2^−/−^ mice had a twofold increase in fasting insulin compared with RC/WT mice ([Fig. 1*C*](#F1){ref-type="fig"}), suggesting that SGLT2 deletion did not completely prevent insulin resistance. Intraperitoneal glucose tolerance tests (IPGTTs) were performed to determine the effect of SGLT2 knockout on glucose homeostasis. Plasma glucose concentrations remained lower in SGLT2^−/−^ than in WT mice for the entire experiment (*P* \< 0.01 by two-way ANOVA; [Fig. 1*D*](#F1){ref-type="fig"}). The area under the curve (AUC) for glucose was 20% lower for SGLT2^−/−^ than for WT mice ([Fig. 1*F*](#F1){ref-type="fig"}). A similar, but more pronounced effect was observed during HFD studies (*P* \< 0.001 by two-way ANOVA; [Fig. 1*D*](#F1){ref-type="fig"}), where AUC glucose was 40% less in SGLT2^−/−^ mice and was indistinguishable from WT/RC controls ([Fig. 1*F*](#F1){ref-type="fig"}). Changes in plasma insulin concentrations roughly mirrored the differences observed for plasma glucose (WT vs. SGLT2^−/−^ RC and HFD *P* \< 0.01, two-way ANOVA; [Fig. 1*E*](#F1){ref-type="fig"}), consistent with appropriate β-cell function. The insulin AUC was 37% less for RC/SGLT2^−/−^ compared with RC/WT mice, while after HFD, the insulin AUC was 53% less for SGLT2^−/−^ mice ([Fig. 1*G*](#F1){ref-type="fig"}). Thus, loss of SGLT2 significantly improved basal and IPGTT-challenged glucose homeostasis in RC- and HFD-fed mice. {#F2} SGLT2 deletion reduces adiposity and improves glucose homeostasis in *db/db* mice. {#s14} ---------------------------------------------------------------------------------- To determine whether loss of SGLT2 was protective in the setting of extreme obesity and insulin resistance, SGLT2^−/−^ mice were bred onto the *db/db* background. The *db/db* mice are leptin receptor--deficient and develop profound hyperphagia, obesity, hypercorticosteronemia, insulin resistance, and eventually, diabetes. At approximately age 18 weeks, body weights were similar for both heterozygous (*db/db*-SGLT2^+/−^) and homozygous (*db/db*-SGLT2^−/−^) SGLT2 deletion compared with controls (*db/db*-SGLT2^+/+^; [Fig. 2*A*](#F2){ref-type="fig"}), whereas body fat was 5% less in *db/db*-SGLT2^−/−^ mice ([Fig. 2*B*](#F2){ref-type="fig"}). There was a progressive effect of SGLT2 deletion on urine volume, with *db/db*-SGLT2^−/+^ having a 2.6-fold and *db/db*-SGLT^−/−^ having a fourfold increase ([Fig. 2*C*](#F2){ref-type="fig"}). The *db/db* mice displayed profound glucosuria, totaling approximately 500 mg/day ([Fig. 2*D*](#F2){ref-type="fig"}). Still, there was a three- and 4.4-fold increase in *db/db*-SGLT2^−/+^ and *db/db*-SGLT2^−/−^ mice, respectively, indicating that lowering the renal glucose threshold had a progressive effect on glucosuria ([Fig. 2*D*](#F2){ref-type="fig"}). Astonishingly, total daily glucose loss for *db/db*-SGLT2^−/−^ mice was 2.2 g or 5% of the animals' body weight. Despite increased glucosuria in *db/db*-SGLT2^+/−^ mice, only the *db/db*-SGLT2^−/−^ mice had significantly reduced plasma glucose concentrations ([Fig. 2*E*](#F2){ref-type="fig"}). Strikingly, this 75-mg/dL reduction normalized glycemia to the same level as RC/WT mice (WT: 131 ± 6 mg/dL vs. db/db-SGLT2^−/−^: 124 ± 8 mg/dL). Plasma insulin levels were 50% lower in *db/db*-SGLT2^−/−^ crosses ([Fig. 2*F*](#F2){ref-type="fig"}), consistent with the lower plasma glucose concentration. Plasma insulin levels were 10-fold higher than RC/WT mice (WT: 9.0 ± 0.6 μU/mL vs. *db/db*-SGLT2^−/−^: 112 ± 13 μU/mL), indicating the persistence of insulin resistance in the *db/db* model. Lastly, SGLT2 deletion had no effect on plasma fatty acid levels in *db/db* mice ([Fig. 2*G*](#F2){ref-type="fig"}). Euglycemic clamp studies were performed to evaluate the effect of SGLT2 deletion on insulin sensitivity and tissue-specific glucose metabolism. Because of extreme insulin resistance in *db/db* mice, plasma glucose concentrations were clamped at 150 mg/dL using a 20 mU · kg^−1^ · min^−1^ insulin infusion ([Supplementary Fig. 4*A*--*D*](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1328/-/DC1)). The glucose infusion rate (GIR) was 2.7-times greater in *db/db*-SGLT2^−/−^ than in *db/db*-SGLT2^+/+^ mice ([Table 2](#T2){ref-type="table"}). Quantitative measurement of glucosuria during the clamp was not possible for control or *db/db*-SGLT2^+/−^ mice because of small urine volumes, but ranged from 0 to 15% of total GIR for *db/db*-SGLT2^−/−^ mice (data not shown). Thus, the higher GIR could only partially be accounted for by loss as glucosuria and suggested improvements in insulin sensitivity. There was no difference in whole-body or tissue-specific glucose uptake in any group ([Table 2](#T2){ref-type="table"}), but clamped endogenous glucose production was modestly reduced in *db/db*-SGLT2^−/−^ compared with *db/db*-SGLT2^+/+^ mice ([Table 2](#T2){ref-type="table"}). ###### Hyperinsulinemic euglycemic clamp studies Mouse Plasma glucose(mg/dL) Glucose infusion rate(mg · kg^−1^ · min^−1^) Endogenous glucose production(mg · kg^−1^ · min^−1^) Whole-body glucose uptake(mg · kg^−1^ · min^−1^) Skeletal muscle glucose uptake(nmol · g^−1^ · min^−1^) White adipose glucose uptake(nmol · g^−1^ · min^−1^) Insulin(μU/mL) ------------- ----------------------- ---------------------------------------------- ------------------------------------------------------ -------------------------------------------------- -------------------------------------------------------- ------------------------------------------------------ ---------------- *db/db*  SGLT2^+/+^ 149 ± 13 2.9 ± 0.8 21.3 ± 1.2 24.1 ± 0.9 135 ± 22 24.7 ± 1.8 1211 ± 138  SGLT2^+/−^ 152 ± 18 3.9 ± 1.2 19.3 ± 1.9 23.3 ± 1.2 134 ± 18 27.1 ± 2.2 1178 ± 105  SGLT2^−/−^ 139 ± 8 8.0 ± 0.8[\*](#t2n1){ref-type="table-fn"} 16.2 ± 2.1[†](#t2n2){ref-type="table-fn"} 24.2 ± 1.9 109 ± 18 25.2 ± 3.4 1268 ± 158 Whole-body and tissue-specific glucose metabolism during hyperinsulinemic infusion. The *db/db*-SGLT2^+/+^ (*n* = 9), *db/db*-SGLT2^+/−^ (*n* = 8), and *db/db*-SGLT2^−/−^ (*n* = 7). Data analyzed by Student *t* test. \**P* \< 0.01; †*P* \< 0.05 compared with *db/db*-SGLT2^+/+^. Pancreatic β-cell function is preserved in *db/db*-SGLT2^−/−^ mice. {#s15} ------------------------------------------------------------------- Because glucose toxicity can lead to β-cell failure, we assessed glucose-stimulated insulin secretion by hyperglycemic clamp. Fasting plasma glucose concentrations varied among genotypes ([Fig. 2*E*](#F2){ref-type="fig"}); therefore, all mice were clamped 200 mg/dL above their basal plasma glucose levels. Similar to euglycemic clamps, the GIR required to maintain hyperglycemia for *db/db*-SGLT2^−/−^ mice was approximately twofold greater than for controls ([Fig. 3*A*](#F3){ref-type="fig"}). The total increase in plasma glucose concentrations above baseline during the clamp was similar for all three groups ([Fig. 3*B* and *C*](#F3){ref-type="fig"}). No first-phase insulin response was observed, consistent with compromised islet function in *db/db* mice ([Fig. 3*D*](#F3){ref-type="fig"}). However, in response to the same net change in plasma glucose concentration (ΔAUC glucose, [Fig. 3*C*](#F3){ref-type="fig"}), *db/db*-SGLT2^−/−^ mice displayed a significant increase in absolute plasma insulin levels during the clamp (*P* \< 0.001, one-way ANOVA; [Fig. 3*D*](#F3){ref-type="fig"}), and ΔAUC insulin was 2.4-fold greater compared with *db/db*-SGLT2^+/+^ mice ([Fig. 3*E*](#F3){ref-type="fig"}). Calculation of the change in plasma insulin (Δ insulin) from baseline also demonstrated an approximate twofold increase in glucose-stimulated insulin secretion for *db/db*-SGLT2^−/−^ compared with *db/db*-SGLT2^+/+^ mice ([Fig. 3*F*](#F3){ref-type="fig"}). Thus, the greater than 10-fold increase in insulin secretion for *db/db*-SGLT2^−/−^ compared with the modest fivefold increase for controls during the hyperglycemic clamp was consistent with improved β-cell function in vivo. {#F3} Isolated pancreatic islets from the *db/db*-SGLT2^+/+^ and *db/db*-SGLT2^−/−^ mice were perifused to determine if changes in individual islet function had occurred. During isolation, islet yield was much greater for *db/db*-SGLT2^−/−^ mice, so islets of similar size were selected for comparison. Surprisingly, there was no difference in glucose-stimulated insulin secretion (633 ± 104 vs. 743 ± 53 AUC insulin; *P* = 0.37; [Fig. 4*A*](#F4){ref-type="fig"}) or glucagon suppression (0.28 ± 0.12 vs. 0.15 ± 0.04 AUC glucagon; *P* = 0.35; [Fig. 4*B*](#F4){ref-type="fig"}) between groups, suggesting that individual islet function, per se, could not account for the observed in vivo differences. Perifusion studies conducted for WT and SGLT^−/−^ mice on RC and HFD also demonstrated no differences in insulin or glucagon secretion ([Supplementary Fig. 5*A*--*D*](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1328/-/DC1)). {#F4} The difference between hyperglycemic clamp and islet perifusion results suggested that changes in β-cell mass might account for the increased insulin secretion observed in vivo. Compared with RC/WT mice, relative β-cell volume was increased 3.3- and 5.4-fold for *db/db*-SGLT2^+/+^ and *db/db*-SGLT2^−/−^ mice, respectively, suggesting compensatory hypertrophy as a result of insulin resistance (1.06 ± 0.12, 3.52 ± 0.52, and 5.77 ± 0.21 islet area/pancreas area; *P* \< 0.001 by one-way ANOVA). Importantly, there was a 63% increase in relative β-cell volume in *db/db*-SGLT2^−/−^ compared with *db/db*-SGLT2^+/+^ mice ([Fig. 4*C* and *G*](#F4){ref-type="fig"}). The difference in islet mass between *db/db* groups appeared to be more a function of increased islet size rather than number (*P* = 0.22; [Fig. 4](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1328/-/DC1)*D*). There was no difference in the frequency of Ki-67--positive β-cells between genotypes ([Fig. 4*E* and *G*](#F4){ref-type="fig"}), suggesting that β-cell proliferation rates were not increased in the *db/db*-SGLT2^−/−^ mice. However, the frequency of TUNEL-positive β-cells was 64% less in *db/db*-SGLT2^−/−^ mice ([Fig. 4*F* and *G*](#F4){ref-type="fig"}), indicating that rates of cell death were slower. Thus, improvements in β-cell function observed in vivo appeared to result from maintained β-cell mass due to reduced frequency of β-cell death. DISCUSSION {#s16} ========== Type 2 diabetes is characterized by impaired glucose-stimulated insulin secretion, which is strongly associated with hyperglycemia in rodent and human studies ([@B8],[@B9],[@B11],[@B26]). Chronic hyperglycemia may shift the balance of β-cell proliferation and apoptosis toward cell death, resulting in β-cell deficiency and impaired insulin secretion ([@B8],[@B10],[@B27]). Although there seem to be some differences between rodents and humans with regard to the importance of β-cell proliferation in response to hyperglycemia, human autopsy studies have demonstrated a decline in β-cell mass in individuals with type 2 diabetes that was associated with increased β-cell death ([@B9]). A major finding of our study was that reducing glucose toxicity in a model of extreme diabetes preserved islet mass and improved insulin secretion in vivo. Increasing renal glucose excretion in *db/db* mice by SGLT2 deletion produced beneficial effects in glucose homeostasis and, importantly, islet function. The lack of difference in islet function during isolation/perifusion studies described here suggested that the primary mechanism by which reduced glucose toxicity improved glucose-stimulated insulin secretion was through preservation of islet mass rather than a change in individual β-cell function. A similar effect was observed after pharmacologic inhibition of SGLT2 in *db/db* and KK-A^y^ mice for 12 and 9 weeks, where total pancreatic insulin content and immunohistologic staining for insulin were increased in treated mice ([@B16],[@B28]). No human studies with SGLT2 inhibitors have addressed islet function directly, and so the potential for SGLT2 inhibition to preserve β-cell mass by reducing glucose toxicity, as it did here in rodents, remains to be determined. Hyperglycemia in insulin-resistant or -deficient models has also been shown to contribute to peripheral insulin resistance ([@B12],[@B29]). Despite a 64% increase in the GIR required to maintain euglycemia in *db/db*-SGLT2^−/−^ mice, equivalent improvements in measured indices of insulin sensitivity were not detected during euglycemic clamps. There was no difference in whole-body or tissue-specific glucose uptake, and endogenous glucose production was modestly improved by 24% in *db/db*-SGLT2^−/−^ mice. It seems likely that unaccounted for differences in GIR could result from glucosuria, but estimating acute glucosuria during the clamp was technically difficult. Furthermore, *db/db* mice possess a complex metabolic phenotype and insulin resistance stemming from multiple factors, including hyperglycemia, hyperlipidemia, and hypercorticosteronemia ([@B30]--[@B32]). An even modest improvement in insulin resistance in this model suggests that reducing glucose toxicity by renal glucose excretion may produce even more beneficial effects on insulin resistance under less severe circumstances. In fact, inhibition of SGLT2 in ZDF rats for 15 days improved insulin sensitivity ([@B15]). In contrast to the subtle improvement in insulin sensitivity, SGLT2 knockout markedly improved glucose intolerance under HFD-induced, insulin-resistant conditions, and reduced fasting plasma glucose and insulin levels. RC/SGLT2^−/−^ mice also displayed improved glucose tolerance and reduced fasting plasma glucose, without hypoglycemia. Interestingly, SGLT2 knockout resulted in substantially increased caloric intake. Whether this was a response to mild, undetected hypoglycemia or for other reasons remains to be determined. Of note, SGLT2 mutations in humans that result in familial renal glucosuria rarely cause hypoglycemia ([@B33]--[@B35]). Likewise, the incidence of hypoglycemia during clinical trials of SGLT2 inhibitors is extremely low ([@B6]--[@B6]). Inhibition of SGLT2 has the potential to promote negative energy balance and, in turn, weight loss. The lack of differences in plasma electrolytes, phosphate, BUN, creatinine, and the BUN/creatinine ratio suggested that volume depletion did not account for the differences in body weight. Lack of volume depletion in SGLT2^−/−^ mice was also noted in a recent study; however, no difference in body weight was detected ([@B6]). Mice in the previous report were studied at between 12 and 20 weeks of age, and group sizes for the body weight data ranged from 6 to 10. Here, mice were studied at 16 weeks, and the group size for body weight data were 16 per group. The breadth of ages cited in the previous report, as well as smaller group sizes, may account for the discrepancy. In support of reduced body weight with loss of SGLT2 function, a recently described SGLT2 mutant mouse with glucosuria similar to that described here was also noted to have reduced body weight ([@B36]). In fact, evidence from the literature supports the notion of reduced body weight with SGLT2 deletion. Short-term studies of approximately 2 weeks have demonstrated no effect of SGLT2 inhibition on body weight in ZDF rats ([@B14],[@B15]), whereas longer-term studies of 8 weeks or more have found reductions in body weight in rodents and humans ([@B13],[@B16],[@B19]--[@B21]). Glucosuria that produced weight loss in humans ranged from 50 to 80 g/day, or 10--16% of a 2,000 kcal/day diet ([@B19],[@B20]). In SGLT2^−/−^ mice, glucosuria averaged 0.48 mg/day or 13% of the daily caloric intake of WT mice in this study. Interestingly, feeding increased about 20% in SGLT2^−/−^ mice, suggesting that an isocaloric diet would have a greater potential for weight loss during SGLT2 deficiency. Previous studies in rodents have also noted concomitant increases in feeding behavior and weight loss during SGLT2 inhibition ([@B13],[@B16]). Although RC/SGLT2^−/−^ mice weighed 10% less than RC/WT mice, there was no significant difference in body weight after 4 weeks HFD, although SGLT2^−/−^ mice weighed about 6% less. RC and HFD mice both had increased urine production during periods of food intake, and there was overall less urine output and glucosuria on the HFD compared with RC. Together, these data suggest that SGLT2 activity is greatest in the postprandial setting and that inhibition will produce greater weight loss on a carbohydrate-rich diet. In conclusion, SGLT2 deletion in mice resulted in profound glucosuria and compensatory behavioral adaptations aimed at replacing water and calories lost in the urine. SGLT2 knockout led to favorable reductions in plasma glucose and insulin during HFD and in the context of genetic obesity. Perhaps the most ominous complication of diabetes is loss of islet function, because this will accelerate the deterioration of glucose homeostasis and increase the exposure to glucose toxicity. Ablation of SGLT2 in *db/db* mice prevented loss of β-cell mass and thus preserved in vivo glucose-stimulated insulin secretion, which partly accounted for the overall improvements in glucose homeostasis. Taken together, these data support SGLT2 inhibition as a viable insulin-independent treatment for type 2 diabetes. This article contains Supplementary Data online at <http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1328/-/DC1>. See accompanying commentary, p. 695. This work was supported by NIH Grants K08-DK-0801420 (R.G.K), R01-DK-40936 (G.I.S.), and U24-DK-07169. This work was also supported by Bristol-Myers Squibb and AstraZeneca. No other potential conflicts of interest relevant to this article were reported. M.J.J. designed and performed experiments, analyzed data, and prepared the manuscript; H.-Y.L., A.L.B., F.R.J., and D.W.F. assisted with euglycemic and hyperglycemic clamp and metabolic cage studies; R.L.P. and X.Z. performed islet isolations, perifusion studies, and β-cell mass calculations; G.W.M. conducted renal histologic analysis; V.T.S. provided technical expertise and reviewed the manuscript; J.M.W., G.I.S., and R.G.K. designed experiments, provided technical expertise, and edited the manuscript.

The World Health Organization (WHO) guidelines call for antiretroviral therapy (ART) for all people living with HIV (PLWH) regardless of their CD4 T cell count, a policy often referred to as 'treat all' [@JVEv4-bib-0001]. 'Treat all' strategies, increasingly common in low-resource settings, are motivated by evidence that treating HIV as soon as possible after infection can improve patient outcomes and reduce transmission [@JVEv4-bib-0002]. Large-scale success of the implementation and scale-up of 'treat all' requires understanding and addressing known barriers to achieving optimal HIV treatment outcomes. Insufficient attention has been paid to identifying and addressing the mental health needs of PLWH, particularly in sub-Saharan Africa (SSA), a region that accounts for more than 70% of the global burden of HIV [@JVEv4-bib-0004]. Because mental health disorders are common among PLWH, often underdiagnosed and undertreated in low-resource settings, and associated with suboptimal HIV treatment outcomes, addressing the mental health needs of those living with HIV should be a critical component of successful implementation, scale-up, and achievement of 'treat all' priorities in SSA and beyond [@JVEv4-bib-0005]. This article seeks to: (1) provide an overview of research regarding the mental health of PLWH in SSA and interventions and strategies to address comorbid mental illness among PLWH and (2) describe key mental health-related research priorities to inform the successful implementation and scale up of 'treat all' policies in SSA and other low-resource settings. Because substance use disorders are the focus of a separate paper in this supplement [@JVEv4-bib-0012], such disorders are not addressed in this article. Mental health disorders among PLWH {#JVEv4-sec-0001} ================================== Mental health disorders (e.g. depression, post-traumatic stress disorder \[PTSD\]) are among the most prevalent comorbidities in PLWH globally and more common among PLWH than the general population [@JVEv4-bib-0005]. It has been estimated that approximately half of PLWH meet criteria for one or more mental health disorder [@JVEv4-bib-0005]. High rates of mental health disorders have been documented among PLWH residing in low- and middle-income countries (LMICs), including SSA [@JVEv4-bib-0014]. Research suggests that these are associated with delayed HIV diagnosis [@JVEv4-bib-0015], and with suboptimal HIV treatment outcomes, including late ART initiation, poor ART adherence, lack of viral suppression, and increased AIDS-related mortality across LMICs [@JVEv4-bib-0015]. It is important to note that most studies focused on the mental health of PLWH in SSA and globally do not distinguish between pre-existing mental health disorders among PLWH and mental health disorders with an onset subsequent to HIV infection or its diagnosis. Two studies from South Africa have revealed that an important proportion of HIV test seekers experience mental health symptoms prior to their diagnosis. In the first, 55% of individuals surveyed displayed depressive symptoms prior to HIV testing and individuals with depressive symptoms prior to HIV diagnosis were less likely to receive CD4 cell count testing after diagnosis [@JVEv4-bib-0017]. In the second, the prevalence of major depressive disorder, generalised anxiety disorder and PTSD among HIV test seekers was 14%, 5% and 5%, respectively [@JVEv4-bib-0018]. Additional longitudinal research is needed to better understand the course and severity of mental health disorders among PLWH in relation to HIV infection, diagnosis, and treatment. Depression {#JVEv4-sec-0002} ---------- Depression is the most prevalent mental health disorder among PLWH globally and in SSA [@JVEv4-bib-0005]. Estimates of depressive symptoms among PLWH on ART in SSA have varied between 14% and 32%, with substantial variability within and between measurement scales [@JVEv4-bib-0021]. A meta-analysis of studies conducted in Western countries found the prevalence of major depressive disorder to be nearly two-fold higher among PLWH compared to those who did not have HIV [@JVEv4-bib-0022]. Little is known about the relationship between depression and delayed HIV diagnosis in SSA. However, depression has been associated with suboptimal HIV treatment outcomes in SSA, including late ART initiation and poor adherence, lack of viral suppression, more rapid decline in CD4 cell count, faster progression to AIDS and increased AIDS-related mortality [@JVEv4-bib-0008]. Similar to other mental health disorders, depression remains underdiagnosed and undertreated throughout SSA, compromising timely ART initiation and treatment adherence at individual and population levels, and attainment of 'treat all' objectives [@JVEv4-bib-0010]. Anxiety disorders {#JVEv4-sec-0003} ----------------- Estimates of the prevalence of anxiety-related symptoms or anxiety disorders among PLWH in SSA have varied between 9% and 34% [@JVEv4-bib-0005] compared to 3--7% among general populations throughout SSA [@JVEv4-bib-0025]. Among PLWH in South Africa, anxiety was significantly associated with delayed engagement in HIV care following diagnosis, but not significantly associated with ART adherence [@JVEv4-bib-0011]. Little is known about the relationship between anxiety symptoms or diagnoses and viral suppression or HIV disease progression in SSA. In high-income settings, anxiety symptoms have been associated with poor adherence to ART, higher viral load and lower CD4 cell count [@JVEv4-bib-0026]. Post-traumatic stress disorder {#JVEv4-sec-0004} ------------------------------ Exposure to traumatic events in childhood and adulthood, including child physical and sexual abuse, intimate partner violence, sexual assault, and war is also prevalent among PLWH and associated with mental health disorders and suboptimal HIV treatment outcomes [@JVEv4-bib-0028]. Estimates of PTSD among PLWH in SSA are limited and vary widely. Studies conducted with PLWH in South Africa estimated PTSD prevalence at 5--20% [@JVEv4-bib-0029] compared to approximately 2% among the general population [@JVEv4-bib-0031]. Little is known about the relationship between PTSD and ART initiation, care engagement, or HIV disease progression globally and throughout SSA [@JVEv4-bib-0005]. Findings regarding the relationship between PTSD and ART adherence remain equivocal [@JVEv4-bib-0033]. Bipolar disorder and psychotic disorders {#JVEv4-sec-0005} ---------------------------------------- Estimates of the prevalence of bipolar disorder and psychotic disorders such as schizophrenia among PLWH in SSA are largely lacking. However, one study of PLWH in South Africa estimated a prevalence of bipolar disorder of 6% while a study with PLWH in Nigeria found a prevalence of psychotic disorders of 6% [@JVEv4-bib-0035]. Estimates of bipolar disorder among the general population in SSA are largely unavailable. However, estimates of bipolar disorder among the general population across 11 countries in the Americas, Europe and Asia range from 0.4% to 2% [@JVEv4-bib-0037]. Little is known about the relationship between bipolar disorder or psychotic disorders and HIV treatment outcomes in SSA. One study with PLWH in Uganda found that serious mental illness at ART initiation was associated with worse retention in HIV care [@JVEv4-bib-0038]. While limited, research in high-income settings has found bipolar disorder to be associated with poor ART adherence [@JVEv4-bib-0039]. Psychiatric multimorbidity {#JVEv4-sec-0006} -------------------------- Among individuals with mental health disorders, psychiatric multimorbidity (i.e. having more than one concurrent mental health or substance use disorder) is common and associated with greater symptom severity and worse health outcomes. Among PLWH in the US with a past-year mental health disorder, half met criteria for multiple mental health diagnoses [@JVEv4-bib-0041]. Co-occurring mood and anxiety disorders and co-occurring mood and substance use disorders were particularly common. In the US, psychiatric multimorbidity has been associated with greater HIV symptomology and worse quality of life [@JVEv4-bib-0042]. Among veterans living with HIV in the US, multimorbidity (co-occurring substance use disorder, psychiatric disorder, and medical disease) was associated with having a detectable viral load [@JVEv4-bib-0043]. Research into the prevalence and impact of psychiatric multimorbidity among PLWH in SSA and other low-resource settings is particularly limited. Mediators and moderators of the relationship between mental health disorders and HIV treatment outcomes {#JVEv4-sec-0007} ======================================================================================================= Several factors have been found to mediate or moderate the relationship between mental health and HIV treatment outcomes among PLWH in SSA including ART adherence, HIV care self-efficacy and motivation, among others. Research with PLWH in Uganda found that cognitive and affective (e.g. depressed mood and loss of interest in activities that are normally pleasurable), but not somatic symptoms of depression (e.g. fatigue, difficulty sleeping) were associated with ART adherence [@JVEv4-bib-0044]. Furthermore, depression alleviation was associated with improved ART adherence and HIV clinic attendance among this population [@JVEv4-bib-0044]. HIV care self-efficacy and adherence motivation have been found to mediate the relationship between depression and ART adherence among PLWH in SSA [@JVEv4-bib-0044]. In high-income settings, integrated interventions that address depression and ART adherence have been effective at improving both depression and ART adherence [@JVEv4-bib-0045]. Priority populations {#JVEv4-sec-0008} ==================== UNAIDS has identified ambitious treatment goals of having 90% of PLWH know their HIV status, 90% of those diagnosed with HIV receiving ART, and 90% of those on ART virally suppressed by 2020 [@JVEv4-bib-0046]. The identification and treatment of mental health disorders may be of particular importance in the successful attainment of 90-90-90 goals among specific priority or underserved populations, including children and adolescents living with HIV, pregnant and postpartum women living with HIV, and additional key populations living with HIV, including men who have sex with men, sex workers and transgender individuals. Children and adolescents living with HIV {#JVEv4-sec-0009} ---------------------------------------- Adolescence represents a period of particular vulnerability among PLWH. Among the general population, most mental health disorders first emerge during adolescence and are associated with poor physical and mental health in adolescence and into adulthood [@JVEv4-bib-0047]. In addition, adolescents living with HIV are often in transition to adult care and may be particularly vulnerable to disruptions and disengagement from HIV care. While limited, research indicates that many adolescents living with HIV experience mental health disorders and may have higher prevalence of mental health disorders than adolescents without HIV [@JVEv4-bib-0048]. Studies of children and adolescents living with HIV in SSA have estimated the prevalence of depression to be 18--25% [@JVEv4-bib-0050]. A study of children and adolescents living with HIV in Kenya found that the prevalence of anxiety disorders to be 32% [@JVEv4-bib-0050]. Comparative research examining the mental health of children and adolescents with and without HIV in SSA is rare. However, a study of children and adolescents in Rwanda found that HIV-affected children (i.e. children who are living with HIV, living with a caregiver who has HIV, or had a caregiver who died from HIV) had a significantly higher likelihood of depression, anxiety, and conduct disorder compared to HIV-unaffected children and adolescents [@JVEv4-bib-0053]. However, there was no significant difference in the likelihood of mental health problems between children who were living with HIV and those who did not have HIV [@JVEv4-bib-0053]. Similar to adults living with HIV, symptoms of depression and anxiety among adolescents living with HIV have been associated with worse HIV treatment outcomes, including poor ART adherence and lack of viral suppression, and greater HIV sexual risk behaviour [@JVEv4-bib-0049]. However, most research on the relationship between adolescent mental health and HIV treatment outcomes has been conducted in high-income countries [@JVEv4-bib-0054]. More research is needed to understand the prevalence and impact of mental health disorders among adolescents living with HIV in SSA and how to effectively identify and address these disorders in this population. Pregnant and postpartum women living with HIV {#JVEv4-sec-0010} --------------------------------------------- Pregnancy and the postpartum period represent periods of vulnerability among women living with HIV during which mental health disorders are common. Mental health disorders are more common among pregnant women living with HIV compared to pregnant women in the general population [@JVEv4-bib-0055]. In non-African settings, perinatal depression (i.e. depression occurring during pregnancy or the postpartum period) has been associated with non-adherence to ART [@JVEv4-bib-0056]. Few studies have examined the effects of perinatal depression on HIV treatment outcomes among women in SSA. Among women newly diagnosed with HIV in the Democratic Republic of Congo, antenatal depression (i.e. depression occurring during pregnancy) was not associated with engagement in HIV care [@JVEv4-bib-0057]. Factors associated with perinatal depression among women living with HIV in SSA remain poorly understood. Identifying and addressing mental health disorders among women during pregnancy and the postpartum period has the potential to improve HIV outcomes among women living with HIV, reduce vertical transmission of HIV, and foster attainment of 'treat all' objectives among mothers and children. Data on other mental health disorders among women living with HIV in SSA during pregnancy and the postpartum period are largely unavailable. One study found that pregnant women living with HIV in South Africa had significantly higher levels of anxiety compared to pregnant women without HIV [@JVEv4-bib-0058]. Additional research on mental disorders beyond depression among women living with HIV during pregnancy and the postpartum period is warranted. Additional key populations living with HIV {#JVEv4-sec-0011} ------------------------------------------ Little is known about the mental health of additional key populations living with HIV in SSA, including sex workers, men who have sex with men and transgender individuals. Research from outside SSA indicates that mental health symptoms and disorders are common among these key populations and more common among young key populations than older key population peers [@JVEv4-bib-0059]. Research with key populations in high-income countries suggests that mental health symptoms are associated with increased risk of HIV acquisition and suboptimal HIV treatment outcomes [@JVEv4-bib-0059]. Little is known about the extent to which poor mental health influences HIV treatment outcomes among key populations living with HIV in SSA. Systems interventions and strategies to address co-morbid mental illness among PLWH {#JVEv4-sec-0012} =================================================================================== Despite the prevalence of mental health disorders among PLWH in SSA and associations with suboptimal HIV outcomes, many HIV providers neither screen nor treat patients for mental health disorders, contributing to underdiagnosis of mental health disorders and a substantial mental health treatment gap among PLWH [@JVEv4-bib-0063]. Numerous factors contribute to challenges identifying and treating PLWH with mental health disorders in SSA including: a substantial mental health workforce shortage, especially in rural areas; limited time available in HIV and primary care settings; limited mental health training of HIV and primary care clinicians; few validated and culturally appropriate screening and diagnostic tools for mental health disorders; limited availability of psychiatric medications; competing priorities; and poor integration of mental health services into HIV care [@JVEv4-bib-0005]. Integrating mental health care into HIV care has been identified as a promising strategy for improving the mental health and HIV treatment outcomes of PLWH in SSA and may facilitate attainment of 'treat all' objectives [@JVEv4-bib-0063]. More research is needed into the feasibility, acceptability and effectiveness of models of integrated mental health and HIV care throughout SSA. The scale up and implementation of 'treat all' objectives adds challenges to already overburdened health systems (e.g. increased workload) in SSA that must be better understood [@JVEv4-bib-0066]. The Mental Health Gap Action Programme (mhGAP), published in 2008 and updated in 2015, provides evidence-based guidelines for diagnosis and management of priority mental health conditions in non-specialised health settings and presents a promising model for integration of mental health care into HIV care in SSA [@JVEv4-bib-0067]. Integration of the mhGAP into primary care in Nigeria was associated with increased identification, treatment, and referral for mental health disorders [@JVEv4-bib-0068]. To the authors' knowledge, the feasibility, acceptability or effectiveness of integrating mhGAP specifically into HIV care settings in SSA has not yet been evaluated. The Programme for Improving Mental Health Care (PRIME) is a multicountry initiative evaluating the implementation and scale up of mental health services in primary and maternal health care settings in Ethiopia, India, Nepal, South Africa and Uganda [@JVEv4-bib-0069]. Results from this work are forthcoming and will contribute to the knowledge base regarding the implementation and scale up of evidence-based mental health care in non-specialty settings in LMICs. Task-shifting and task-sharing have emerged as promising strategies to increase access to evidence-based mental health care in SSA. Evidence suggests that mental health screening, evaluation and pharmacological interventions can be effectively implemented by non-specialists in SSA when appropriate training, supervision and mentorship are available [@JVEv4-bib-0070]. Evidence also indicates that psychological interventions can be effectively delivered in non-specialty settings and by lay health workers in SSA. Randomised trials of the Friendship Bench intervention in Zimbabwe in which trained, supervised lay health workers delivered individual problem-solving therapy in primary care, found that the intervention was associated with significant improvement in symptoms of depression and other common mental health disorders [@JVEv4-bib-0072]. While integration of this intervention into HIV care has not yet been evaluated, previous trials were conducted among a population with high HIV prevalence [@JVEv4-bib-0074]. Although this intervention has been shown to improve depressive symptoms, the extent to which this intervention impacts HIV treatment outcomes remains unknown. Individual- and group-level interventions to improve outcomes among PLWH with mental health disorders {#JVEv4-sec-0013} ===================================================================================================== Evidence-based, individual- and group-level mental health interventions offer promising strategies to manage mental health disorders among PLWH and improve health and HIV treatment outcomes. Their implementation and scale up in SSA may begin to address the mental health treatment gap among PLWH. Depression {#JVEv4-sec-0014} ---------- Pharmacological interventions have been implemented by non-specialists in SSA. A pilot trial of a measurement-based care (MBC) approach to antidepressant medication management trained non-specialists to screen and monitor depression symptoms in an HIV treatment setting in Cameroon. This study found that the intervention was associated with improvements in depression symptoms and HIV treatment outcomes [@JVEv4-bib-0071]. A cluster randomised trial in Uganda compared two task-shifting models of pharmacological depression care: a structured protocol model and a model focused on clinical acumen [@JVEv4-bib-0070]. The two models performed similarly in the prescription of antidepressants to clinically depressed participants. However, those who screened positive for depressive symptoms were significantly more likely to receive a diagnostic evaluation in the structured protocol arm as compared to the clinical acumen arm [@JVEv4-bib-0070]. Group interpersonal psychotherapy (IPT-G), recommended by the WHO, has been successfully adapted for delivery in LMICs and has demonstrated effectiveness at reducing depression [@JVEv4-bib-0077]. The effectiveness of IPT-G should be evaluated with PLWH in SSA. A group support psychotherapy intervention for depression among PLWH in Uganda has also been associated with lower mean depression scores; changes in HIV treatment outcomes were not reported [@JVEv4-bib-0079]. Cognitive behavioural interventions have also demonstrated effectiveness in LMICs [@JVEv4-bib-0080]. Research in the US found that integrating evidence-based treatment for depression and evidence-based adherence counselling improved adherence and depression among PLWH [@JVEv4-bib-0045]. Similar research is needed with PLWH in SSA. Anxiety disorders {#JVEv4-sec-0015} ----------------- Although selective serotonin reuptake inhibitors (SSRIs) are a common and effective treatment for anxiety disorders in middle- and high-income countries, access to SSRIs and health professionals trained to prescribe SSRIs are limited in SSA. One US-based study reported 66% of medications prescribed for anxiety among PLWH were benzodiazepines [@JVEv4-bib-0081]. Benzodiazepines should be used cautiously due to their potential for abuse. To our knowledge, there are no published studies of pharmacological, psychotherapeutic or behavioural interventions for anxiety disorders among PLWH in SSA. Post-traumatic stress disorder {#JVEv4-sec-0016} ------------------------------ Little is known regarding the effectiveness of interventions for PTSD among PLWH in LMICs. A review of psychological interventions for PTSD among PLWH in resource-poor settings identified seven such studies, six of which used cognitive behavioural therapy and none of which was conducted in SSA [@JVEv4-bib-0082]. Children and adolescents living with HIV {#JVEv4-sec-0017} ---------------------------------------- Few evidence-based mental health interventions have been studied with children or adolescents living with HIV in SSA. One intervention that shows promise is the VUKA family-based programme, which has been implemented with pre-adolescents living with HIV and their caregivers in South Africa [@JVEv4-bib-0083]. A pilot found that the intervention was associated with improved mental health and ART adherence [@JVEv4-bib-0083]. Pregnant and postpartum women living with HIV {#JVEv4-sec-0018} --------------------------------------------- Similarly, few evidence-based mental health interventions have been studied with pregnant or postpartum women living with HIV in SSA. A randomised controlled trial (RCT) of a group counselling intervention which used a problem-solving therapy approach was associated with a marginally significant reduction in depression compared to standard of care (i.e. pre- and post-test voluntary counselling and testing for HIV and information on how to access prevention of vertical transmission of HIV services) among pregnant women living with HIV in Tanzania [@JVEv4-bib-0084]. Additional key populations living with HIV {#JVEv4-sec-0019} ------------------------------------------ The effectiveness of mental health interventions with additional key populations living with HIV in SSA remains largely unknown. However, an RCT of a cognitive behaviour therapy intervention for ART adherence and depression was conducted with PLWH who had depression and were in treatment for injection drug use in the US [@JVEv4-bib-0085]. The intervention was significantly associated with improvements in depression, ART adherence, and CD4 cell count post treatment. Similar research is needed with key populations living with HIV in SSA. Key research priorities to improve the mental health and HIV treatment outcomes of PLWH with mental health disorders in SSA {#JVEv4-sec-0020} =========================================================================================================================== Numerous research gaps exist in our understanding of how to effectively identify and manage mental health needs and optimise HIV treatment outcomes of PLWH in SSA. The authors recommend the following mental health-related research priorities to inform effective and efficient scale up and implementation of 'treat all' in SSA and beyond. •Research is needed to advance understanding of the prevalence and incidence of mental health multimorbidities among PLWH and their impact on HIV treatment outcomes. Greater understanding of the prevalence of mental health symptoms and disorders among PLWH in SSA compared to people without HIV is also needed. Longitudinal studies that examine the onset and trajectory of mental health symptoms and disorders in relation to HIV infection, diagnosis and treatment are needed in SSA.•Factors, such as ART adherence, that mediate or moderate the relationship between mental health and HIV treatment outcomes should be evaluated as potential intervention targets to improve mental health and HIV treatment outcomes among PLWH with mental health disorders. Additional mediators and moderators of the relationship between mental health and HIV treatment outcomes should be identified and evaluated as potential intervention targets.•Research is needed to understand the prevalence, incidence, impact and treatment of mental health disorders among children and adolescents living with HIV in SSA. Such research should include the examination of whether, and in what ways, the burden and impact of mental health disorders among children and adolescents living with HIV varies between children and adolescents who acquired HIV perinatally or behaviourally. In addition, research is needed to understand the burden and impact of mental health disorders among children and adolescents living with HIV compared to both HIV-affected (but not infected) children and adolescents in SSA as well as HIV-unaffected and uninfected children and adolescents in SSA.•Research to examine the prevalence of and factors associated with mental health disorders during pregnancy and the postpartum period among women living with HIV is needed. Interventions to optimise mental health and HIV treatment outcomes among this population should be developed, implemented and evaluated. Such research should examine mental health disorders beyond perinatal depression, including perinatal anxiety disorders.•Research to examine the prevalence of and factors associated with mental health disorders among key populations living with HIV is needed. Research that examines the extent to which mental health disorders influence HIV treatment outcomes among key populations living with HIV in SSA is needed. Interventions to optimise mental health and HIV treatment outcomes among sex workers, men who have sex with men, transgender individuals and other key populations should be developed, implemented and evaluated. The effectiveness of mental health interventions with such key populations living with HIV in SSA warrants investigation.•The effectiveness of promising strategies to address psychiatric multimorbidity, such as a common elements treatment approach, a transdiagnostic intervention developed to treat mood and/or anxiety disorders in low-resource settings, needs to be evaluated among PLWH at critical points throughout HIV treatment.•Intervention research is needed to understand the extent to which improving the mental health of PLWH improves HIV treatment outcomes. Research has consistently found that mental health disorders are associated with suboptimal HIV treatment outcomes. However, less is known about whether improvement in one\'s mental health is associated with subsequent improvement in HIV treatment outcomes. Research is needed that examines the relationship between improvements in symptoms and severity of mental health disorders among PLWH and improvements in HIV treatment outcomes including uptake in HIV care, adherence to ART, immunological response and sustained viral suppression. Research that examines mechanisms through which changes in mental health are associated with changes in HIV treatment outcomes is also warranted. Such research should incorporate longer-term follow-up when possible to examine intervention sustainability and long-term effectiveness.•Promising models of integrated mental health and HIV care should be implemented and evaluated. Although screening and treatment for mental health conditions are limited in many healthcare settings in SSA and globally [@JVEv4-bib-0086], the integration of mental health care into HIV testing and care settings must be a priority. The International AIDS Society supports integrated healthcare systems as an important element of not only strengthening the HIV response, but also advancing global health [@JVEv4-bib-0087]. There is a critical need for research to identify effective and efficient strategies to integrate mental health interventions into HIV service delivery programmes in the context of 'treat all' implementation. Implementation research is needed to more fully understand multilevel (e.g., patient-, provider-, and systems-level) barriers and facilitators to integrating mental health care into HIV care in the context of 'treat all' and to develop and evaluate strategies to address identified barriers. To address barriers to ART adherence and reach the 90-90-90 objectives, screening and treatment of mental health disorders is necessary at HIV diagnosis and throughout the patients' life. Screening and treatment protocols for mental health disorders that can be integrated into HIV treatment and implemented by non-specialists need to be developed, implemented and evaluated. Strategies to effectively supervise non-specialists delivering mental health interventions with PLWH and to strengthen health systems to effectively integrate mental health care into HIV are also needed. Conclusion {#JVEv4-sec-0021} ========== Key mental health-related recommendations have been identified to advance understanding and treatment of mental health disorders among PLWH and attainment of 'treat all' objectives in SSA and beyond. Key recommendations include a call for epidemiological research to examine the prevalence and impact of mental health multimorbidities on HIV treatment outcomes, intervention research to examine the extent to which improving the mental health of people living with HIV improves HIV treatment outcomes, and implementation research to evaluate promising models of integrated mental health and HIV care. The authors wish to thank the IeDEA Mental Health Working Group for their support in development of this manuscript. **Asia-Pacific** The TREAT Asia HIV Observational Database and pediatric Observational Database are initiatives of TREAT Asia, a programme of amfAR, The Foundation for AIDS Research, with support from the US National Institutes of Health\'s National Institute of Allergy and Infectious Diseases, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Cancer Institute, the National Institute of Mental Health, and the National Institute on Drug Abuse, as part of the International Epidemiology Databases to Evaluate AIDS (IeDEA; U01AI069907). The Kirby Institute is funded by the Australian Government Department of Health and Ageing, and is affiliated with the Faculty of Medicine, UNSW Sydney. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of any of the governments or institutions mentioned above. Adult site investigators and study teams: PS Ly\* and V Khol, National Center for HIV/AIDS, Dermatology & STDs, Phnom Penh, Cambodia; FJ Zhang\* ‡, HX Zhao and N Han, Beijing Ditan Hospital, Capital Medical University, Beijing, China; MP Lee\*, PCK Li, W Lam and YT Chan, Queen Elizabeth Hospital, Hong Kong, China; N Kumarasamy\*, S Saghayam and C Ezhilarasi, Chennai Antiviral Research and Treatment Clinical Research Site (CART CRS), YRGCARE Medical Centre, VHS, Chennai, India; S Pujari\*, K Joshi, S Gaikwad and A Chitalikar, Institute of Infectious Diseases, Pune, India; TP Merati\*, DN Wirawan and F Yuliana, Faculty of Medicine Udayana University & Sanglah Hospital, Bali, Indonesia; E Yunihastuti\*, D Imran and A Widhani, Faculty of Medicine Universitas Indonesia - Dr. Cipto Mangunkusumo General Hospital, Jakarta, Indonesia; J Tanuma\*, S Oka and T Nishijima, National Center for Global Health and Medicine, Tokyo, Japan; JY Choi\*, Na S and JM Kim, Division of Infectious Diseases, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea; BLH Sim\*, YM Gani, and R David, Hospital Sungai Buloh, Sungai Buloh, Malaysia; A Kamarulzaman\*, SF Syed Omar, S Ponnampalavanar and I Azwa, University Malaya Medical Centre, Kuala Lumpur, Malaysia; R Ditangco\*, E Uy and R Bantique, Research Institute for Tropical Medicine, Manila, Philippines; WW Wong\* †, WW Ku and PC Wu, Taipei Veterans General Hospital, Taipei, Taiwan; OT Ng\*, PL Lim, LS Lee and Z Ferdous, Tan Tock Seng Hospital, Singapore; A Avihingsanon\*, S Gatechompol, P Phanuphak and C Phadungphon, HIV-NAT/Thai Red Cross AIDS Research Centre, Bangkok, Thailand; S Kiertiburanakul\*, A Phuphuakrat, L Chumla and N Sanmeema, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; R Chaiwarith\*, T Sirisanthana, W Kotarathititum and J Praparattanapan, Research Institute for Health Sciences, Chiang Mai, Thailand; P Kantipong\* and P Kambua, Chiangrai Prachanukroh Hospital, Chiang Rai, Thailand; KV Nguyen\*, HV Bui, DTH Nguyen and DT Nguyen, National Hospital for Tropical Diseases, Hanoi, Vietnam; DD Cuong\*, NV An and NT Luan, Bach Mai Hospital, Hanoi, Vietnam; AH Sohn\*, JL Ross\* and B Petersen, TREAT Asia, amfAR - The Foundation for AIDS Research, Bangkok, Thailand; MG Law\*, A Jiamsakul\* and R Bijker, The Kirby Institute, UNSW Australia, Sydney, Australia. \* TAHOD Steering Committee member; † Steering Committee Chair; ‡ co-Chair. Paediatric site investigators and cohorts: PS Ly\*, and V Khol, National Centre for HIV/AIDS, Dermatology and STDs, Phnom Penh, Cambodia; J Tucker, New Hope for Cambodian Children, Phnom Penh, Cambodia; N Kumarasamy\*, and E Chandrasekaran, YRGCARE Medical Centre, CART CRS, Chennai, India; DK Wati\*, D Vedaswari, and IB Ramajaya, Sanglah Hospital, Udayana University, Bali, Indonesia; N Kurniati\*, and D Muktiarti, Cipto Mangunkusumo -- Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia; SM Fong\*, M Lim, and F Daut, Hospital Likas, Kota Kinabalu, Malaysia; NK Nik Yusoff\*‡, and P Mohamad, Hospital Raja Perempuan Zainab II, Kelantan, Malaysia; TJ Mohamed\* and MR Drawis, Pediatric Institute, Hospital Kuala Lumpur, Kuala Lumpur, Malaysia; R Nallusamy\*, and KC Chan, Penang Hospital, Penang, Malaysia; T Sudjaritruk\*, V Sirisanthana, and L Aurpibul, Department of Pediatrics, Faculty of Medicine, and Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand; R Hansudewechakul\*, P Ounchanum, S Denjanta, and A Kongphonoi, Chiangrai Prachanukroh Hospital, Chiang Rai, Thailand; P Lumbiganon\*†, P Kosalaraksa, P Tharnprisan, and T Udomphanit, Division of Infectious Diseases, Department of Pediatrics, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; G Jourdain, PHPT-IRD UMI 174 (Institut de recherche pour le développement and Chiang Mai University), Chiang Mai, Thailand; T Puthanakit\*, S Anugulruengkit, W Jantarabenjakul and R Nadsasarn, Department of Pediatrics, Faculty of Medicine and Research Unit in Pediatric and Infectious Diseases, Chulalongkorn University, Bangkok, Thailand; K Chokephaibulkit\*, K Lapphra, W Phongsamart, and S Sricharoenchai, Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; KH Truong\*, QT Du, and CH Nguyen, Children\'s Hospital 1, Ho Chi Minh City, Vietnam; VC Do\*, TM Ha, and VT An Children\'s Hospital 2, Ho Chi Minh City, Vietnam; LV Nguyen\*, DTK Khu, AN Pham, and LT Nguyen, National Hospital of Pediatrics, Hanoi, Vietnam; ON Le, Worldwide Orphans Foundation, Ho Chi Minh City, Vietnam; AH Sohn\*, JL Ross, and C Sethaputra, TREAT Asia/amfAR -- The Foundation for AIDS Research, Bangkok, Thailand; MG Law\* and A Kariminia, The Kirby Institute, UNSW Australia, Sydney, Australia; (\*Steering Committee members; † Current Steering Committee Chair; ‡ co-Chair). **Central Africa (CA-IeDEA)** Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number U01AI096299 (PI: Anastos and Nash). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Site investigators and cohorts: Nimbona Pélagie, ANSS, Burundi; Patrick Gateretse, Jeanine Munezero, Valentin Nitereka, Théodore Niyongabo, Christelle Twizere, Centre National de Reference en Matiere de VIH/SIDA, Burundi; Hélène Bukuru, Thierry Nahimana, CHUK, Burundi; Jérémie Biziragusenyuka, Risase Scholastique Manyundo, HPRC, Burundi; Tabeyang Mbuh, Kinge Thompson Njie, Edmond Tchassem, Kien-Atsu Tsi, Bamenda Hospital, Cameroon; Rogers Ajeh, Mark Benwi, Anastase Dzudie, Akindeh Mbuh, Marc Lionel Ngamani, Victorine Nkome, CRENC & Douala General Hospital, Cameroon; Djenabou Amadou, Eric Ngassam, Eric Walter Pefura Yone, Jamot Hospital, Cameroon; Alice Ndelle Ewanoge, Norbert Fuhngwa, Chris Moki, Denis Nsame Nforniwe, Limbe Regional Hospital, Cameroon; Catherine Akele, Faustin Kitetele, Patricia Lelo, Martine Tabala, Kalembelembe Pediatric Hospital, Democratic Republic of Congo; Emile Wemakoy Okitolonda, Landry Wenzi, Kinshasa School of Public Health, Democratic Republic of Congo; Merlin Diafouka, Martin Herbas Ekat, Dominique Mahambou Nsonde, CTA Brazzaville, Republic of Congo; Adolphe Mafou, CTA Pointe-Noire, Republic of Congo; Fidele Ntarambirwa, Bethsaida Hospital, Rwanda; Yvonne Tuyishimire, Busanza Health Center, Rwanda; Theogene Hakizimana, Gahanga Health Center, Rwanda; Josephine Ayinkamiye, Gikondo Health Center, Rwanda; Sandrine Mukantwali, Kabuga Health Center, Rwanda; Henriette Kayitesi, Olive Uwamahoro, Kicukiro Health Center, Rwanda; Viateur Habumuremyi, Jules Ndumuhire, Masaka Health Center, Rwanda; Joyce Mukamana, Yvette Ndoli, Oliver Uwamahoro, Nyarugunga Health Center, Rwanda; Gallican Kubwimana, Pacifique Mugenzi, Benjamin Muhoza, Athanase Munyaneza, Emmanuel Ndahiro, Diane Nyiransabimana, Jean d'Amour Sinayobye, Vincent Sugira, Rwanda Military Hospital, Rwanda; Chantal Benekigeri, Gilbert Mbaraga, WE-ACTx Health Center, Rwanda. Coordinating and data centres: Adebola Adedimeji, Kathryn Anastos, Madeline Dilorenzo, Lynn Murchison, Jonathan Ross, Albert Einstein College of Medicine, USA; Diane Addison, Margaret Baker, Ellen Brazier, Heidi Jones, Elizabeth Kelvin, Sarah Kulkarni, Grace Liu, Denis Nash, Matthew Romo, Olga Tymejczyk, Institute for Implementation Science in Population Health, Graduate School of Public Health and Health Policy, City University of New York (CUNY), USA; Batya Elul, Columbia University, USA; Xiatao Cai, Don Hoover, Hae-Young Kim, Chunshan Li, Qiuhu Shi, Data Solutions, USA; Robert Agler, Kathryn Lancaster, Marcel Yotebieng, Ohio State University, USA; Mark Kuniholm, University at Albany, State University of New York, USA; Andrew Edmonds, Angela Parcesepe, University of North Carolina at Chapel Hill, USA; Olivia Keiser, University of Geneva; Stephany Duda; Vanderbilt University School of Medicine, USA; April Kimmel, Virginia Commonwealth University School of Medicine, USA; Margaret McNairy, Weill Cornell Medical Center. **Caribbean, Central and South America network for HIV epidemiology (CCASAnet)** This work was supported by the NIH-funded Caribbean, Central and South America network for HIV epidemiology (CCASAnet), a member cohort of the International Epidemiology Databases to Evaluate AIDS (leDEA) (U01AI069923). This award is funded by the following institutes: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Cancer Institute (NCI), National Institute of Allergy And Infectious Diseases (NIAID), National Institute of Mental Health (NIMH), and the Office of the Director, National Institutes of Health (OD). Acknowledgements list Fundación Huésped, Argentina: Pedro Cahn, Carina Cesar, Valeria Fink, Omar Sued, Emanuel Dell'Isola, Hector Perez, Jose Valiente, Cleyton YamamotoInstituto Nacional de Infectologia-Fiocruz, Brazil: Beatriz Grinsztejn, Valdilea Veloso, Paula Luz, Raquel de Boni, Sandra Cardoso Wagner, Ruth Friedman, Ronaldo Moreira.Universidade Federal de Minas Gerais, Brazil: Jorge Pinto, Flavia Ferreira, Marcelle Maia.Universidade Federal de São Paulo, Brazil: Regina Célia de Menezes Succi, Daisy Maria Machado, Aida de Fátima Barbosa GouvêaFundación Arriarán, Chile: Marcelo Wolff, Claudia Cortes, Maria Fernanda Rodriguez, Gladys Allendes Les Centres GHESKIO, Haiti: Jean William Pape, Vanessa Rouzier, Adias Marcelin, Christian Perodin. Hospital Escuela Universitario, Honduras: Marco Tulio Luque.Instituto Hondureño de Seguridad Social, Honduras: Denis Padgett.Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico: Juan Sierra Madero, Brenda Crabtree Ramirez, Paco Belaunzaran, Yanink Caro Vega.Instituto de Medicina Tropical Alexander von Humboldt, Peru: Eduardo Gotuzzo, Fernando Mejia, Gabriela Carriquiry.Vanderbilt University Medical Center, USA: Catherine C McGowan, Bryan E Shepherd, Timothy Sterling, Karu Jayathilake, Anna K Person, Peter F Rebeiro, Mark Giganti, Jessica Castilho, Stephany N Duda, Fernanda Maruri, Hilary Vansell. **East Africa IeDEA** Research reported in this publication was supported by the National Institute Of Allergy And Infectious Diseases (NIAID), Eunice Kennedy Shriver National Institute Of Child Health & Human Development (NICHD), National Institute On Drug Abuse (NIDA), National Cancer Institute (NCI), and the National Institute of Mental Health (NIMH), in accordance with the regulatory requirements of the National Institutes of Health under Award Number U01AI069911East Africa IeDEA Consortium. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Site investigators and cohorts Diero L, Ayaya S, Sang E, MOI University, AMPATH Plus, Eldoret, Kenya; Bukusi E, Charles Karue Kibaara, Elisheba Mutegi, KEMRI (Kenya Medical Research Institute), Kisumu, Kenya; John Ssali, Mathew Ssemakadde, Masaka Regional Referral Hospital, Masaka, Uganda; Mwebesa Bosco Bwana, Michael Kanyesigye, Mbarara University of Science and Technology (MUST), Mbarara, Uganda; Barbara Castelnuovo; John Michael Matovu, Infectious Diseases Institute (IDI), Mulago, Uganda; Fred Nalugoda, Francis X. Wasswa, Rakai Health Sciences Program, Kalisizo, Uganda; G.R. Somi, Joseph Nondi, NACP (National AIDS Control Program) Dar es Salaam, Tanzania; Rita Elias Lyamuya, Francis Mayanga, Morogoro Regional Hospital, Morogoro, Tanzania; Kapella Ngonyani, Jerome Lwali, Tumbi Regional Hospital, Pwani, Tanzania; Mark Urassa, Denna Michael, Richard Machemba, National Institute for Medical Research (NIMR), Kisesa HDSS, Mwanza, Tanzania; Kara Wools-Kaloustian, Constantin Yiannoutsos, Rachel Vreeman, Beverly Musick, Indiana University School of Medicine, Indiana University, Indianapolis, IN, USA; Batya Elul, Columbia University, New York City, NY, USA; Jennifer Syvertsen, Ohio State University, Columbus, OH, USA; Rami Kantor, Brown University/Miriam Hospital, Providence, RI, USA; Jeffrey Martin, Megan Wenger, Craig Cohen, Jayne Kulzer, University of California, San Francisco, CA, USA; Paula Braitstein, University of Toronto, Toronto, Canada **NA-ACCORD** The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was supported by National Institutes of Health grants U01AI069918, F31DA037788, G12MD007583, K01AI093197, K23EY013707, K24AI065298, K24AI118591, K24DA000432, KL2TR000421, M01RR000052, N01CP01004, N02CP055504, N02CP91027, P30AI027757, P30AI027763, P30AI027767, P30AI036219, P30AI050410, P30AI094189, P30AI110527, P30MH62246, R01AA016893, R01CA165937, R01DA011602, R01DA012568, R01 AG053100, R24AI067039, U01AA013566, U01AA020790, U01AI031834, U01AI034989, U01AI034993, U01AI034994, U01AI035004, U01AI035039, U01AI035040, U01AI035041, U01AI035042, U01AI037613, U01AI037984, U01AI038855, U01AI038858, U01AI042590, U01AI068634, U01AI068636, U01AI069432, U01AI069434, U01AI103390, U01AI103397, U01AI103401, U01AI103408, U01DA03629, U01DA036935, U01HD032632, U10EY008057, U10EY008052, U10EY008067, U24AA020794,U54MD007587, UL1RR024131, UL1TR000004, UL1TR000083, UL1TR000454, UM1AI035043, Z01CP010214 and Z01CP010176; contracts CDC-200-2006-18797 and CDC-200-2015-63931 from the Centers for Disease Control and Prevention, USA; contract 90047713 from the Agency for Healthcare Research and Quality, USA; contract 90051652 from the Health Resources and Services Administration, USA; grants CBR-86906, CBR-94036, HCP-97105 and TGF-96118 from the Canadian Institutes of Health Research, Canada; Ontario Ministry of Health and Long Term Care; and the Government of Alberta, Canada. Additional support was provided by the National Cancer Institute, National Institute for Mental Health and National Institute on Drug Abuse. Site investigators and cohorts: Constance A. Benson and Ronald J. Bosch, AIDS Clinical Trials Group Longitudinal Linked Randomized Trials; Gregory D. Kirk, AIDS Link to the IntraVenous Experience; Stephen Boswell, Kenneth H. Mayer and Chris Grasso, Fenway Health HIV Cohort; Robert S. Hogg, P. Richard Harrigan, Julio SG Montaner, Angela Cescon and Karyn Gabler; HAART Observational Medical Evaluation and Research; Kate Buchacz and John T. Brooks, HIV Outpatient Study; Kelly A. Gebo and Richard D. Moore, HIV Research Network; Richard D. Moore, Johns Hopkins HIV Clinical Cohort; Benigno Rodriguez, John T. Carey Special Immunology Unit Patient Care and Research Database, Case Western; Benigno Rodriguez, Reserve University; Michael A. Horberg, Kaiser Permanente Mid-Atlantic States; Michael J. Silverberg; Kaiser Permanente Northern California; Jennifer E. Thorne, Longitudinal Study of Ocular Complications of AIDS; Charles Rabkin, Multicenter Hemophilia Cohort Study--II; Lisa P. Jacobson and Gypsyamber D'Souza, Multicenter AIDS Cohort Study; Marina B. Klein, Montreal Chest Institute Immunodeficiency Service Cohort; Sean B. Rourke, Anita R. Rachlis, Jason Globerman and Madison Kopansky-Giles, Ontario HIV Treatment Network Cohort Study; Robert F. Hunter-Mellado and Angel M. Mayor, Retrovirus Research Center, Bayamon Puerto Rico; M. John Gill, Southern Alberta Clinic Cohort; Steven G. Deeks and Jeffrey N. Martin, Study of the Consequences of the Protease Inhibitor Era; Pragna Patel and John T. Brooks, Study to Understand the Natural History of HIV/AIDS in the Era of Effective Therapy; Michael S. Saag, Michael J. Mugavero and James Willig, University of Alabama at Birmingham 1917 Clinic Cohort; Joseph J. Eron and Sonia Napravnik, University of North Carolina at Chapel Hill HIV Clinic Cohort; Mari M. Kitahata, Heidi M. Crane and Daniel R. Drozd, University of Washington HIV Cohort; Timothy R. Sterling, David Haas, Peter Rebeiro, Megan Turner, Sally Bebawy and Ben Rogers, Vanderbilt Comprehensive Care Clinic HIV Cohort; Amy C. Justice, Robert Dubrow and David Fiellin, Veterans Aging Cohort Study; Stephen J. Gange and Kathryn Anastos, Women\'s Interagency HIV Study Study administration: Richard D. Moore, Michael S. Saag, Stephen J. Gange, Mari M. Kitahata, Keri N. Althoff, Rosemary G. McKaig and Aimee M. Freeman, Executive Committee; Richard D. Moore, Aimee M. Freeman and Carol Lent, Administrative Core; Mari M. Kitahata, Stephen E. Van Rompaey, Heidi M. Crane, Daniel R. Drozd, Liz Morton, Justin McReynolds and William B. Lober, Data Management Core; Stephen J. Gange, Keri N. Althoff, Alison G. Abraham, Bryan Lau, Jinbing Zhang, Jerry Jing, Sharada Modur, Cherise Wong, Brenna Hogan, Fidel Desir and Bin Liu and Bin YouHealth, Family Medicine, University of Cape Town, South Africa, Epidemiology and Biostatistics Core **IeDEA Southern Africa** Research reported in this publication was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number U01AI069924. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Site investigators and cohorts: Gary Maartens, Aid for AIDS, South Africa; Michael Vinikoor, Centre for Infectious Disease Research in Zambia (CIDRZ), Zambia; Monique van Lettow, Dignitas, Malawi; Robin Wood, Gugulethu ART Programme, South Africa; Nosisa Sipambo, Harriet Shezi Clinic, South Africa; Frank Tanser, Africa Centre for Health & Population Studies (Hlabisa), South Africa; Andrew Boulle, Khayelitsha ART Programme, South Africa; Geoffrey Fatti, Kheth'Impilo, South Africa; Sam Phiri, Lighthouse Clinic, Malawi; Cleophas Chimbetete, Newlands Clinic, Zimbabwe; Karl Technau, Rahima Moosa Mother and Child Hospital, South Africa; Brian Eley, Red Cross Children\'s Hospital, South Africa; Josephine Muhairwe, SolidarMed Lesotho; Anna Jores, SolidarMed Mozambique; Cordelia Kunzekwenyika, SolidarMed Zimbabwe, Matthew P Fox, Themba Lethu Clinic, South Africa; Hans Prozesky, Tygerberg Academic Hospital, South Africa. Data centres: Nina Anderegg, Marie Ballif, Lina Bartels, Julia Bohlius, Frédérique Chammartin, Benedikt Christ, Cam Ha Dao Ostinelli, Matthias Egger, Lukas Fenner, Per von Groote, Andreas Haas, Taghavi Katayoun, Eliane Rohner, Lilian Smith, Adrian Spörri, Gilles Wandeler, Elizabeth Zaniewski, Kathrin Zürcher, Institute of Social and Preventive Medicine, University of Bern, Switzerland; Andrew Boulle, Morna Cornell, Mary-Ann Davies, Victoria Iyun, Leigh Johnson, Mmamapudi Kubjane, Nicola Maxwell, Tshabakwane Nembandona, Patience Nyakato, Ernest Mokotoane, Gem Patten, Michael Schomaker, Priscilla Tsondai, Renee de Waal, School of Public Health and Family Medicine, University of Cape Town, South Africa. **West Africa** Research reported in this publication was supported by the US National Institutes of Health (NIAID, NICHD, NCI and NIMH) under Award Number U01AI069919 (PI: Dabis). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Site investigators and cohorts: Adult cohorts: Marcel Djimon Zannou, CNHU, Cotonou, Benin; Armel Poda, CHU Souro Sanou, Bobo Dioulasso, Burkina Faso; Fred Stephen Sarfo & Komfo Anokeye Teaching Hospital, Kumasi, Ghana; Eugene Messou, ACONDA CePReF, Abidjan, Cote d'Ivoire; Henri Chenal, CIRBA, Abidjan, Cote d'Ivoire; Kla Albert Minga, CNTS, Abidjan, Cote d'Ivoire; Emmanuel Bissagnene, & Aristophane Tanon, CHU Treichville, Cote d'Ivoire; Moussa Seydi, CHU de Fann, Dakar, Senegal; Akessiwe Akouda Patassi, CHU Sylvanus Olympio, Lomé, Togo. Paediatric cohorts: Sikiratou Adouni Koumakpai-Adeothy,\_CNHU, Cotonou, Benin; Lorna Awo Renner, Korle Bu Hospital, Accra, Ghana; Sylvie Marie N'Gbeche, ACONDA CePReF, Abidjan, Ivory Coast; Clarisse Amani Bosse, ACONDA_MTCT+, Abidjan, Ivory Coast; Kouadio Kouakou, CIRBA, Abidjan, Cote d'Ivoire; Madeleine Amorissani Folquet, CHU de Cocody, Abidjan, Cote d'Ivoire; François Tanoh Eboua, CHU de Yopougon, Abidjan, Cote d'Ivoire; Fatoumata Dicko Traore, Hopital Gabriel Toure, Bamako, Mali; Elom Takassi, CHU Sylvanus Olympio, Lomé,Togo Coordinating and data centres: François Dabis, Elise Arrive, Eric Balestre, Renaud Becquet, Charlotte Bernard, Shino Chassagne Arikawa, Alexandra Doring, Sophie Desmonde, Patricia Dumazert, Antoine Jaquet, Julie Jesson, Valeriane Leroy, Karen Malateste, Elodie Rabourdin, Thierry Tiendrebeogo, ADERA, Isped & INSERM U1219, Bordeaux, France. Didier Koumavi Ekouevi, Jean-Claude Azani, Patrick Coffie, Abdoulaye Cissé, Guy Gnepa, Apollinaire Horo, Christian Kouadio, Boris Tchounga, PACCI, CHU Treichville, Abidjan, Côte d'Ivoire

Introduction {#s1} ============ *Anoectochilus roxburghii*, a member of the Orchidaceae, is a valued plant species in many Asian countries, where it is used for ornamental, culinary, and medicinal purposes. Because of its unique medicinal properties, such as its notable curative effects of clearing heat and cooling blood, eliminating dampness, and detoxification, *A. roxburghii* has been called "the king of medicine" [@pone.0085996-Zhang1], [@pone.0085996-Du1]. Recent research has demonstrated that the entire plant possesses medicinal properties, such as antioxidant, anti-inflammatory, and antitumor activities [@pone.0085996-He1], [@pone.0085996-Zhang2]. *A. roxburghii* has been traditionally harvested mainly from wild populations. The species has become endangered, however, as a result of human overexploitation coupled with its specific environmental growth requirements. As a consequence, artificial cultivation is beginning to be carried out in various locations in China. Light has long been known to be the most important factor influencing plant growth, with changes in irradiance having impacts on plant growth, morphology, and anatomy, various aspects of physiology and cellular biochemistry, and, ultimately, flowering time and plant productivity [@pone.0085996-Dai1]--[@pone.0085996-Deng1]. In the light reactions of photosynthesis, light energy is used to produce ATP and NADPH, which are then used for carbon fixation to carbohydrates and production of oxygen during the light-independent phase. Shading effects are not just about the plants' growth and development, but through which, it also has a major impact on plant photosynthesis. Normal plant growth needs optimal light irradiance because excessively high and low irradiances would result in photoinhibition and light deficiency respectively, and therein the growth of plant was restricted severely. Under high irradiance conditions, photoinhibition takes place: the photosynthetic apparatus absorbs excessive light energy, resulting in the inactivation or impairment of the chlorophyll-containing reaction centers of chloroplasts and consequent depression of photosynthetic activity [@pone.0085996-Bertaminia1], [@pone.0085996-Chen1]. In contrast, under low irradiance conditions, insufficient ATP is produced to allow for carbon fixation and carbohydrate biosynthesis. This leads to a reduction in plant growth. Chloroplasts are the sole organelles of photosynthesis. Although many authors have reported a close relationship among photosynthesis, chlorophyll content, and chlorophyll fluorescence in different species under shading conditions, little work has been performed on the association between photosynthesis-related parameters and chloroplast ultrastructure and physiology [@pone.0085996-Raveh1]. A comprehensive understanding of changes in chloroplast ultrastructure and physiology during leaf development under different irradiance conditions is needed. The objectives of the present study were to quantify the influences of different shading levels on photosynthetic characteristics, chloroplast ultrastructure, and physiology of *A. roxburghii*, to determine optimum light intensity for plant growth, and to consequently expand our understanding of its shade-tolerance abilities and mechanisms. In addition, we wished to establish a sound foundation for improving the cultivation and breeding of this important plant species. Materials and Methods {#s2} ===================== Plant Materials and Growth Conditions {#s2a} ------------------------------------- *A. roxburghii* plants were obtained from Lin'an commercial plantations and were maintained in a greenhouse at the Baicaoyuan test site of Zhejiang A & F University, China (30°15′N, 119°43′E). The photoperiod (day/night) and air relative humidity in the greenhouse were 14/10 h and 75% respectively. Plants were subjected to four different shade treatments for 40 days, beginning on June 1, 2012. Treatments consisted of 50%, 30%, 20%, or 5% natural irradiance, and were conducted in net houses (2.5 m high, 3 m long, and 2 m wide) covered with one or two layers of commercial plastic shading nets. Each treatment involved 10 pots with three replications ([Figure 1](#pone-0085996-g001){ref-type="fig"}). Diurnal variations of photosynthetically active radiation (400--700 nm wavelengths) were measured under all shade treatments with a TES-1332 digital lux meter (TES, Taiwan). Recorded data are displayed in [Figure 2](#pone-0085996-g002){ref-type="fig"}. All plants were kept well-irrigated and protected from bacterial pathogens and weed competition. {#pone-0085996-g001} {#pone-0085996-g002} Photosynthetic Parameters {#s2b} ------------------------- Photosynthetic parameters were investigated using a LI-6400XT CO~2~/H~2~O porometer (Li-Cor, Lincoln, NE, USA). The parameters measured were net photosynthetic rate (P~n~, µmol m^−2^ s^−1^), stomatal conductance (G~s~, mmol m^−2^ s^−1^), and intercellular CO~2~ concentration (C~i~, µmol mol^−1^). During the treatment period, data were recorded between 9∶30 and 11∶30 am on days 0, 10, 20, 30, and 40. Air cuvette irradiance, temperature, and CO~2~ concentration were maintained at 1000 µmol m^−2^ s^−1^, 30°C and 380 µmol l^−1^ respectively, and the assimilation was recorded following a 10 min acclimation period [@pone.0085996-Dai1]. At each conducted time point, five representative plants were randomly selected from each treatment and analyzed for the above parameters. Chlorophyll Fluorescence {#s2c} ------------------------ Chlorophyll fluorescence of the same leaves used for determination of photosynthetic parameters was measured with a MINIPAM fluorometer (Walz, Effeltrich, Germany). Leaves were light-adapted for approximately 15 min prior to measurements of effective quantum yield of photochemical energy conversion (Yield) and photochemical (qP) and nonphotochemical (NPQ) quenching of chlorophyll fluorescence. The effective quantum yield of photochemical energy conversion at steady-state photosynthesis was calculated as Yield = (F~m~′ − F~s~)/F~m~′, where F~s~ and F~m~′ were fluorescence at steady-state photosynthesis and maximum fluorescence in the light, respectively. qP was calculated as (F~m~ − F~m~′)/(F~m~′ − F~0~), and NPQ was calculated as (F~m~ − F~m~′)/F~m~′ [@pone.0085996-Genty1]. The relative rate of electron transport through PSII (ETR) was calculated as Yield × photosynthetically active radiation × 0.84 × 0.5 [@pone.0085996-Schreiber2]. Chlorophyll Content {#s2d} ------------------- Following measurement of chlorophyll fluorescence as described above, leaves were collected for determination of chlorophyll content (Chl a, Chl b, and Chl a+b). Chlorophyll pigments were extracted by grinding leaves in 80% acetone in the dark at room temperature, with their concentrations expressed as mg g^−1^ FW based on the equations of Porra [@pone.0085996-Porra1]. Chloroplast Ultrastructure {#s2e} -------------------------- To examine chloroplast ultrastructure of mesophyll cells, sampled leaves described above were immediately fixed in 2.5% (v/v) glutaraldehyde (0.1 mol l^−1^ phosphate buffer, pH 7.2) for at least 48 h after removal from plants. The samples were then immersed in 1% (v/v) osmium acid for post-fixation, resin embedding, and ultrathin sectioning for transmission electron microscopy (H7650, Hitachi, Tokyo, Japan). Physiological and Biochemical Assays {#s2f} ------------------------------------ Approximately 0.5 g leaf samples were collected on treatment days 0, 10, 20, 30, and 40 frozen immediately at −80°C. Protein content was determined based on the Bradford method [@pone.0085996-Bradford1], with bovine serum albumin employed as a standard. Crude enzyme preparation for superoxide dismutase (SOD) and peroxidase (POD) assays involved tissue homogenization in 5 ml of 100 mM potassium phosphate buffer (pH 7.0) following the procedure of He et al. [@pone.0085996-He2]. Assays used for POD, SOD, and catalase (CAT) were those described by Argandona et al. [@pone.0085996-Argandona1] and Yin et al. [@pone.0085996-Yin1]. Soluble sugar (SS) content was determined using anthrone colorimetry as described by Li [@pone.0085996-Li1], and malondialdehyde (MDA) content was measured by the thiobarbituric acid method according to Deng et al. [@pone.0085996-Deng3]. Statistical Analysis {#s2g} -------------------- One-way analysis of variance (ANOVA) was carried out using SPSS software version 16.0 (SPSS, Chicago, IL, USA). Duncan's multiple range test was employed to detect differences between means (with *P* set to 0.05). Results {#s3} ======= Photosynthesis {#s3a} -------------- P~n~ values of *A. roxburghii* varied significantly (*P*\<0.05) among light intensities and treatment periods ([Figure 3a](#pone-0085996-g003){ref-type="fig"}). The P~n~ value was always highest at 30% irradiance, followed (in descending order) at most time points by 20%, 5%, and 50% irradiance treatments. P~n~ values significantly increased during the first 20 days of treatment, with the highest P~n~ value measured at 30% irradiance on day 20. Subtle changes were observed within treatments between days 30 and 40, but P~n~ values under 50% irradiance were always higher than those under 5% irradiance. {#pone-0085996-g003} G~s~ values varied significantly among various light levels and exposure periods ([Figure 3b](#pone-0085996-g003){ref-type="fig"}). G~s~ values of plants subjected to 50% and 5% irradiance were always lower than those of 30%- and 20%-irradiance treated plants; on days 10 to 30, 50%-irradiance treated plants always exhibited the lowest values. The highest G~s~ values under 30%, 20%, and 5% irradiance were observed on the 20th day of treatment. C~i~ values displayed slight increases over time ([Figure 3c](#pone-0085996-g003){ref-type="fig"}). On treatment day 10, C~i~ values from the 20% irradiance treatment were higher than those from other treatments. The highest values, however, were observed under 30% irradiance on days 20--40, and were higher than values measured during other treatments. Values measured from plants under 5% irradiance treatment were always the lowest. Chlorophyll Fluorescence {#s3b} ------------------------ The 50% irradiance treatment resulted in a significant (*P*\<0.05) reduction in apparent ETR and qP, and an increase in NPQ on the 40th day of treatment ([Figure 4a--c](#pone-0085996-g004){ref-type="fig"}). On day 10, the highest ETR value was recorded in leaves under 30% irradiance, while the lowest was found in plants grown under 50% irradiance. The trend observed for qP upon variation in light intensities and treatment times was similar to that of ETR. The highest and lowest qP values were observed in plants under 30% and 50% irradiance treatments, respectively. The highest NPQ values were always measured in 50% irradiance plants. {#pone-0085996-g004} Chlorophyll Contents {#s3c} -------------------- Chlorophyll content was significantly affected by different light intensities ([Figure 5](#pone-0085996-g005){ref-type="fig"}). Chl a and Chl b contents were increased and Chl a/b was decreased on days 20--40 of reduced irradiance treatments. The highest Chl a, Chl b, and Chl a+b contents were observed in 5%-treated plants on day 40. The shade treatments caused a decrease in Chl a/b over time, with the highest Chl a/b values observed in plants under 50% irradiance on day 40. {#pone-0085996-g005} Chloroplast Ultrastructure {#s3d} -------------------------- Chloroplast sizes and numbers were obviously influenced by light levels of *A. roxburghii* ([Figure 6](#pone-0085996-g006){ref-type="fig"}). The number of chloroplasts, grana, and grana lamellae generally increased as light irradiance was reduced. Most chloroplasts in leaves grown under 30% and 20% irradiance conditions exhibited normal ultrastructural organization, possessing a typical arrangement of grana and stroma thylakoids. Grana of plants grown under 30%, 20%, and 5% shading generally contained more thylakoids than those from plants grown under 50% irradiance. In addition, the number and size of osmiophilic globules was reduced in plants under 30% and 20% shading treatments compared with those from leaves grown under 50% and 5% treatments. {#pone-0085996-g006} Physiological and Biochemical Assays {#s3e} ------------------------------------ Under 50% irradiance conditions, leaf protein content remained relatively stable during the first 20 days of treatment, but increased rapidly thereafter. The other shaded plants reacted rather differently, however: during the first 10 days of treatment, leaf protein content increased, and subsequently declined ([Figure 7a](#pone-0085996-g007){ref-type="fig"}). A pronounced fall in leaf POD activity occurred after 20 days of treatment. On days 30--40, POD activity in 50% irradiance plants was significantly lower than in other shade-treated plants. Up to day 20, SOD activity in 50% irradiance plants was higher than in other shade-grown plants ([Figure 5c](#pone-0085996-g005){ref-type="fig"}). By day 40, relative SOD levels were reversed, with the highest expression (at 30% irradiance) being approximately double that of the 50% irradiance treatment. Shading had little influence on CAT activity up to the 20th day of treatment, but significant increases were observed thereafter. Up to day 20, 50% irradiance plants displayed the lowest CAT activity; on days 30--40, however, they exhibited the highest CAT activity levels. SS contents were positively correlated with the irradiance levels ([Fig. 7e](#pone-0085996-g007){ref-type="fig"}). SS content decreased at all levels of shade treated plants. Similarly, the response of MDA contents were also positively correlated with the irradiance levels. But its contents increased at all levels of treatments. {#pone-0085996-g007} Discussion {#s4} ========== Photosynthetic Response of *A. roxburghii* to Shading {#s4a} ----------------------------------------------------- C~i~ changed only slightly in *A. roxburghii* grown under different shade treatments, with the data suggesting that CO~2~ concentration was not the main factor reducing photosynthetic rate in leaves of plants under 50% and 5% irradiance treatments ([Figure 3c](#pone-0085996-g003){ref-type="fig"}). At the same time, values of G~s~ decreased significantly in plants grown under these treatments ([Figure 3b, d](#pone-0085996-g003){ref-type="fig"}). Under the high light environment, the observed reductions in G~s~ indicate that stomatal closure was due to light saturation and functioned to decrease water loss. When net CO~2~ assimilation became light saturated, transpiration constantly decreased with the declined photosynthetic photon flux density [@pone.0085996-Schapendonk1]. Thus under 5% irradiance treatment, plants also exhibit a similar behaviour as that in high light condition, that is, closing stomata due to water saturation to adapt to low light. Chlorophyll Fluorescence Response of *A. roxburghii* to Shading {#s4b} --------------------------------------------------------------- Chlorophyll fluorescence measurement is a mainstay of studies of photosynthetic regulation and plant responses to the environment because of its sensitivity, convenience, and nonintrusive characteristics [@pone.0085996-Schreiber1], [@pone.0085996-Rascher1]. ETR represents the relative quantity of electrons passing through PSII during steady-state photosynthesis [@pone.0085996-Tezara1]. Exposure to the high irradiance conditions of 50% irradiance resulted in a greatly reduction in ETR value ([Fig. 4a](#pone-0085996-g004){ref-type="fig"}). Reductions in ETR may be due to the loss of chlorophyll via and the reduction in the efficiency of excitation capture, which most likely as a result of photoinhibition [@pone.0085996-Flowers1]. qP is an indicator of the proportion of open PSII reaction centers [@pone.0085996-Maxwell1]. A high qP is advantageous for the separation of electric charge in the reaction center, and is also beneficial to electron transport and PSII yield [@pone.0085996-Guo1], [@pone.0085996-Mao1]. In this experiment, observed differences in qP values revealed that *A. roxburghii* had significant differences in PSII electron transport activities when plants were grown under varied shade treatments. Electric charge separation in the reaction center, electron transport ability, and quantum yield of PSII were enhanced under 30% irradiance and weakened under 50% irradiance. NPQ is a reflection of the amount of unused energy from photosynthetic electron transport that is dissipated harmlessly as heat energy from PSII antennae [@pone.0085996-Muller1], [@pone.0085996-Veres1]. The low NPQ measured for 30% irradiance treatment plants indicates that these plants were able to effectively reduce irradiance heat and efficiently utilize the energy absorbed by PSII antenna pigments [@pone.0085996-Guo1]. In contrast, the high NPQ observed in plants under 50% irradiance demonstrates that the energy absorbed in the physiologically usable range of light was much higher than the quantity photochemically usable, which would cause inhibition of photosynthetic capacity. Chlorophyll Content Response of *A. roxburghii* to Shading {#s4c} ---------------------------------------------------------- Leaf chlorophyll content is an important determinant of photosynthetic rate [@pone.0085996-Mao1] and dry matter production [@pone.0085996-Ghosh1]. Naidu et al. [@pone.0085996-Naidu1] have suggested that reduced rates of photosynthesis may be due to reduced levels of chlorophyll, particularly Chl a, which is more directly involved in determining photosynthetic activity [@pone.0085996-Sestak1]. Decreases in Chl a and Chl b contents may thus reflect destruction of pigments by excessive irradiance. We observed significant (*P*\<0.05) decreases in chlorophyll contents (Chl a, Chl b, and Chl a+b) under 50% irradiance conditions, suggesting that high irradiance may seriously impair the photosynthetic system. Plants grown under shaded conditions are known to optimize their light absorption efficiency by increasing pigment density per unit leaf area [@pone.0085996-Wittmann1]. The reductions we observed in Chl a/b ratios in leaves of 30%, 20% and 5% irradiance plants were due primarily to significant (*P*\<0.05) increases in Chl b content, most likely as a result of changes in the organization of both light harvesting and electron transport components [@pone.0085996-Schiefthaler1]. The marked increase in leaf Chlorophyll content under 5% irradiance conditions demonstrates the ability of plants to maximize their light-harvesting capacity under low-light growth conditions [@pone.0085996-Lei1]. Chloroplast Ultrastructure Response of *A. roxburghii* to Shading {#s4d} ----------------------------------------------------------------- Leaves from 30%, 20%, and 5% shade treatments possessed grana containing more thylakoids than those of leaves grown under high (50%) irradiance conditions, and, as a consequence, had higher photosynthetic rates and pigment contents. Leaves grown in 30% irradiance environments exhibited better-developed chloroplasts, grana, and stroma lamellae. This result implies that 30% irradiance treatments were somewhat conducive to plant growth. Modulation of chloroplast development through increases in numbers of thylakoids, grana, and grana lamellae may be an important shade-tolerance mechanism in *A. roxburghii*. The number and size of osmiophilic globules can also be used as an indicator of photosynthetic efficiency [@pone.0085996-Helle1], [@pone.0085996-Liu1]. Chloroplasts in leaves of 30%- and 20%-treated plants contained the fewest and smallest osmiophilic globules; this result also suggests that moderate shading, to some extent, was beneficial, whereas 50% and 5% shade treatments were harmful to plant growth. Physiological and Biochemical Response of *A. roxburghii* to Shading {#s4e} -------------------------------------------------------------------- Performances of leaf protein content and antioxidant enzyme activities revealed that these traits were under strong genetic control, whereas SS and MDA contents were largely determined by the degree of shading. Exposure to high (50%) irradiance conditions greatly increased total protein content. Activity profiles of the various antioxidant enzymes were not uniform. After 40 days of shading, POD and SOD levels were significantly higher in 30%, 20%, and 5% irradiance plants than in 50% irradiance plants, whereas CAT activity remained lower. During these experiments, we carefully maintained consistent moisture availability and temperature conditions. Consequently, it is clear that POD, SOD, and CAT activity levels were not only markedly affected by the degree of imposed shading, but were also heavily influenced by the developmental status of the experimental plants. As SSs are an important carbon source and osmoregulator of plant growth, SS levels reflect plant nutritional status [@pone.0085996-Dong1]. In our study, plant SS content was lowest under high irradiance treatments, and was responsive to shading severity. This result suggests that *A. roxburghii* responds differently to light with respect to carbohydrate metabolism. MDA content is considered to be an indicator of cellular membrane lipid peroxidation [@pone.0085996-Sharma1], [@pone.0085996-Deng2]. The significantly high MDA content observed in our study under high irradiance indicates the occurrence of damage due to excessive irradiance. Conclusions {#s5} =========== Our study, which measured photosynthetic characteristics associated with light stress sensitivity in *A. roxburghii*, has contributed to an understanding of this species' shade tolerance. Our results demonstrate that shading is necessary for its normal growth, although different degrees and durations of shading treatments significantly influence photosynthetic activity, chlorophyll content, chlorophyll fluorescence, chloroplast ultrastructure, and physiological and biochemical indexes. Plants subjected to 50% irradiance conditions suffer photoinhibition because of excess light exposure, whereas those grown under 5% irradiance suffer from light deficiency. *A. roxburghii* adapts to shade conditions through increased levels of chloroplasts, grana, and grana lamellae, and higher POD and SOD activitities. We thank Dr. Jie Zhang for assistance in improving the written English. In addition, we express our gratitude to the anonymous reviewers who carefully reviewed our manuscript and put forth many valuable suggestions. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: QS. Performed the experiments: QS HW HG. Analyzed the data: AZ YH. Contributed reagents/materials/analysis tools: ML. Wrote the paper: QS YS.

{#sp1 .870} {#sp2 .871} {#sp3 .872} {#sp4 .873}

Background {#Sec1} ========== Steroidal drugs are the second largest category in the pharmaceutical market. More than 400 kinds of steroid drugs for a wide range of diseases are selling with an annual sale of 100 billion dollars \[[@CR1]\]. Modifying the mycobacterial metabolic pathway for accumulating high value-added steroid intermediates \[[@CR2]\] is the most important step of the latest upgraded semi-synthetic route in steroidal pharmaceutical industry \[[@CR3]\]. By the conversion of low value-added phytosterols, environment friendly extracts from the vegetable oil processing waste, sustainable pine tree bioresource and waste products in papermaking \[[@CR4]\], C19 steroids (androst-4-ene-3,17-dione, AD; boldenone, BD; 9α-hydroxy-androst-4-ene-3,17-dione, 9-OHAD) \[[@CR5], [@CR6]\] and C22 steroids (22-hydroxy-23,24-bisnorchol-4-ene-3-one, 4-HBC) \[[@CR7]\] can be respectively accumulated. Then, almost all kinds of steroid drugs, including adrenocortical and progestational hormones, can be produced by the combinational chemical modifications \[[@CR8]\]. For instance, 9-OHAD is a core intermediate and has been used as a cost-effective precursor to synthesize C21 adrenocortical hormone drugs \[[@CR8]\]. However, the unsatisfying yield and productivity of the currently used strains has prompted researchers to intensively investigate more efficient and stable strategies for the biosynthesis of important steroidal intermediates \[[@CR9], [@CR10]\]. Sterols can be catabolized as the sole carbon and energy source for maintaining the balance of basic physiological metabolism in mycobacteria \[[@CR9]\]. The uptake of sterols in cells may be divided into two distinguished stages: (I) the mass transfer stage of sterol molecules and particles to cell surface and (II) the diffusion stage of sterols across the cell wall and membrane. Stage I is mainly depends on the direct contact with the substrates dispersed in the extracellular environment. Early studies on material transfers demonstrated that in the presence of hydroxypropyl-β-cyclodextrin \[[@CR11]\], the use of biocompatible water-immiscible organic phase \[[@CR12]\] could largely improve the solubilization of sterol substrates in the transformation system. As a result, the cells contacted with the sterols more efficiently. The substrate transfer was enhanced and the conversion productivity was increased accordingly. In addition, the β-cyclodextrin possibly improved the permeability due to the alteration of mycobacterial cell wall structure \[[@CR13]\]. Thus, with the addition of glycine and vancomycin, which were inhibitors to the synthesis of mycobacterial cell wall, the cell permeability displayed a marked improvement \[[@CR14]\]. However, these strategies employing massive additives are seldom used in the industrial process because of the high costs and low effects. It is noteworthy that most of the aforementioned methods possibly lead to some defects of the cell wall. The mycobacteria cell wall contains extremely rich mycolic acids \[[@CR15]\]. This component accounts for 40--60% of the cell dry weight and are probably responsible for the crucial cell permeability characteristic \[[@CR16], [@CR17]\]. Rational modifications of the mycolic acid biosynthesis pathway might be reasonable ways to alter the permeability performance of the steroidal conversion microbial cell factories. Mycolic acids are synthesized originally from acetyl-CoA and malonyl-CoA \[[@CR18]\]. The C16--C18 and C24--C26 α-alkyl chain is elongated based on Claisen condensation catalyzed by the fatty acid synthase I (FAS-I). The resulting short chain is synthesized by β-ketoacyl-ACP synthases (FabH) to form β-ketoacyl-ACP. Then, a long mero chain can be obtained by the repetitive reductive cycles due to the catalysis of multienzyme fatty acid synthase II complex (FAS-II). Additional elongation cycles are subsequently catalyzed by the two β-ketoacyl-ACP synthase KasA and KasB. After the mero-chain and α-chain are coupled together by the acyl-AMP ligase FadD32 and the polyketide synthase Pks13 and then deoxidized by the mycolate reductase CmrA, the mature mycolate (trehalose monomycolate, TMM) can be synthetized in the mycobacterial cytoplasm. Next, the TMM is transported to the cell periplasm and participates in the subsequent assembly of mycolic acid-related structures, including the polar TDM and mycolic acid methyl esters in the core mycolyl-arabinogalactan-peptidoglycan (MAMEs-AG-PG) complex of cell wall \[[@CR17]\]. The FAS-I synthesis gene *fas* is required in *M. smegmatis* \[[@CR19]\] and *M. tuberculosis* \[[@CR20]\] and the fatty acid synthase II (FASII) enzymes InhA \[[@CR21]\], MabA \[[@CR22]\], HadB \[[@CR23]\], and KasA \[[@CR24]\] are also required. The inactivation of these indispensable genes could lead to the lysis of mycobacterial cells \[[@CR21]--[@CR24]\]. The disruption of nonessential genes possibly caused some stable defects only in the cell wall. Thus, the loss of the dispensable genes, such as *hadA*, *hadC* and *kasB* in the mero-mycolic acid synthesis pathway, are worth investigation in the model steroid transformation cells (Fig. [1](#Fig1){ref-type="fig"}a) \[[@CR16], [@CR18]\].Fig. 1Rational disruption of the mycolic acid synthesis disturbed the sterol conversion. **a** Profile of the mycolic acid synthesis pathway in mycobacteria cells \[[@CR18]\]. FAS-I, fatty acid synthase I; FabD, malonyl CoA-acyl carrier protein (ACP) transacylase; FabH, β-ketoacyl-ACP synthase III; MabA, β-ketoacyl-ACP reductase; HadABC, β-hydroxyacyl-ACP dehydratase subunits A, B and C; InhA, enoyl-ACP reductase; KasA, β-ketoacyl-ACP synthase 1; KasB, β-ketoacyl-ACP synthase 2; PcaA, proximal cyclopropanation of alpha-MAs enzyme; MmaA1-4, methyl mycolic acid synthase; CmaA2, cyclopropyl mycolic acid synthase; AccD4, propanoyl-CoA carbon dioxide ligase; AccD5, propionyl-CoA carboxylase; FadD32, long-chain-fatty-acid-AMP synthetase, Pks13, polyketide synthase. **b** Transcription changes in the dispensable genes involved in mycolic acid synthesis. All data indicate log2 fold change ratio of the gene expression. Mn, the wild type *M. neoaurum* was cultured in MYC/02 medium. Mn + C, the wild type strain was cultivated in the presence of phytosterol. MnΔ*kstD1 *+ C, the primary 9-OHAD-producing strain MnΔ*kstD1* was cultured in MYC/02 medium with phytosterol addition. Data were from two independent analyzes. **c** The alternation of sterol utilization rate caused by the targeted gene deletion in 72 h sample time. Data represent the mean standard deviation of three measurements The biotransformation process is a rate-limiting step in the microbes producing steroid intermediates. It usually takes 120 to 144 h to realize a satisfactory conversion rate of the substrate to target steroid intermediates in the microbes \[[@CR5], [@CR6], [@CR25]\]. However, it only takes about 48 to 72 h in most of other prokaryotic microorganisms \[[@CR26]--[@CR28]\]. The long conversion time is primarily attributed to the low permeability of sterol substrates into the cell wall \[[@CR2]\]. Promoting the substrate to enter microbial cells by modifying the cell wall may shorten the time required by the bioconversion process and improve the integral production capacity of mycobacterial cells. Increasing the sterol biotransformation efficiency in *M. neoaurum* through a systemic cell wall engineering technique was rarely reported \[[@CR2]\]. The disruption of the genes involved in mycolic acid synthesis in mycobacterial cells was not directly assessed. In the study, the annotated nonessential mycolic acid synthetic genes were inactivated individually. The modification which significantly altered the sterol conversion was further investigated. The result revealed the roles of accessory genes in the formation of mycolic acids and provided an alternative evolution strategy for the microbial transformation of steroidal intermediates. Methods {#Sec2} ======= Strains, plasmids and primers {#Sec3} ----------------------------- All strains used in this study are described below (Table [1](#Tab1){ref-type="table"}). *Escherichia coli* DH5α (TIANGEN Biotech. Co., Ltd., Shanghai, China) was used for plasmid amplification. The wild type *M. neoaurum* ATCC 25795 (Mn) was purchased from American Type Culture Collection (ATCC). The C19 steroidal intermediate 9-OHAD producers MnΔ*kstD1* and MnΔ*kstD1*Δ*kstD2*Δ*kstD3* (WI) were constructed by Kang Yao \[[@CR6]\]. The C22 steroidal intermediate 4-HBC-producing strain MnΔ*kshA*Δ*hsd4A*Δ*kstD1*Δ*kstD2*Δ*kstD3* (WIII) was constructed by Xu \[[@CR7]\]. Others were all derived from the above three *M. neoaurum* strains. Common plasmids (Additional file [1](#MOESM1){ref-type="media"}: Table S1) and primers (Additional file [1](#MOESM1){ref-type="media"}: Table S2) were used for constructing the mutants.Table 1Strains used in this studyNameDescriptionSource*E. coli* DH5α*E. coli* strain for cloningTIANGEN CO., LTD.*M. neoaurum* ATCC 25795 (Mn)Wild type strain, the starting strainATCCMnΔ*kstD1kstD1* deleted in *M. neoaurum* ATCC 25795\[[@CR6]\]WI*kstD1*, *kstD2* and *kstD3* deleted in *M. neoaurum* ATCC 25795, 9-OHAD producing strain\[[@CR6]\]WIΔ*hadAhadA* deleted in WI strainThis studyWIΔ*hadChadC* deleted in WI strainThis studyWIΔ*kasBkasB* deleted in WI strainThis studyWIΔ*mmaANmmaAN* (N represents 1, 2, 3, 4) deleted in WI strainThis studyWIΔ*pks13pks13* deleted in WI strainThis studyMnΔ*kasBkasB* deleted in *M. neoaurum* ATCC 25795This studyMnΔ*kasB *+ *kasBkasB* complemented in MnΔ*kasB* strainThis studyWIII*kshA1*, *kshA2*, *hsd4A*, *kstD1, kstD2* and *kstD3* deleted in *M. neoaurum* ATCC 25795, 4-HBC producing strain\[[@CR7]\]WIIIΔ*kasBkasB* deleted in WIII strainThis study Media and culture conditions {#Sec4} ---------------------------- Media and culture conditions were the same as the previously described conditions \[[@CR2], [@CR29]\]. *E. coli* DH5α was inoculated at 37 °C in 5 mL of Luria--Bertani (LB) medium. Kanamycin (50 mg/L) or hygromycin (100 mg/L) was added to the culture medium as required. Mycobacterial strains were firstly cultivated in 5 mL of LB until OD~600~ was between 1.2 and 1.8. Then, according to an inoculum volume ratio of 1:10 (v/v), the cell suspension was inoculated into 30 mL of MYC/01 medium (20.0 g/L glycerol, 2.0 g/L citric acid, 2.0 g/L NH~4~NO~3~, 0.5 g/L K~2~HPO~4~, 0.5 g/L MgSO~4~·7H~2~O, and 0.05 g/L ammonium ferric citrate, pH 7.5) in 250-mL flasks to obtain the mycobacterial seed suspension (OD600 = 1.2--1.8). For phenotypic identification, according to an inoculum volume ratio of 1:10 (v/v), the cultivated cells were then transferred into 30 mL of minimal medium (MM) (2.0 g/L NH~4~NO~3~, 0.5 g/L K~2~HPO~4~, 0.5 g/L MgSO~4~·7H~2~O, and 0.05 g/L ammonium ferric citrate) with 1 g/L glycerol or 1 g/L cholesterol (purity \> 95.0%, Adamas Reagent, Ltd., Shanghai, China). Cells were harvested by the centrifugation at 4000*g* for 10 min. For the bioconversion in growth cells, according to an inoculum volume ratio of 1:10 (v/v), the cultivated seed cells were inoculated into 30 mL of MYC/02 medium (10.0 g/L glucose, 2.0 g/L citric acid, 2.0 g/L NH~4~NO~3~, 0.5 g/L MgSO~4~·7H~2~O, and 0.05 g/L ferric ammonium citrate, pH 7.5) with 5 g/L phytosterols (purity \> 95.0%, every 100 g of phytosterol contained 47.5 g of β-sitosterol, 26.4 g of campesterol, 17.7 g of stigmasterol, 3.6 g of brassicasterol and 4.8 g of undetermined components) (Zhejiang Davi Pharmaceutical Co., Ltd., Zhejiang, China) \[[@CR29]\]. Cholesterol (100.0 g/L) and phytosterol (100.0 g/L) was emulsified in Tween 80 (5% w/v) aqueous solution at 121 °C for 60 min before use. The shake flask experiments of *M. neoaurum* strain were carried out at 30 °C and 200 rpm. For resting cell conversion, according to an inoculum volume ratio of 1:10 (v/v), the cultivated cells were transferred into 150 mL of MYC/02 medium in 1000-mL shake flasks for the growth at 30 °C and 200 rpm. The cells were harvested by the centrifugation at 8000*g* for 15 min, washed with 20 mM KH~2~PO~4~, and diluted into 200 g/L of cell suspensions. The subsequent conversion step was performed in 250-mL flasks containing 100 g/L mycobacterial cells, 20 g/L phytosterols and 80 g/L hydroxypropyl-β-cyclodextrin (HP-β-CD, RSC Chemical Industries Co., Ltd., Jiangsu, China) in at 30 °C and 200 rpm \[[@CR30]\]. Standard 9-OHAD (99%) was purchased from J&K Scientific Ltd. (Beijing, China). Standard reference 4-HBC (97%) was purified and identified by ourselves \[[@CR7]\]. Construction of genetically modified strains {#Sec5} -------------------------------------------- Target gene-deleted strains were obtained through allelic homologous recombination in mycobacteria as previously described \[[@CR31]\]. p2NIL and pGOAL19 were used for the construction of the homologous recombination plasmids (Additional file [1](#MOESM1){ref-type="media"}: Table S1). The knockout-plasmids p19-gene, including p19-*hadA*, p19-*mmaA2*, p19-*hadC*, p19-*mmaA1*, p19-*mmaA3*, p19-*mmaA4*, p19-*pks13* and p19-*kasB*, was transferred into mycobacterial cells via electroporation, respectively. Then, the target gene deficient strain can be obtained following the two-step screening process \[[@CR32]\]. To complement the deficient-gene function, the complete gene sequence of *kasB* was firstly amplified from the wild type strain with the primer pairs (C-*kasB*-F & C-*kasB*-R) (Additional file [1](#MOESM1){ref-type="media"}: Table S2). After double digestion with EcoRI and HindIII, the enzyme-digested fragment was inserted into the pMV261 to create a recombinant p261-*kasB* plasmid. This constructed recombination plasmid could be used to overexpress the carried *kasB* in multiple copies. Moreover, the expression cassette of the target *kasB* containing a heat shock promoter *hsp60* was obtained from the recombinant p261-*kasB* through double-digestion with XbaI and HindIII then integrated into the pMV306 to create a complemental plasmid p306-*kasB*. The constructed plasmid could be integrated into chromosomal DNA in single copy to complement the disrupted gene function. Analysis of cell permeability and steroid uptake performance {#Sec6} ------------------------------------------------------------ The permeability change of cell envelope was estimated by measuring the fluorescence intensity of cells labeled by fluorescein diacetate (FDA, Aladdin Reagents (Shanghai) Co., Ltd., Shanghai, China) according to previous procedures with some minor amendments \[[@CR33]\]. The same wet weight of mycobacterial cells were suspended in 4.5 mL of phosphate buffer (cell density reached 10^6^ cells/mL), mixed with 0.5 mL of FDA acetone solution (2 mg/mL) and then vibrated at 32 °C for 10 min before the detection with a Fluoroskan Ascent fluorescence spectrophotometer (Thermo Labsystems Inc., PA, USA). Maximum excitation wavelength for the detection was 485 nm, and the emission wavelength was 538 nm. The quantity of cholest-4-en-3-one (purity \> 95.0%, Shanghai TITAN Scientific Co., Ltd., China) entering mycobacterial cells per unit time was determined to check for the cell permeability change. This steroid was emulsified in Tween 80 (5% w/v) aqueous solution at 121 °C for 60 min in advance for use. The cultivated cells were inoculated into 30 mL of MYC/02 medium with 1.0 g/L cholest-4-en-3-one. After 12-h growth, 5 mL of culture solution was sampled, centrifuged at 12,000*g* for 10 min, washed with 1.0 mL of ddH~2~O for two times, and then washed with 1.0 mL of the mixture of petroleum ether and ethyl acetate (6:4, v/v) to remove the cholest-4-en-3-one from the media. The cells (50 mg, wet weight) were then suspended in 1.0 mL of the mixture of acetonitrile and ddH~2~O (7:3, v/v). Then, 0.8 g of glass beads were added in the suspension. The cells were destroyed with FastPrep-24 instrument (MP Biomedicals, CA, USA) and centrifuged at 12,000*g* for 10 min. Cholest-4-en-3-one entering cells could be released and dissolved in acetonitrile. The extracts were analyzed with a reversed-phase C18-column (250 mm × 4.6 mm) at 254 nm with the Agilent 1100 series HPLC system. The mixture of methanol and water (8:2, v/v) was used as the mobile phase. Analysis of mycolic acid methyl esters (MAMEs) {#Sec7} ---------------------------------------------- The MAMEs were extracted and analyzed as previously described \[[@CR2], [@CR17], [@CR34]\]. Briefly, 50 mg (in wet weight) of mycobacterial cells were collected at 12,000*g* for 10 min. After adding 0.5 mL of the mixture of methanol and chloroform (2:1, v/v), the homogenized mixture was incubated at 60 °C for 2 h and centrifuged at 12,000*g* for 10 min. The polar lipids including TMM and TDM were dissolved in the supernatant. Next, 500 μL of 10% tetrabutylammonium hydroxide (Sigma-Aldrich LLC., MO, USA) was added to the above defatted cells or 50 mg of whole cells and heated at 100 °C overnight. After cooling, 500 μL of ddH~2~O, 250 μL of dichloromethane, and 62.5 μL of iodomethane (Sigma-Aldrich LLC., MO, USA) were added into the mixture. Then, the diluted mixture was stirred for 30 min and centrifuged at 12,000*g* for 10 min to remove the upper layer. The lower organic layer was washed with 1.0 mL of 1 M hydrochloric acid, followed by 1.0 mL of ddH~2~O. The reaction solution was dried under a stream of nitrogen. The residue was dissolved in a mixture of toluene (0.2 mL) and acetonitrile (0.1 mL), followed by the addition of acetonitrile (0.2 mL) for 1-h incubation at 4 °C. The MAMEs were centrifuged at 12,000*g* for 10 min and then re-suspended in 200 μL of dichlormethane. The extracted mycolic acids were analyzed by silica gel TLC plates in a solvent system (chloroform: methanol, 90:10, v/v). The mean grayscale intensity of spots in the TLC plate was analyzed with Quantity One (Version 4.6.6, Bio-Rad Laboratories, CA, USA) The relative abundances of the polar mycolic acids (TMM and TDM) and MAMEs were calculated, respectively. The keto-MA spots on preparative silica gel TLC were purified for MALDI-TOF--MS (Xevo G2, Waters, Ltd., MA) analysis as described \[[@CR16]\]. Sterol bioconversion and the extraction and analysis of steroidal intermediates {#Sec8} ------------------------------------------------------------------------------- Both vegetative cells and resting cells were determined to assess the sterol conversion capability \[[@CR2], [@CR30]\]. Firstly, the vegetative cell biotransformation medium (0.5 mL) was extracted with the same volume of ethyl acetate. Then the sample containing steroidal intermediates from resting cell transformation system was extracted with ten times of volume of ethyl acetate. A gas chromatography (GC) system 7820A (Agilent Technologies, CA, USA) was used for the quantitative determination of cholesterol and phytosterols. The ethyl acetate extracts (5 μL) were injected into a DB-5 column (30 m × 0.25 mm (i.d.) × 0.25 μm film thickness, Agilent Technologies, CA, USA). The oven temperature was programmed as follows: 200 °C for 2 min, 200 °C to 280 °C within 4 min, 280 °C for 2 min, 280 °C to 305 °C within 1.5 min, and 305 °C for 10 min. Inlet and flame-ionization detector temperatures were maintained at 320 °C. Nitrogen carrier gas flow was 2 mL/min at 50 °C. The sum of three major components (*β*-sitosterol, campesterol and stigmasterol) was calculated to assess the utilization of phytosterols as previously described \[[@CR29]\]. A 1100 series high-performance liquid chromatography system (HPLC) (Agilent Technologies, CA, USA) was employed to analyze the extracts containing 9-OHAD or 4-HBC. The prepared samples were analyzed with a reversed-phase XDB-C18-column (250 mm × 4.6 mm, 30 °C) (Agilent Technologies, CA, USA) at 254 nm. The mixture of methanol and water (8:2, v/v) was used as the mobile phase. The mass concentration of 9-OHAD was calculated using the standard calibration curve constructed at the same time. The mass concentration of 4-HBC produced by the WIII and WIIIΔ*kasB* strain was calculated using the 4-HBC standard calibration curve. Results and discussion {#Sec9} ====================== Disruption of the mycolic acid synthesis genes disturbed the sterol conversion {#Sec10} ------------------------------------------------------------------------------ Mycolic acids, as the main cell wall constituent, are generally synthesized in the cytoplasm (Fig. [1](#Fig1){ref-type="fig"}a) \[[@CR17], [@CR18]\]. The interference with the nonessential gene, such as the (3R)-hydroxyacyl-ACP dehydratase *hadA* and methyl mycolic acid synthase 1 *mmaA1,* etc., involved in the synthesis of mycolic acids might reduce the tightness of the cell wall and lead to a stable change in cell permeability. For further studies, the genes involved in the synthesis of mycolic acids were preliminarily evaluated by the comparative transcriptome analysis between the wild type strain and its primary derivative 9-OHAD-producing strain (MnΔ*kstD1*) \[[@CR31]\]. We planned to screen some genes whose transcription levels were remarkably fluctuated during the accumulation of 9-OHAD. However, the transcriptional levels of most of the annotated genes showed discrete variations in the bioconversion of sterols to 9-OHAD (Fig. [1](#Fig1){ref-type="fig"}b, Additional file [1](#MOESM1){ref-type="media"}: Table S3). Next, we had to randomly select some dispensable genes and obtained the targeted deletion of the mycolic acid synthesis pathway in the final 9-OHAD-producing strain WI. Interestingly, the inactivation of most of the accessary genes resulted in a slight alteration of sterol utilization rate in all the strains except the WIΔ*kasB* strain (Fig. [1](#Fig1){ref-type="fig"}c). As expected, the deletion of the gene remarkably increased the sterol utilization by 143% at the 72-h sampling time. Early studies demonstrated that the *kasB* was a nonessential gene responsible for the extension to full-length mero-mycolic acids in *M. tuberculosis* \[[@CR16]\]. The result indicated that a meaningful permeability change might occur in the mutant strain. Functional KasB maintained the cell permeability and the balance of steroid uptake in *M. neoaurum* {#Sec11} --------------------------------------------------------------------------------------------------- The possible *kasB* genome region in *M. neoaurum* ATCC 25795 (GenBank Accession No. NZ_JMDW00000000.1) was re-confirmed by comparing the homologous regions in *Mycobacterium tuberculosis* H37Rv (GenBank Accession No. NC_000962), *Mycobacterium smegmatis* mc2 155 (GenBank Accession No. NC_008596) and *Mycobacterium neoaurum* VKM Ac-1815D (GenBank Accession No. CP006936.2). The *kasB* gene (GeneBank: NZ_JMDW01000013.1; Region: 177334...178587, 1254-bp) in *M. neoaurum* shared high sequence identity with its homologs (Additional file [2](#MOESM2){ref-type="media"}: Figure S1), indicating its conserved function in mycobacteria. In addition, the flanking genes of *kasB* also had the similar frame. These results proved that the annotation and position of the *kasB* gene was correct (Additional file [2](#MOESM2){ref-type="media"}: Figure S1; Additional file [1](#MOESM1){ref-type="media"}: Table S4). The allelic homologous recombination was employed to delete the *kasB* cassette in the wild type *M. neoaurum*. A 1171-bp upstream sequence and 1111-bp downstream sequence were amplified to construct the plasmid vector for gene knockout (Additional file [2](#MOESM2){ref-type="media"}: Figure S2). PCR and electrophoresis analysis results of the *kasB* region in genomic DNA confirmed the occurrence of allelic replacement in *M. neoaurum* (Fig. [2](#Fig2){ref-type="fig"}a).Fig. 2Effects of the deficiency of *kasB* on the cell permeability. **a** Validation of allelic replacement at the *kasB* locus in *M. neoaurum* ATCC 25795. The wild type (WT) 3260-bp was replaced by a 2282-bp fragment ligate with the upstream and downstream homologous arm. **b** Growth characteristic of the *kasB* mutant strain. The wide-type *M. neoaurum* (Mn), the *kasB*-deficient strain (MnΔ*kasB*) and the *kasB*-complemented strain (MnΔ*kasB *+ *kasB*) were cultured in MM containing 1.0 g/L cholesterol. **c** Determination of the cell permeability in the *kasB* mutant strain. The cells were stained with FDA, incubated at 32 °C for 10 min, and analyzed by a fluorescence spectrophotometer. The mutant strain MnΔ*kasB* displayed about two times penetrated FDA compared with that in its parental Mn strain after 30 min of incubation. **d** Influences of the deficiency of *kasB* on the steroid (cholest-4-en-3-one) uptake. The cholest-4-en-3-one entering the cells after 12-h growth in MM containing 1.0 g/L cholest-4-en-3-one was determined. The uptake of cholest-4-en-3-one in the strain MnΔ*kasB* showed about 2.3 times improvement than that of the wild type Mn strain In mycobacteria, *kasA* and *kasB* encode two distinct fatty acid synthase II complexes. KasA is responsible for the initial elongation of mycolic acids less than 40 carbons, whereas KasB is involved in the extension from 40 carbons to 54 carbons \[[@CR18]\]. The subsequent deletion of *kasB* in the mutant strain WI might be disadvantage to test the phenotype. In order to assess the effect of *kasB* on the cell permeability, the MnΔ*kasB* mutant strain and the complemented strain MnΔ*kasB *+ *kasB* were generated for subsequent experiments. The deletion of *kasB* led to an obvious alteration of cell growth in the presence of cholesterol and the MnΔ*kasB* strain growth was much faster than that of its parental wild type strain and the complemented strain (Fig. [2](#Fig2){ref-type="fig"}b). The acceleration in growth rate of the MnΔ*kasB* strain was similarly to the result of *mmpL3* deletion in *M. neoaurum* \[[@CR2]\]. The enhanced cell permeability might raise the supplement of steroids in the cell wall deficient strain. Subsequently, the permeability of *kasB*-deficient strain was assessed through determining the fluorescence intensity of the cells after labeling with fluorescein diacetate (FDA) (Fig. [2](#Fig2){ref-type="fig"}c). The result showed that the MnΔ*kasB* mutant strain had the more permeable cell wall than that of the wild type strain. The penetrated FDA of MnΔ*kasB* strain was about two times compared to the parental Mn strain after 30 min of incubation. This wild type property could be restored in the mutant strain upon the introduction of the complete functional *kasB* gene. To further confirm this, the analog of cholesterol, cholest-4-en-3-one was employed as a label to check for the cell permeability to steroids \[[@CR2]\]. The analysis indicated that the improved the cell wall permeability indeed resulted in about 2.3 times enhancement in the uptake of cholest-4-en-3-one in the *kasB*-deficient strain after 12 h of growth (Fig. [2](#Fig2){ref-type="fig"}d). The improvement might be interpreted as a chain effect caused by the enhanced cell permeability. These results further confirmed that the observed enhancement of sterol conversion and utilization was probably attributed to the improved cell permeability through the inactivation of *kasB* function. Deletion of *kasB* changed the composition of cell wall mycolic acids {#Sec12} --------------------------------------------------------------------- Previous studies demonstrated that *kasB* was dispensable for normal mycobacterial growth in *M. marinum* and *M. smegmatis* \[[@CR24], [@CR35]\]. The *kasB* in *M. neoaurum* was proved to play a similar role in mycobacterial growth. The mechanism for the alternation of cell permeability with respect to the *kasB* deficiency in *M. neoaurum* remains unclear. Notably, KasB is responsible for the extension of mero-mycolic acid carbon chain \[[@CR16]\]. This function indicated that the increased permeability was likely attributed to the changed KasB-responsible cell wall mycolic acid synthesis in the mutant strain. In the TLC analysis results, the polar TMM and TDM showed no obvious difference, whereas the mycolic acid methyl esters (MAMEs) displayed a slight decrease in the MnΔ*kasB* mutant strain (Fig. [3](#Fig3){ref-type="fig"}a; Additional file [2](#MOESM2){ref-type="media"}: Figure S3). The relative abundances of the α-MA, methoxy-MA and keto-MA were respectively 25.1%, 23.5%, and 51.4% in the MnΔ*kasB* strain and 23.5%, 22.6%, and 53.9% in its parental strain Mn (Fig. [3](#Fig3){ref-type="fig"}b). The decrease in keto-MA content was similar to the trend of the *kasB*-deleted *M. tuberculosis* \[[@CR16]\]. Next, the keto-MA spot was purified and analyzed by MALDI-TOF MS. The spectrogram showed a changed keto-MA in MnΔ*kasB* strain compared with that of the wild type Mn strain (Additional file [2](#MOESM2){ref-type="media"}: Figure S4). Considering the function of *kasB* in other mycobacteria, the inactivation of the *kasB* was most likely shortened the length of the keto-MA, the specific changes of MA need to be further determined.Fig. 3Effects of *kasB* on the component of cell wall mycolic acids in *M. neoaurum*. **a** The strain carrying the wild type *kasB* (Mn) or the deficient *kasB* (MnΔ*kasB*) was cultivated in the presence of 1.0 g/L phytosterols. MAMEs (α-, methoxy- and keto- forms of mycolic acids) were isolated from *M. neoaurum* cells. TLC plates were revealed with cupric sulfate (10% w/v in an 8% v/v phosphoric acid solution). **b** Relative intensity of the mycolate compared to the total mycolates was calculated. The deletion of *kasB* caused a slight disturbance of MAMEs components in MnΔ*kasB* (α-: 25.1%, methoxy-: 23.5%, and keto-: 51.4%) compared with that of the Mn strain (α-: 23.5%, methoxy-: 22.6%, and keto-: 53.9%) Loss of *kasB* led to a remarkable improvement in steroid intermediate productivity {#Sec13} ----------------------------------------------------------------------------------- To determine the effect of altered MAMEs and permeability on the production of steroidal intermediates, the transformation phenotype of the 9-OHAD-producing strain WI and WIΔ*kasB* was determined. The result showed that the growth speed of the mutant strain WIΔ*kasB* was not changed obviously under the sterol-free culture conditions (Additional file [2](#MOESM2){ref-type="media"}: Figure S5). In addition, the cell morphology of mutant strain was unaffected apparently (Fig. [4](#Fig4){ref-type="fig"}a). This phenomenon was different with the deletion of *kasB* in *M. tuberculosis* \[[@CR16]\]. These results indicated that the *kasB* was possibly not the sole functional enzyme involved in the specific elongation step of mero-MAs in *M. neoaurum* ATCC 25795. Despite the deficiency of *kasB*, the stability of cellular structure could be still maintained in *M. neoaurum*. In view of the enhanced uptake of sterols resulted from the altered cell permeability, the accumulation capability of target steroids was preliminary analyzed. The vegetative cell transformation led to a remarkably increased 9-OHAD yield in the WIΔ*kasB* strain compared to its parental strain (Fig. [4](#Fig4){ref-type="fig"}b). The deletion of *kasB* increased the target steroid by 137.7% from 0.61 to 1.45 g/L after 72-h conversion. However, the increase precipitously declined to 28% after 96-h of biotransformation.Fig. 4Enhancement of the 9-OHAD productivity in *M. neoaurum*. **a** Cell morphologies of the engineered mutant strains revealed by a scanning electron microscope. The cell morphology of the WIΔ*kasB* stain showed no obvious defects compared to that of its parental strain WI. **b** Assessment of 9-OHAD yield for the deficiency of *kasB*. Quantitative analyses of the 9-OHAD yield in the vegetative cell transformation of 5 g/L phytosterols. **c** Determination of the C19 intermediate 9-OHAD productivity in the constructed 9-OHAD-producing strain WIΔ*kasB* by a resting cell system containing 20 g/L of phytosterols. **d** Measurement of the C22 intermediate 4-HBC productivity in the engineered producer WIIIΔ*kasB* by resting cell conversion in the presence of 20 g/L phytosterols Next, a resting cell bioconversion system widely applied in the industry was used to further assess the enhancement effect of C19 steroid intermediate 9-OHAD generated by the *kasB* deletion (Fig. [4](#Fig4){ref-type="fig"}c). The highest increase was detected in WIΔ*kasB* strain after 72-h transformation with the production of 9.8 g/L, which was 48.5% higher than that of its parental WI strain (6.6 g/L). Ultimately, the WIΔ*kasB* strain yielded 10.9 g/L 9-OHAD with a molar yield of 69.5%, whereas its parental strain WI only produced 8.9 g/L with a molar yield of 56.7%. In addition, if the bioconversion time was extended by 48 h, the 9-OHAD production of WI strain would increase to about 10.3 g/L, which was still lower than that of the WIΔ*kasB* strain. In other words, the modification of *kasB* gene shortened the conversion time by more than 33%. The screened *kasB* stably remodeled the cell wall mycolic acid component, thus resulting in an increase of 22.5% in the production of C19 steroidal 9-OHAD. The enhancement effect of *kasB* deficiency had been tested in another typical C22 steroidal intermediate 4-HBC producing strain WIII \[[@CR7]\]. Similarly, an obvious improvement in the target intermediate was detected in the vegetative WIIIΔ*kasB* cell (Additional file [2](#MOESM2){ref-type="media"}: Figure S6), indicating that the strategy of disrupting the mycolic acid synthesis might be efficient for the stable evolution towards target steroidal producer. Accordingly, the assessment of resting cells showed that the 4-HBC production in the WIIIΔ*kasB* strain was increased by 34.5% from 5.8 g/L to 7.8 g/L after 96-h conversion (Fig. [4](#Fig4){ref-type="fig"}d). In addition, the 4-HBC yield was improved by 37.5% from 6.4 to 8.8 g/L after 120-h biotransformation \[[@CR2]\]. Thus, the modification of *kasB* is highly effective for the self-enhancement of steroid intermediate conversion in *M. neoaurum*. Conclusions {#Sec14} =========== This study aimed to develop a gentle and stable self-excitation strategy of steroid intermediate conversion by the disruption of cell wall components in mycobacterial cells. To understand the important role of MAs in cell permeability related to the uptake of sterol substrate, the dispensable genes of MA synthesis in *M. neoaurum* were deleted respectively. The modification of *kasB* showed a striking increase in sterol conversion rate, indicating a meaningful change in the cell wall mycolic acids. The deficiency of the screened *kasB* gene significantly changed the cell wall permeability by altering the constitution of MAMEs and shortening the length of mycolic acids in the cell wall, thus resulting in an efficient self-enhancement of steroidal intermediate conversion. Supplementary information ========================= {#Sec17} **Additional file 1: Table S1.** Plasmids used in this study. **Table S2.** Primers used in this study. **Table S3.** Identification and annotation of the mycolic acid synthesis related genes. **Table S4.** Comparisons of *kasB* region in mycobacteria. **Additional file 2: Figure S1.** Comparison of the localization of *kasB* homologous gene in mycobacteria. **Figure S2.** In-frame deletion of *kasB* in *M. neoaurum* ATCC 25795. **Figure S3.** Absolute intensity of the mycolate in *M. neoaurum*. **Figure S4.** MALDI-TOF mass spectra of the keto-MAMEs of *M. neoaurum* strains. **Figure S5.** Growth curve of the *kasB* mutant strain. **Figure S6.** Assessment of 4-HBC production for the deletion of *kasB* in the typical 4-HBC-producing strain MnΔ*kshA*Δ*hsd4A*Δ*kstD1*Δ*kstD2*Δ*kstD3* (WIII). KasB : β-Ketoacyl-acyl carrier protein synthase MA : Mycolic acids 9-OHAD : 9*α*-Hydroxy-4-androstene-3,17-dione 4-HBC : 22-Hydroxy-23,24-bisnorchol-4-ene-3-one AD : Androst-4-ene-3,17-dione BD : Boldenone MAMEs-AG-PG : Mycolyl-arabinogalactan-peptidoglycan FASII : Fatty acid synthase II FDA : Fluorescein diacetate TMM : Trehalose monomycolate TDM : Trehalose dimycolate MAMEs : Mycolic acid methyl esters LB : Luria--Bertani MM : Minimal medium HPLC : High performance liquid chromatography GC : Gas chromatography FAS-I : Fatty acid synthase I FabD : Malonyl CoA-acyl carrier protein (ACP) transacylase FabH : β-Ketoacyl-ACP synthase III MabA : β-Ketoacyl-ACP reductase HadABC : β-Hydroxyacyl-ACP dehydratase subunits A, B and C InhA : Enoyl-ACP reductase KasA : β-Ketoacyl-ACP synthase 1 PcaA : Proximal cyclopropanation of alpha-MAs enzyme MmaA1-4 : Methyl mycolic acid synthase CmaA2 : Cyclopropyl mycolic acid synthase AccD4 : Propanoyl-CoA carbon dioxide ligase AccD5 : Propionyl-CoA carboxylase FadD32 : Long-chain-fatty-acid-AMP synthetase Pks13 : Polyketide synthase **Publisher\'s Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Liang-Bin Xiong and Hao-Hao Liu contributed equally to this work Supplementary information ========================= **Supplementary information** accompanies this paper at 10.1186/s12934-020-01335-y. We sincerely thank T. Parish (Department of Infectious and Tropical Diseases, United Kingdom) for providing the plasmids, p2NIL and pGOAL19, and W. R. Jacobs Jr. (Howard Hughes Medical Institute) for providing the plasmids, pMV261 and pMV306. LBX, HHL, MZ, YJL, LS and ZYX carried out the experiments. LBX and FQW analyzed the data. LBX, YXX, FQW and DZW conceived the study and reviewed the manuscript. All authors read and approved the final manuscript. This work was supported by the National Natural Science Foundation of China (Nos. 81830052, 31370080), the construction project of Shanghai Key Laboratory of Molecular Imaging (18DZ2260400), Shanghai Municipal Education Commission (Class II Plateau Disciplinary Construction Program for Medical Technology of SUMHS, 2018-2020), the Training Subsidy Scheme of Young Teachers in Colleges and Universities of Shanghai (No. ZZJKYX19011) and the Hundred Teachers' Bank of Shanghai University of Medicine and Health Sciences. All data generated or analyzed during this study are included in this published article and its additional files. Not applicable. Not applicable. The authors declare that they have no competing interests.

Introduction ============ Expansive cervical laminoplasty began its evolution in the 1970s in Japan for the treatment of cervical spondylotic myelopathy secondary to ossification of posterior longitudinal ligament or cervical spondylosis. The goal of the procedure is to reduce the complications associated with the inherent destabilization associated with laminectomy while preserving cervical spine range of motion.[@JR02053-1] It has also been established in the literature that preoperative kyphotic deformity is a risk factor for poor surgical outcome and neurological recovery.[@JR02053-2],[@JR02053-3] We hypothesize that meticulous preservation of the interspinous ligaments may help prevent iatrogenic kyphosis. Methods ======= A retrospective review was conducted of patients who underwent laminoplasty from 2003 through January 2011 at UC-Davis Medical Center in Sacramento, Calif, USA. Patients who had simultaneous or staged anterior fusion in addition to laminoplasty were excluded. Additional exclusion criteria were inadequate preoperative or 3-month postoperative x-rays. Cervical lordosis was measured on preoperative, 3-month postoperative, and 1-year postoperative (when available) x-rays. Additional data collected include patient age, surgeon performing the procedure, and preoperative/postoperative Nurick scores. There were technical differences that were surgeon dependent and are illustrated in [Table 1](#TB02053-1){ref-type="table"}. Statistical analysis was performed. Results ======= Fifty-seven people met inclusion criteria and had presurgery and 3-month postsurgery cervical x-rays. Of the 57 patients, 32 also had postsurgery x-rays at 12 months. The average age of patients was 58.4 years (SD = 13.3 years), and the average presurgery Nurick score was 1.7 (SD = 1.5). There were 3 surgical techniques used: surgeon 1 (n = 21), surgeon 2 (n = 15), and surgeon 3 (n = 21) ([Table 1](#TB02053-1){ref-type="table"}). The average cervical lordosis measured was the following: presurgery, 8.4 (SD = 11.9); at 3 months, 4.0 (SD = 13.2); and at 12 months, 4.8 (SD = 15.2). The average change in cervical lordosis measured presurgery to 3 months postsurgery was −4.4 (SD = 9.7) and the average additional change in cervical lordosis measured from 3 months to 12 months postsurgery was −0.38 (SD = 5.8) ([Fig. 1](#FI02053-1){ref-type="fig"}). ###### Surgical technique by surgeon. Surgeon Fixation Interspinous ligaments --------- --------------------- ------------------------ 1 Plate and allograft Variably preserved 2 Plate and allograft Meticulously preserved 3 Suture anchor Not preserved ###### Estimates of cervical lordosis measures and the variation in change among surgeons. Parameter Estimate Confidence interval *P* value Total *P* value -------------------------------------------------------------- ---------- --------------------- ----------- ----------------- Average change in lordosis, 3 mo after surgery for surgeon 1 −3.71 −7.5, 0.1 .06 \< .01 Average change in lordosis, 3 mo after surgery for surgeon 2 −1.33 −5.9, 3.2 .56 Average change in lordosis, 3 mo after surgery for surgeon 3 −7.19 −11.0, −3.4 \< .01 {#FI02053-1} Conclusions =========== Preventing kyphosis after laminoplasty allows decompression of the spinal cord by allowing it to float posteriorly due to the enlarged spinal canal. Decreased surgical outcomes have been observed when cervical kyphosis is more than 13° without cord signal change and more than 5° when cord signal change is present.[@JR02053-3] Our study demonstrates that the use of a plate for stabilization and meticulous preservation of the interspinous ligaments limits postoperative kyphosis. Additionally, the best predictor of postoperative sagittal alignment is preoperative sagittal alignment and the change in alignment is independent of the preoperative alignment. This Study was IRB approved. Synthes ARCH Fixation System is FDA approved.

Background {#Sec1} ========== Genes that are not interrupted by introns are called intronless genes or single-exon genes. Depending on their structure, genes in eukaryotes fall into two categories: intronless genes and intron-containing genes. Intronless genes can serve as beacons in analyses of gene function and evolution. For example, intronless genes as a model, compared with intron-containing homologs, can enable an inverse approach to studying the considerable roles of introns, which are only found in eukaryotes \[[@CR1]\]. Furthermore, studies on intronless genes should help to unravel some evolutionary issues including: (i) the major factors that have contributed to the emergence of intronless genes (gene duplications, inheritance from ancient prokaryotes, retroposition or other mechanisms); (ii) the evolutionary significance of retroposition (retrogenes are considered to be intronless); and (iii) the biological origins of introns (i.e., which hypothesis is more correct: the introns-early hypothesis or introns-late hypothesis?) \[[@CR2]\]. Therefore, related studies and the construction of databases for intronless genes have received great attention in recent years. IGD \[[@CR3]\] and PIGD \[[@CR4]\] are two existing intronless gene databases that contain information about intronless genes in human and Poaceae, respectively. The identification methods and knowledge of intronless genes in these two databases are worthy of reference. However, limitation still persists as they still are not the centralized platforms for intronless genes. Furthermore, IGD is derived from GenBank, which does not contain all the sequences available from genome projects; thus, it does not provide representative intronless gene sets. Compared with IGD, PIGD has an improved methodology, but contains less evolutionary information in its gene annotations. To build a centralized platform, we developed IGDD---a comprehensive and integrated database for intronless genes that covers five dicots, including *Arabidopsis thaliana* (Arabidopsis), *Carica papaya* (papaya), *Populus trichocarpa* (poplar), *Salix suchowensis* (willow) and *Vitis vinifera* (grape). These plants were chosen as representatives not only because of their burgeoning sets of genome sequencing, but also because of their obvious patterns of genome duplication \[[@CR5]\]. Specifically, the *Arabidopsis thaliana* genome has undergone two whole genome duplication events (α and β) within the crucifer lineage and one more ancient genome triplication event (γ) shared with most dicots \[[@CR6]\], *Carica papaya* and *Vitis vinifera* have each experienced only the γ triplication and no subsequent polyploidies; whereas *Populus trichocarpa* and *Salix suchowensis* have undergone a salicoid-specific duplication (ρ) besides the γ triplication \[[@CR7]\]. The large number of paralogous genes present in these species offers a unique opportunity to help us elucidate the evolutionary consequences of intronless genes. IGDD not only contains comprehensive knowledge and links to authoritative external datasets, but also includes the important features within and among species. In addition to basic information on intronless genes, the annotations in IGDD include predicted protein domains, KEGG pathways, GO items, functional descriptions and subcellular localizations. Moreover, IGDD also lists paralogs, orthologs, syntenic blocks, and expression patterns across different developmental stages and tissues. By integrating massive functional and evolutionary information and developing valuable tools, we aim to provide a useful resource and versatile platform that will benefit the related research community. Construction and content {#Sec2} ======================== Data source {#Sec3} ----------- The genome sequences of Arabidopsis, papaya, poplar, and grape were acquired from the publicly available database Phytozome \[[@CR8]--[@CR11]\]. In our database, the latest versions of data in use are TAIR10 for *Arabidopsis*, ASGPBv0.4 for papaya, v3.0 for poplar, and Genoscope.12X for grape separately. The genome of *Salix suchowensis* is a most updated in-house database developed by our research group \[[@CR7]\]. Identification of intronless genes {#Sec4} ---------------------------------- Intronless genes were identified by Perl scripts. First, the scripts extracted the information for mRNAs that were not interrupted by "introns" from a GFF file, and genes containing only one continuous exon were considered putative intronless genes. Subsequently, the longest transcript was retained if two or more transcripts represented a gene that met the criteria in the first step. Considering their biological functions, only proteins encoded by intronless genes with lengths ≥ 30aa were selected. Annotation of intronless genes {#Sec5} ------------------------------ To provide informative clues for further functional analysis, we systematically annotated each intronless gene. Initially, we obtained the standard gene information from Phytozome \[[@CR12]\], including PFAM \[[@CR13]\], GO annotation \[[@CR14]\] and PANTHER data \[[@CR15]\]. Functional description was then added using the InterproScan program (Version 5.44.0) \[[@CR16]\]. Pathway annotations from the KEGG database \[[@CR17]\] were also included in IGDD. In addition, we added subcellular localization prediction information from WoLF PSORT \[[@CR18]\]. Putative homolog annotation {#Sec6} --------------------------- To predict paralogs of the intronless genes, the method described by Blanc and Wolfe was used \[[@CR19]\]. We performed all-against-all nucleotide sequence similarity searches among the transcribed sequences in each genome using the BLASTN software \[[@CR20]\]. Sequences with strict cutoffs of alignment length ≥ 300 bp and identity ≥ 40 % were defined as paralogs. We also downloaded the paralogs of intronless genes annotated in Ensembl using Biomart \[[@CR21]\]. The results obtained from both methods were included for accurate prediction of paralogs. Orthologs of intronless genes across the five dicots were identified by OrthoMCL with the default parameters \[[@CR22]\]. Syntenic analysis {#Sec7} ----------------- To detect intronless genes that had arisen from whole-genome duplication (WGD), syntenic blocks within and between species were identified using BLAST, OrthoMCL and the MCscanX software \[[@CR20], [@CR22], [@CR23]\]. We then retrieved the syntenic blocks containing intronless genes. Finally, to show an overview of all blocks, the intra-genome and inter-genome syntenic relationships of the intronless genes were visualized with Circos \[[@CR24]\]. The nonsynonymous (Ka) and synonymous (Ks) substitution rates of gene pairs in each block were determined by KaKs_Calculator 2.0 \[[@CR25]\]. Expression patterns of intronless genes {#Sec8} --------------------------------------- An expression-based resource can provide an important bridge between genotype and phenotype through transcript profiles; therefore, we integrated a wide variety of expression data to explore the spatial and temporal expression patterns of intronless genes during development. Comprehensive microarray data for *A. thaliana* was acquired from TAIR across 63 tissues \[[@CR26]\], for *P.trichocarpa* from NCBI-GEO (GSE13990) covering nine tissues \[[@CR27]\], and for *V.vinifera* from NCBI-GEO (GSE36128), which included 54 samples representing green and woody tissues \[[@CR28]\]. Expression data for *S. suchowensis* was obtained using RNA-Seq from five tissues \[[@CR7]\]. The expression data of the intronless genes was imported into R and Bioconductor, and then the pheatmap package was used to make heatmaps. Utility and discussion {#Sec9} ====================== IGDD collects sequence information from multiple resources to enrich the data for intronless genes. At present, IGDD contains 28,016 intronless genes from five dicots (Table [1](#Tab1){ref-type="table"}). In IGDD, different categories of datasets are classified and compared within and among species with user-friendly graphics. Additionally, analytical tools such as BLAST are embedded in IGDD to help predict the putative orthologous groups of the intronless genes. Thus, IGDD is a solid web platform for searching, browsing, visualizing and downloading intronless genes (Fig. [1a](#Fig1){ref-type="fig"}). Below, we discuss the four main functional units in IGDD: (1) the comprehensive individual gene information, (2) the integration of data sources, (3) the BLAST sequence search engine, and (4) the interactive platform.Table 1List of five dicots currently served by IGDDSpeciesRelease versionIdentified Intronless GenesPercentage of Intronless Genes*Arabidopsis thaliana*TAIR 10573320.9 %*Carica papaya*ASGPB V0.4779128.1 %*Populus trichocarpa*V3.0720917.4 %*Salix suchowensis*V 1.0565721.3 %*Vitis vinifera*Genoscope.12X16266.2 %Fig. 1An overview of the IGDD website. **a** The home page. **b** Comprehensive individual gene information (e.g., AT2G21910). **c** The browse page. **d** The comparison page. **e** The BLAST pageComprehensive individual gene informationIGDD provides detailed annotations for every putative intronless gene (Fig. [1b](#Fig1){ref-type="fig"}). Users can access the webpage in multiple ways, such as by clicking "Browse" on the main navigation bar or importing a gene name into the "Search" section directly. The annotation information for individual genes includes: (i) basic information, (ii) protein sequence features, (iii) homolog groups including paralogs and orthologs, (iv) syntenic blocks, and (v) expression profiles. In detail, basic information consists of the gene identifier, location, strand and annotations for the corresponding coding protein (such as length, isoelectric point (PI), and molecular weight (Mw)). The protein sequence features display protein identities (IDs) for PFAM, GO, PANTHER and KEGG, and functional descriptions. These IDs are hyperlinked to external databases to access more details. Additionally, subcellular localization prediction is shown on the page because it gives an important clue to the protein's role(s). Gene duplication followed by rapid sequence divergence between paralogous pairs is considered to be the major mechanism for the emergence of new genes \[[@CR29]\]. To determine the contribution of gene duplication to the amplification of intronless genes, we provide information on paralogous pairs among intronless genes, and between intronless genes and intron-containing genes. To explore cross-species evolutionary study, ortholog groups between intronless genes were identified. Among the syntenic blocks shown on the webpage, the corresponding intronless gene is highlighted in blue font to facilitate searching. The Ka and Ks values of paralogous pairs are also provided to determine their evolutionary distances. Knowing the expression patterns of intronless genes in different developmental stages and tissues is essential to illustrate whether they have corresponding biological functions. In IGDD, we extracted microarray and RNA-seq data to assess the temporal and spatial expression of intronless genes. When an intronless gene is found to be expressed, the expression data is visualized by a line diagram that allows users to view the expression changes across different tissues and developmental stages.Integration of data sourcesBased on the collection of available data as aforementioned, individual genes were further analyzed and compared within and among species. We dedicated two main functions of the interface to this: the "Browse" and "Comparison" sections. Specifically, the comparative data for intronless genes within species was analyzed from a variety of perspectives, and visualized on the "Browse" webpage (Fig. [1c](#Fig1){ref-type="fig"}). From the interface, basic information on the classification under different attributes is displayed, such as the distribution of intronless genes on chromosomes, subcellular localization, distribution of protein length, PI and Mw. In particular, users can browse and download detailed data by clicking the corresponding section of the figure to view the differences. Apart from basic information, we also collected functional annotations through GO associations, PFAM domain information, and KEGG data. The "Browse" page shows the proportion of genes and gene product attributes associated with cellular components, biological processes, molecular functions, gene families, and pathways. To study genome organization and evolution, colinearity information within species can be applied to analyze segmental and WGD events. The Circos software \[[@CR24]\] was employed to enable browsing of the syntenic relationships between intronless genes and other genes using different colored curves. In addition, to better understand whether intronless genes are associated with plant phenotypes, the gene expression in different tissues is clearly visible from the heat map. The heat map provides not only an overview of the global gene expression trends, but also conclusive evidence that these intronless genes are truly transcribed to mRNA.The "Comparison" section compares gene characteristics across species and provides detailed information by clicking on the image (Fig. [1d](#Fig1){ref-type="fig"}). We found that proportions of intronless genes against the total number of genes differed significantly across the five investigated species, with 20.9, 28.1, 17.4, 21.3, and 6.2 % in Arabidopsis, papaya, poplar, willow, and grape, respectively (Chi-square test, *P* \< 0.05). The average protein sizes were 279, 182, 229, 333, and 178aa in Arabidopsis, papaya, poplar, willow, and grape, respectively. In pairwise comparisons, the differences in the average protein sizes were remarkable (μ-test; *P* \< 0.05) except between papaya and grape. Conversely, we noticed that intronless genes had similar characteristics across the five dicots. For the protein features, the peak Mw value ranged from 8 to 13 kDa, and the PI distribution showed two main peaks at 6 and 9.The BLAST sequence search engineThe BLAST (Basic Local Alignment Search Tool) interface provides a flexible way to search for homologous genes of every intronless gene stored in IGDD. Users can submit query sequence(s) and adjust the BLAST parameters including database and search program. To make viewing the result page easier, we set the following two parameters: e-value cutoff of 1e-5 and retaining the top 100 hits based on e-values.The interactive platformTo expand the annotation scope of IGDD, reliable functional and evolutionary information was retrieved from the well-known databases PFAM, PANTHER, KEGG, GO and Expasy. Once plant genomics websites release the latest versions of the four dicots genomes, the datasets in IGDD would be updated synchronously. Compared with comparative genomics websites, IGDD focuses on the relationship among intronless genes, which is helpful to explore functional and evolutionary features on intronless genes across dicots. All of the information in IGDD is available for researchers at an FTP (File Transfer Protocol) site. In addition, IGDD encourages users to submit fully annotated intronless genes from other dicots to the database. When the quality of the submitted information meets our requirements, the IGDD curator will import the data into the database. Overall, the interactive platforms, including links to external databases, and download and submission components, make IGDD a comprehensive and systematic platform for the research community. Conclusions {#Sec10} =========== Our main goal with IGDD is to construct a comprehensive platform for the exploration of intronless genes in dicots. IGDD integrates various types of data and links with multiple external databases to provide rich annotation information, which can be browsed and retrieved through user-friendly web interfaces. Biological tools such as BLAST and a comparison platform are also provided to facilitate investigations into the functional and evolutionary consequences of intronless genes. In future, the authors will regularly review authoritative databases for new dicot genomes, and expand the content of IGDD. Moreover, we intend to add new functions and integrate multiple data sources to enhance the IGDD database. For example, expression profiles will focus not only on different tissues and developmental stages, but also experimental treatments, such as biotic and abiotic stress. Our ultimate goal is to construct a co-expression network that provides informative clues to the regulation of intronless gene expression. In conclusion, we hope that IGDD will serve as a useful resource and fundamental platform for studying intronless genes, especially their occurrence and evolution. Availability and requirements {#Sec11} ============================= Database: IGDD Database homepage: <http://bio.njfu.edu.cn/igdd> Operating system(s): Unix Programming language: C+, JavaScript, Perl Other requirements: MySQL, Apache, PHP These data are freely available without restrictions for use by academics. Inquiries concerning the database may be directed to IGDD\@163.com. Abbreviations {#Sec12} ============= IGDD, intronless genes database in dicots WGD, whole-genome duplication PI, isoelectric point Mw, molecular weight BLAST, basic local alignment search tool FTP, file transfer protocol Special thanks go for the anonymous reviewers for their enlightening comments in reformulating this paper. Funding {#FPar1} ======= This work was supported by the National Basic Research Project (2012CB114505), the Natural Science Foundation of China (31570662), and Anhui provincial Natural Science Foundation (1608085QC65). It was also enabled by China postdoctoral science foundation (2015 M581806), the Innovative Research Team of the Educational Department of China and the PAPD (Priority Academic Program Development) program at Nanjing Forestry University. Availability of data and materials {#FPar2} ================================== The database is available at <http://bio.njfu.edu.cn/igdd/>, and no restrictions to its use by non-academics. Authors' contribution {#FPar3} ===================== HY performed integration, constructed the database platform, and wrote the manuscript. XD, KF and QM helped with the design of database platform and update of the database, and provided scientific suggestions and criticisms for improving the manuscript and website. TY participated in the design, helped write the manuscript and supervised the whole project. All authors read and approved the final manuscript. Competing interests {#FPar4} =================== The authors declare that they have no competing interests. Consent for publication {#FPar5} ======================= Not applicable. Ethics approval and consent to participate {#FPar6} ========================================== Not applicable.

Introduction ============ In recent years, morbidity and mortality of nosocomial infections have increased, at least partly due to the increased frequency of antibiotic resistance in both Gram-positive and Gram-negative organisms. In the past, Gram-negative aerobes were the most frequently reported pathogens, but *Staphylococcus aureus* has been increasing in frequency, particularly methicillin-resistant *S. aureus* (MRSA) in intensive care units.[@b1-idr-10-049] Over 70% of *S. aureus* isolates are resistant to methicillin in the Middle East and with \>85% of *S. aureus* pneumonia cases being MRSA pneumonia in the Arabian peninsula.[@b2-idr-10-049] In Saudi Arabia, 76% of health-care workers have tested positive for MRSA colonization despite being asymptomatic.[@b3-idr-10-049] On a separate note, a systematic review on the epidemiology of *S. aureus* in the Middle East indicates high incidence of community-acquired MRSA carried by healthy individuals, with positive MRSA samples obtained from random wound, throat, and nasal specimens.[@b4-idr-10-049] Glycopeptide antibiotics, vancomycin and teicoplanin, have been considered drugs of choice in treatment for infections due to suspected or documented MRSA.[@b5-idr-10-049]--[@b8-idr-10-049] Burgeoning glycopeptide prescribing is believed to have led to the emergence of vancomycin-resistant enterococci and, more recently, *S. aureus* with reduced glycopeptide susceptibility. Treatment resistance of clinical MRSA has also been shown in this region against both β-lactam and non-β-lactam antibiotics (erythromycin, clindamycin, ciprofloxacin, and tetracycline), chloramphenicol, gentamicin, and sulfamethoxazole--trimethoprim.[@b4-idr-10-049] Even though there is a recent debate surrounding the use of vancomycin,[@b9-idr-10-049]--[@b11-idr-10-049] it is still a common antibiotic for the treatment of nosocomial pneumonia and is recommended in the most recent guideline endorsed by the American Thoracic Society and the Infectious Disease Society of America.[@b1-idr-10-049] Despite guidelines, real-world practice patterns are known to vary substantially from those recommended. Furthermore, little is known in low- and middle-income countries of the Middle East regarding practice patterns and resource associated with MRSA pneumonia. The objectives of this study were to describe the microbiologic profile of MRSA pneumonia in Lebanon and Saudi Arabia and assess the treatment received by these patients, in terms of drug selection, dosing, timing, and sensitivity of the infection pathogen, and to document hospital resource utilization and clinical outcomes among patients with this infection. Methods ======= Study design ------------ This study is a retrospective, observational medical chart review that took place at a total of five study sites in Saudi Arabia (two sites) and Lebanon (three sites). Per the study protocol, the study investigator (local physician responsible for data collection) assessed the need for ethics/IRB application at each site, IRB waiver or approval was completed at all sites based on local requirements. Specifically, approval was obtained from National Guard Health Affairs, King Abdalaziz Medical City- Jeddah and Riyadh, Kingdom of Saudi Arabia; Ain Wazein Hospital IRB; and Makassed General Hospital, Riad El-Solh Beirut, Lebanon. Inclusion and exclusion criteria -------------------------------- In order to be included, patients had to have suspected or culture-proven MRSA pneumonia with appropriate respiratory symptoms. In the absence of culture results, suspected cases needed to have at least two of the following symptoms for inclusion: fever (body temperature ≥38.5°C), hypothermia (body temperature ≤35.5°C), respiratory rate (\>30 breaths/min), systolic hypotension (\<90 mmHg), heart rate (\>120 beats/min), elevated peripheral white blood cell count (\>10,000/mm^3^), leukopenia with total white blood cell count (\<4,500 cells/mm^3^), and elevated inflammatory markers (eg, procalcitonin or C-reactive protein). Patients were excluded if they had inadequate data on the treatments and outcomes of interest or hospital resource utilization for the study, as judged by the investigators. Data collection --------------- Data were collected from patient medical records by hospital-based infectious disease specialists, medical microbiologists, internal medicine specialists, surgeons, and intensivists. All data collection instruments were translated into local language and, prior to roll out, pilot tested to ensure relevance and clarity of data points to be collected. Extracted chart data included demographics and comorbid conditions; antibiotic treatment patterns, including initial and subsequent inpatient antibiotic regimens prescribed and antibiotic therapy prescribed at discharge; health-care resource utilization, including diagnostic procedures, laboratory tests, and length of hospital stay; and clinical outcomes, including clinical response at discharge and rehospitalization. Pneumonia was classified into one of the four categories by the abstracting physician as follows: Community-acquired pneumonia: Symptom onset prior to hospital admission but with no known patient history of 1) another hospital visit, 2) visit to another health-care institution, or 3) residence in a nursing home within 90 days prior to the present hospital admissionHealth-care-associated pneumonia (HCAP): Symptom onset during 1) another hospital visit, 2) visit to another health-care institution, or 3) residence in a nursing home within 90 days prior to the present hospital admissionNosocomial pneumonia: Symptom onset at least 48 hours after hospital admissionUnknown Antibiotics received during hospitalization were categorized as MRSA active and not MRSA active. MRSA-active antibiotics as defined by guidelines included vancomycin (intravenous only), teicoplanin (intravenous only), clindamycin, doxycycline, trimethoprim/sulfamethoxazole, linezolid, fusidic acid, rifampin, and tigecycline.[@b12-idr-10-049] Antibiotics categorized as not MRSA active included beta-lactams (by definition), and in the absence of institution-specific antibiograms, fluoroquinolones and carbapenems were only categorized as MRSA active if culture results indicated sensitivity to the respective antibiotic. Statistical analysis -------------------- Unless otherwise listed, binary and categorical end points were summarized in terms of percentages in each category, and continuous variables were presented using number of observations (when less than the entire sample), arithmetic mean, and standard deviation (SD). Results ======= Demographic and clinical characteristics ---------------------------------------- Chart-level data were collected for 93 patients with MRSA pneumonia, 50 in Saudi Arabia and 43 in Lebanon. Of the 93 patients in the sample, 20% of the MRSA infections were community acquired, 32% health-care associated, 35% nosocomial, and 12% of unknown origin. Underlying chronic pulmonary disease was present in 28% of patients ([Table 1](#t1-idr-10-049){ref-type="table"}). However, the most common comorbidities among these patients were diabetes (39%) and congestive heart failure (30%). Antibiotic treatment patterns ----------------------------- Ninety of the 93 patients in the sample (97%) had at least one culture taken, and 82 of these (91%) had at least one culture positive for MRSA. As would be expected, only one-third of the isolates tested were sensitive to ciprofloxacin and only 15% were sensitive to beta-lactams. Patients most frequently had positive cultures from pulmonary (87%) and blood (27%) samples; nasal sites were also culture positive in 16% of patients. [Table 2](#t2-idr-10-049){ref-type="table"} shows the susceptibility profile for MRSA isolates from patients with pneumonia. All isolates tested were sensitive to vancomycin (n=80) and teicoplanin (n=32). Only ten isolates were tested for linezolid sensitivity, and all were found to be sensitive. Only one-third of the isolates tested were sensitive to ciprofloxacin. Drug selection for MRSA pneumonia, expressed as the percentage of patients receiving each antibiotic (with potential for multiple antibiotics per line of therapy), is summarized in [Figure 1](#f1-idr-10-049){ref-type="fig"}. Overall, 79 patients (85%) received at least one antibiotic considered to be MRSA active. The 93 MRSA pneumonia patients received a total of 140 antibiotic orders designated by study investigators to be first-line therapy (as some regimens consisted of multiple antibiotics). Of these, 28% were beta-lactams (thus representing inactive therapy), and the most frequently prescribed MRSA-active antibiotic was vancomycin (22% of orders). Among the 48 patients (52%) who were prescribed a total of 66 second-line antibiotic orders, vancomycin remained the most commonly prescribed antibiotic (39% of orders), followed by teicoplanin (12%) and linezolid (9%). Of the eleven third-line drugs received by eight patients, linezolid was the most commonly used (36% of orders), followed by vancomycin (27%) and ciprofloxacin (18%). Other health-care resource utilization -------------------------------------- Overall, the mean total length of stay (LOS) was 32.4 days, with the majority (20.4 days) spent in general wards ([Figure 2](#f2-idr-10-049){ref-type="fig"}). Thirty-six patients (39%) required mechanical ventilation, for a mean ± SD of duration of 18.7±24.4 days per patient. Four patients (4%) required hemodialysis, for a mean of 9.8 ± 11.9 days per patient. Of these patients, two patients were reported to have moderate-to-severe renal disease on admission, but whether these patients were receiving dialysis prior to admission is unknown. Other interventions required among patients with MRSA pneumonia included peripheral catheters (52%), central venous catheters (52%), and peripherally inserted central catheters (6%). Clinical outcomes ----------------- Over one-fourth of the patients were considered treatment failures at the time of discharge ([Table 3](#t3-idr-10-049){ref-type="table"}). The overall in-hospital mortality rate was 30%, with nearly one-third of deaths being MRSA related. Of the 28 patients who died, treatment response at discharge was failure in 24 (86%) cases, improved in one (3.5%), cure in two (7%), and indeterminate in one (3.5%). Relapse of MRSA pneumonia (3%) and rehospitalization for MRSA pneumonia (2%) were rare; however, all-cause rehospitalization occurred in 20% of patients. Discussion ========== This real-world burden and treatment patterns study of MRSA pneumonia in the Middle East indicates a resource-intensive disease with a high LOS and mortality, consistent with other studies in high-income countries. Inappropriate/inactive therapy for MRSA nosocomial pneumonia was prescribed more than a quarter of the time over the entire course of therapy, and in first-line treatment was prescribed 42% of the time. The relatively high mortality rate in this study may have been influenced by the high proportion receiving suboptimal therapy as first-line treatment. In a multinational observational study in Asia and a randomized clinical trial in Canada, the frequency of prescribing inadequate initial antibiotic therapy in pneumonia may be \>50%, and this significantly increases mortality rates.[@b13-idr-10-049],[@b14-idr-10-049] MRSA-targeted antibiotics were more frequently prescribed in second-line (vancomycin 54%) and third-line (linezolid 51%) treatments. Potential reasons for the administration of inappropriate antibiotics may be due to lack of physician knowledge, delay in receiving culture results, or availability of medications that cover MRSA. While the exact reason for prescribing inappropriate antibiotics cannot be determined from this study, a body of literature has focused on inadequate initial therapy as a driver of added morbidity and mortality.[@b14-idr-10-049],[@b15-idr-10-049] Administration of inappropriate empiric antibiotic therapy can increase mortality in patients with hospital-acquired pneumonia (defined in this study as occurring within 48 hours of admission) and ventilator-associated pneumonia (VAP, defined as occurring within 48 hours of endotracheal intubation). Results from a prospective surveillance study conducted through the Asian Network for Surveillance of Resistant Pathogens (ANSORP) found that of the 15.8% of patients with *S. aureus* hospital-acquired pneumonia or VAP, 50.4% received discordant therapy.[@b13-idr-10-049] Patients receiving discordant therapy had higher all-cause (44% vs 35%) and pneumonia-related (32% vs 21%) mortality than patients who received concordant therapy. Multivariate analysis results showed that discordant therapy was a risk factor for pneumonia-related mortality (OR, 1.542; 95% CI, 1.127--2.110; *P*=0.007). Administration of inappropriate empiric antibiotic therapy can also increase mortality in patients with HCAP, defined as pneumonia in a patient admitted from a nursing home or other long-term care setting, who was hospitalized within the last 12 months, receiving dialysis or infusion therapy requiring regular visits to a hospital-based clinic, or who is immunocompromised. In a US-based study, patients with HCAP compared to community acquired pneumonia had higher mortality (24.6% vs 9.1%, *P*\<0.001) and were more likely to receive inappropriate antimicrobial treatment (28.3% vs 13.0%, *P*\<0.001).[@b16-idr-10-049] Mortality was higher for patients who received inappropriate compared to appropriate antibiotic therapy (32.2% vs 15.7%, *P*\<0.001). Inappropriate initial antimicrobial therapy was identified as an independent risk factor for hospital mortality (OR, 2.19; 95% CI, 1.27--3.78; *P*\<0.005). Results from separate studies conducted at the same institution found that treatment escalation did not decrease mortality in patients with HCAP treated with inappropriate empiric antibiotic therapy[@b17-idr-10-049] and that initial therapy for HCAP should include an antibiotic that targets MRSA.[@b16-idr-10-049] The resource use findings were quite disparate between countries in this study where the mean LOS was 30 days longer in Saudi Arabia than in Lebanon. It is unclear whether this difference results from an underlying disparity in the severity of infection between groups, or a difference in health-care infrastructure. A surveillance study in Saudi Arabia looking at 2,800 patients admitted to the medical-surgical intensive care unit suggests that long LOS is not uncommon for patients that are severely ill. They found that patients with VAP had a longer mean LOS of 85 days versus 61 days in those without VAP.[@b18-idr-10-049] This population had primarily Gram-negative pneumonias, but 20% were attributed to Gram-positive infection. The study is naturally limited by its retrospective design and reliance on data that are documented in the medical chart. Since inclusion criteria required that the patients were diagnosed with MRSA pneumonia, we do not have details on other patients that may have been colonized and not developed active infection. However, the information gathered from the charts represents a snapshot of the real-world treatment patterns and supplements the limited data we have in this indication and region of the world. Conclusion ========== MRSA pneumonia in Saudi Arabia and Lebanon may be treated with antibiotics that are inactive against MRSA in a majority of patients for first-line therapy, resulting in an added resource use burden and mortality. The information gathered in this Middle East population provides important real-world perspectives on the current treatment patterns and opportunities to improve care. Future studies should more closely examine the appropriateness of initial therapy in MRSA pneumonia patients. The authors thank Monica Katyal for her assistance with data collection and analysis. **Author contributions** Adel F Alothman, Abdulhakeem O Althaqafi, Madonna J Matar, Rima Moghnieh, Nirvana Raghubir, Seema Haider, Caitlyn T Solem, and Jennifer M Stephens contributed to study design. Cynthia Macahilig, Adel F Alothman, Abdulhakeem O Althaqafi, Madonna J Matar, Rima Moghnieh, Thamer H Alenazi, and Fayssal Farahat were involved in data acquisition. Caitlyn T Solem and Shelby Corman undertook data analysis. All authors contributed to data interpretation, manuscript drafting, and approved the final manuscript. **Disclosure** This study was sponsored by Pfizer. Shelby Corman, Jennifer M Stephens, and Caitlyn T Solem are employees of Pharmerit International, who were paid consultants to Pfizer in connection with the development of this manuscript and study design, management, and statistical analysis for the study. Cynthia Macahilig is an employee of Medical Data Analytics, a subcontractor to Pharmerit International, who was a paid consultant to Pfizer in the study design, management, and data collection for the study. Nirvana Raghubir and Seema Haider are employees and shareholders of Pfizer. Adel F Alothman received honoraria for several presentations from Pfizer, MSD, and Al Hikma; received travel support from Pfizer, MSD, Gilead, and Al Hikma to attend symposia; and received honoraria for patient data collection related to this study from Pfizer. Abdulhakeem O Althaqafi received a research grant (RR13/248/J) sponsored by Pfizer and received honoraria for patient data collection related to this study from Pfizer. Fayssal Farahat received honoraria for patient data collection related to this study from Pfizer. Madonna J Matar received travel support for attending meetings and received honoraria for patient data collection related to this study from Pfizer. Rima Moghnieh received honoraria for patient data collection related to this study from Pfizer and sponsorship for attending medical meetings from Pfizer and MSD. Thamer H Alenazi received travel support for attending medical meetings from MSD and Pfizer. The authors report no other conflicts of interest in this work. {#f1-idr-10-049} {#f2-idr-10-049} ###### Patient profile -- MRSA pneumonia -------------------------------------------------------------------------------------------------------------------------------------------------- Overall,\ Saudi Arabia,\ Lebanon,\ N=93 N=50 N=43 --------------------------------------------------------------------------------------------------------- ----------- ---------------- ----------- Male, % 60 64 56 Age at hospital admission, mean ± SD 56.0±26.8 56.1±26.0 55.9±28.0 Weight at hospital admission, kg Patients with information, n 67 38 29 Mean ± SD 63.5±25.4 61.9±24.1 65.6±27.2 Body mass index, mg/m^2^ Patients with information, n 44 29 15 Mean ± SD 26.0±5.8 25.8±6.3 26.4±4.8 Number of reported comorbidities, mean ± SD 2.5±2.0 2.5±2.0 2.4±1.9 Diabetes, % 39 38 40 Congestive heart failure, % 30 26 35 Coronary artery disease, % 29 22 37 Chronic pulmonary disease, % 28 20 37 Cerebrovascular disease, % 18 22 14 Peripheral vascular disease, % 9 10 7 Dementia, % 8 12 2 Moderate-to-severe renal disease, % 8 4 12 MRSA pneumonia symptoms, % Fever 65 52 79 Hypothermia 12 16 7 Respiratory rate \>30 breaths/min 47 66 26 Systolic hypotension 29 28 30 Heart rate \>120 beats/min 46 54 37 Elevated peripheral WBC \>10,000/mm^3^ 76 74 79 Leukopenia with total WBC \<4,500 cells/mm^3^ 6 2 12 Elevated appropriate inflammatory markers (eg, PCT or CRP) 59 34 88 Cultures taken to confirm MRSA, % 97 100 93 Any positive MRSA culture, %[a](#tfn1-idr-10-049){ref-type="table-fn"} 91 100 80 1 positive culture 57 66 45 2 positive cultures 19 12 28 3+ positive cultures 16 22 8 Site of culture, %[a](#tfn1-idr-10-049){ref-type="table-fn"},[b](#tfn2-idr-10-049){ref-type="table-fn"} Pulmonary 79 90 65 Blood 24 18 33 Nasal 14 20 8 Other (pus, urine, catheter tip) 5 8 3 Timing of MRSA infection,% Community-acquired pneumonia 20 14 28 Health-care-associated pneumonia 32 30 35 Nosocomial pneumonia 35 44 26 Unknown 12 12 12 -------------------------------------------------------------------------------------------------------------------------------------------------- **Notes:** Denominator is patients with a culture taken to confirm MRSA. Patients may have had MRSA confirmed by more than one type of culture. Groups not mutually exclusive. **Abbreviations:** MRSA, methicillin-resistant *Staphylococcus aureus*; SD, standard deviation; WBC, white blood cell; PCT, procalcitonin; CRP, C-reactive protein. ###### MRSA pneumonia susceptibility profile Antibiotics N tested \% sensitive ------------------------------- ---------- -------------- Vancomycin 80 100 Teicoplanin 32 100 Linezolid 10 100 Rifampin 29 76 Erythromycin 12 75 Gentamicin 8 75 Fusidic acid 9 67 Moxifloxacin 3 67 Levofloxacin 16 63 Trimethoprim/sulfamethoxazole 53 66 Clindamycin 73 53 Tetracycline 4 50 Tigecycline 4 50 Ciprofloxacin 18 33 Beta-lactam antibiotic 54 15 **Abbreviation:** MRSA, methicillin-resistant *Staphylococcus aureus*. ###### MRSA pneumonia clinical outcomes ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Outcome Overall,\ Saudi Arabia,\ Lebanon,\ N=87,% N=49, % N=38, % -------------------------------------------------------------------------------------------------------------------------------- ----------- ---------------- ----------- Clinical response at discharge Cure (resolution of all signs and symptoms/improvement to such an extent that further antimicrobial therapy was not necessary) 41 54 26 Improvement (improvement in signs and symptoms) 24 10 40 Failure (persistence, incomplete clinical resolution, or worsening in signs and symptoms) 27 28 26 Indeterminate (inability to determine an outcome) 9 8 9 In-hospital mortality 30 34 26 Cause of death[a](#tfn5-idr-10-049){ref-type="table-fn"} Underlying disease-related complication 54 71 27 MRSA related 32 18 55 Cardiac arrest 7 12 0 Another hospital-acquired pneumonia 4 6 0 Other 18 29 0 Unknown 11 6 18 Outcomes 30 days[b](#tfn6-idr-10-049){ref-type="table-fn"} post-discharge Relapse of MRSA pneumonia 3 3 3 Rehospitalization for MRSA pneumonia 2 3 0 Rehospitalization, any reason 20 21 19 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Notes:** Multiple causes of death could be listed. only measured in patients who survived to discharge (n=65; Saudi Arabia =33 and Lebanon =32). **Abbreviation:** MRSA, methicillin-resistant *Staphylococcus aureus*.

1. INTRODUCTION {#sec1} =============== Knowledge of proper names is an aspect of semantic memory that is particularly vulnerable to aging. The susceptibility of proper names to forgetting has been associated with their peculiar nature. Proper names refer to unique entities, such as persons, animals, places, buildings, and so on \[[@r1]\], with each name entailing unique semantics. Thus, they lack lexical meanings characteristic for common nouns, which in contrast refer to categories of objects, *i.e*. non-unique entities. The peculiar nature of proper names has been debated across disciplines, with the major debates revolving around their special status in grammar \[[@r1]-[@r2]\], elusiveness of their meaning \[[@r3]-[@r8]\], their susceptibility to forgetting and the way they are organized and stored in long-term memory \[[@r9]-[@r13]\]. Remembering proper names is the most common memory concern in older neurologically intact people, while a deficit in proper name retrieval accompanies a wide range of neurological conditions. Regardless of age, cognitively healthy people typically find proper names more difficult to retrieve than common nouns \[[@r14]-[@r16]\]. Surprisingly, this is the case even in name-occupation homophones (*e.g*. *Baker-baker*, *Potter-potter*) \[[@r11]\]. Sometimes proper name retrieval is associated with so-called Tip-Of-The-Tongue (TOT) states \[[@r10], [@r17]\]. TOT states are metacognitive states in which a person cannot retrieve a specific word at a given moment, despite a strong feeling that the word is within reach; crucially, the person is fully aware of this temporary word retrieval failure \[[@r10], [@r18], [@r19]\]. In TOT states, we typically remember information about the entity in question, *i.e*. semantic information about that entity is available, but the name eludes us, despite sometimes recalling a part of the name. Put differently, TOT states differ from mere failures to retrieve a name (we can fail to retrieve a name without having the impression that the target name is on the tip-of-the-tongue) and from experiences of retrieving an incorrect name without realizing that the retrieved name does not coincide with the target name. TOTs are complex states, which require multiple cognitive processes and implicate a range of brain areas \[[@r20]\]. Recent evidence suggests that cognitively healthy middle-Aged Adults (MA) have more problems remembering familiar proper names than Young Adults (YA) \[[@r19]\]. More specifically, MA individuals experience more TOT states than YA. They also retrieve more incorrect names for which they believe are the correct ones. Furthermore, the higher incidence of TOTs in midlife has been associated with significantly less cortical thickness in left BA 45 (pars triangularis) and right BA 44 (pars opercularis) as well as with less grey matter density in BA 40 (supramarginal gyrus) relative to young adulthood. These findings raise the possibility that the anatomical link between BAs 44 and 40 may have a role in name retrieval processes leading to TOTs \[[@r19]\]. This hypothesis deserves attention, because the white matter tract connecting inferior frontal and inferior parietal areas, *i.e*. the Superior Longitudinal Fasciculus (SLF) is implicated in the control of phonological processing \[[@r21]\]. This is relevant because TOTs in cognitively healthy people have been interpreted in terms of an interrupted access to phonological processing. In addition, this complex system of fibers, whose functionality is yet to be fully described, may play a role in higher cognitive processes \[[@r22]\], including those that support awareness about failures to retrieve the target word and possibly with the generation of alternative strategies for word retrieval. This tract could therefore contribute to name retrieval and associated TOT experiences in more than one way. So far, proper name retrieval has been typically associated with white matter tracts supporting the ventral processing stream for language, in particular, the left Uncinate Fasciculus (UF) \[[@r23]-[@r25]\]. An alternative proposal, however, suggests that the uncinate fasciculus is redundant for language \[[@r26], [@r27]\]: together with the inferior longitudinal fasciculus, it represents only a back-up pathway for the inferior fronto-occipital fasciculus, which is the main ventral tract for language. Possible further modifications of the model of white matter supporting name retrieval pertain to the role of dorsal white matter. In the present study, we investigated whether dorsal white matter would be relevant for proper name retrieval and whether there would be differences in the ability to retrieve proper names as well as in white matter integrity in middle-aged cognitively healthy subjects relative to young controls. Testing the hypothesis on the relevance of anatomical connectivity between right BAs 44 and 40 for proper name retrieval \[[@r19]\], we investigated whether the microstructural integrity of the right Superior Longitudinal Fasciculus (SLF) would be associated with middle-aged subjects' ability to retrieve proper names. Previous research has suggested that anatomical connections between pars opercularis (BA 44) and the supramarginal gyrus (BA 40) *via* one component of the Superior Longitudinal Fasciculus (SLF III) may have a role in phonological processing \[[@r21], [@r22]\], while the anatomical connections between the orbitofrontal and anterior temporal areas *via* uncinate fasciculus is implicated in semantic/ conceptual knowledge. Thus, the anatomical links implicated in proper name retrieval go beyond the uncinate fasciculus. If SLF is implicated in proper name retrieval, and possibly related to TOTs, we would find significant associations between the indices of white matter integrity of this tract and the ability to retrieve proper names, and possibly TOTs in the MA group. To test this hypothesis, we used Diffusion Tensor Imaging (DTI) and assessed white matter microstructural integrity in 131 cognitively healthy individuals, including 72 middle-aged and 59 young adults. This *in vivo* MR imaging technique quantifies integrity of white matter microstructure through diffusion parameters, based on micron-scale displacement of water molecules \[[@r28]\]. DTI indices of white matter microstructural integrity are defined based on the directional variability of water diffusion in white matter. Importantly, they are more sensitive indicators of age-related differences in white matter microstructure than white matter lesions \[[@r29], [@r30]\]. Thus, we examined *in vivo* the following indices of microstructural integrity of white matter: Fractional Anisotropy (FA), Mean Diffusivity (MD), axial diffusivity (AD), Radial Diffusivity (RD), and Mode of Anisotropy (MO). These diffusion parameters are sensitive to age-related changes of white matter and white matter pathology, indicating for instance abnormalities in crossing fibers regions (MO), myelin injury or glial cell damage (RD), axonal injury or changes in intracellular space (AD) \[[@r29], [@r31]\]. Normally, water molecules spread in parallel to the axon and myelin sheath, leading to anisotropic diffusion. However, abnormalities in fiber tracts lead to a more heterogeneous diffusion. For example, lower FA values and higher MD values in white matter tracts in Alzheimer's disease patients indicate tracts' deterioration due to Alzheimer's pathology \[[@r32], [@r33]\]. Still, these indices are far from being unambiguous. As an example, changes in FA values may indicate changes in myelination, axon density and diameter, to intravoxel incoherence of fiber deterioration \[[@r34]\]. Thus, simultaneously assessing multiple indices of white matter microstructural integrity is more informative regarding the possible effects of aging than focusing on only one of them \[[@r35]\]. Since microstructural integrity of white matter tracts deteriorates differentially due to aging \[[@r35]\] and given that aging differentially affects associations between DTI parameters of white matter integrity and cognitive functions \[[@r36], [@r37]\], in addition to comparing the two groups' values in the five DTI parameters described above we conducted a correlation analysis, testing for possible associations between these parameters' values in the tract under study (SLF) and MA group's naming scores. 2. MATERIALS AND METHODS {#sec2} ======================== Data used in the preparation of this work were obtained from the Cambridge Center for Ageing and Neuroscience (Cam-Can) data repository \[[@r38], [@r39]\]. The protocol for the CamCan study has been approved by their institution's ethics committee (reference: 10/H0308/50), as stated in \[[@r39]\]. We obtained permission to use the data in December of 2016, within a data sharing initiative launched by the Center earlier that year. For more information about the CamCan study, see <http://www.mrc-cbu.cam.ac.uk/datasets/camcan/>. 2.1. Participants {#sec2.1} ----------------- The sample in the present study (N = 131) consisted of a group of Middle-Aged Adults (MA) (n = 72), who were between 45 and 55 years old, and a group of Young Adults (YA) (n = 59), who were between 18 and 30 years old (Table **[1](#t1){ref-type="table"}**). All participants were cognitively healthy individuals, with a Mini Mental State Exam score of 24 or better, with no history of substance abuse, dementia, stroke, motor neuron disease, multiple sclerosis, encephalitis, epilepsy or other neurological or psychiatric (schizophrenia, bipolar disorder, psychosis) problems that could affect cognition \[[@r39]\]. 2.2. Behavioral data {#sec2.2} -------------------- For the present study, we retrieved behavioral data on a picture-naming task. In this task, pictures of 50 famous people such as actors, musicians, and politicians were presented to participants, who were required to look at one picture at a time and name the person on it. Each picture appeared after a fixation cross, which was presented for 1000 milliseconds, and it remained on the screen for 5000 milliseconds. Participants named a person on the picture if they could, they responded as "don't know" if they did not know the name of the person, or said that they were experiences a TOT state if they knew the name, but could not recall it. For analysis, the correct responses were divided into "know-correct" and "know-incorrect" responses (*i.e*. the retrieved name was correct *vs*. incorrect) \[[@r39]\]. 2.3. DTI Data Acquisition and Preprocessing {#sec2.3} ------------------------------------------- DTI data retrieved for the present study were collected on a single Siemens TIM Trio 3 T scanner with a 32 channel head coil and twice-refocused spin echo sequence. The data included b = 1000/ 2000 scans (TR = 9100 ms; TE = 104 ms; Field Of View (FOV) = 192x192; voxel size: 2 x 2 x 2; 30 directions), and a set of b = 0 scans with the same parameters, but without applying the diffusion-coding gradients, as described in \[[@r38]\]. We also retrieved high-resolution structural T1-weighted images for anatomical reference in data processing and analysis. These images were obtained using a Magnetization Prepared RApid Gradient Echo (MPRAGE) sequence, with TR = 2250 ms, TE = 2.99 ms, flip angle = 9 degrees, FOV = 256 x 240 x 192 mm, voxel size 1 x 1 x 1 mm, GRAPPA acceleration factor = 2, acquisition time = 4 minutes and 32 seconds \[[@r38]\]. DTI data preprocessing was performed following the standard FSL protocol (<http://www.fmrib.ox.ac.uk/fsl>), including correction for head motion and eddy currents, removal of non-brain voxels with the Brain Extraction Tool, and fitting of diffusion tensors to the data with DTIfit \[[@r40]\]. A tensor model fit in FSL was used to calculate quantities of FA, MD, AD, RD, and MO. 2.4. Statistical Analyses {#sec2.4} ------------------------- We used Tract-Based Spatial Statistics (TBSS) in FSL (<http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FSL>) (Smith *et al*., 2006) to calculate group differences in the five diffusion parameters separately, correcting for multiple comparisons (*p* \< .05, TFCE) at the cluster level. Furthermore, we extracted the values for the right SLF from the skeletonized images of all MA participants using the Johns Hopkins University white matter atlas (<http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/Atlases>). In the next step, these values were entered into SPSS 22 to test for significant associations between the microstructural properties of this tract and the naming scores in the MA group, using Spearman test of correlation. An alpha level of .05 was set for all tests. All tests were two-tailed. 3. RESULTS {#sec3} ========== 3.1. Demographics and Behavioral Tests' Results {#sec3.1} ----------------------------------------------- The two groups differed statistically in age, with the MA group being significantly older than the YA group, as shown in Table **[1](#t1){ref-type="table"}**. There were no statistically significant differences in gender distribution across the groups. The two groups differed in their behavioral scores. We summarize subjects' characteristics and behavioral data in Table **[1](#t1){ref-type="table"}**. The MA group had significantly more "know-correct" scores and significantly less "don't-know" scores relative to the YA group, indicating that they were more familiar with the tested names than the young adults. However, MA also had significantly more "know-incorrect" answers and experienced considerably more TOT states than YA, as shown in Fig. (**[1](#f1){ref-type="fig"}**). 3.2. Results of TBSS Analysis {#sec3.2} ----------------------------- Statistically significant group differences were found in a range of tracts supporting cognition, including the superior longitudinal fasciculus, the inferior longitudinal fasciculus, inferior fronto-occipital fasciculus, and uncinate fasciculus bilaterally, alongside anterior thalamic radiation and corticospinal tracts, among others Fig. (**[2](#f2){ref-type="fig"}**). More specifically, compared to YA, the MA group had significantly lower FA values in a range of tracts, including the right superior longitudinal fasciculus Fig. (**[2a](#f2){ref-type="fig"}**). The MA group also had lower MO values in the superior longitudinal fasciculus, uncinate fasciculus, inferior fronto-occipital fasciculus, and inferior longitudinal fasciculus bilaterally Fig. (**[2e](#f2){ref-type="fig"}**). In contrast, they had higher RD values than the YA group in the superior longitudinal fasciculus, involving the portion that links inferior frontal lobe to the supramarginal gyrus Fig. (**[2d](#f2){ref-type="fig"}**), as well as higher MD values in this tract and in the major ventral language tracts bilaterally, including the uncinate fasciculus, inferior longitudinal fasciculus, and inferior fronto-occipital fasciculus Fig. (**[2b](#f2){ref-type="fig"}**). Finally, lower AD values in the MA group relative to the YA group were found in the superior longitudinal fasciculus, uncinate fasciculus and inferior longitudinal fasciculus Fig. (**[2c](#f2){ref-type="fig"}**). Thus, the results of TBSS analysis suggest significant group differences in the microstructural integrity of a range of white matter tracts, including the tract of interest (SLF). There was a significant direct correlation between MA group's FA values in the right SLF and their "don't know" responses (*r~s~* = .287, *p* = .014). No significant correlations were found between their TOT scores and this tract's DTI values. 4. DISCUSSION {#sec4} ============= A recent study on proper name retrieval in cognitively healthy subjects reported more TOT states associated with proper name retrieval in midlife relative to young adulthood as well as an association between reduced grey matter density in the right supramarginal gyrus (BA 40) as well as cortical thinning in right pars opercularis (BA 44) and TOT states \[[@r19]\]. Assuming that the right hemisphere becomes more involved in name retrieval with increased age \[[@r41]\], in the present study we tested the hypothesis on a possible involvement of the right SLF in retrieval of proper names in midlife. The SLF is a complex fiber system, whose one component connects inferior frontal and inferior parietal areas \[[@r21], [@r22]\]. Previous research has emphasized the role of the uncinate fasciculus in proper name retrieval \[[@r23], [@r24]\], which is consistent with the evidence on the role of the anterior temporal lobe in naming \[[@r12]\], given the fact that the uncinate fasciculus links the temporal pole with orbito-frontal areas \[[@r27], [@r42], [@r43]\]. Thus, a possible association of the SLF and naming would provide evidence for the relevance of the dorsal white matter to name retrieval. Consistent with previous work on proper name retrieval in midlife \[[@r19]\], our behavioral data revealed that, relative to young participants, the middle-aged participants retrieved considerably more incorrect names while believing that those were the corrects names, and experienced more TOT states during proper name retrieval. Taken together, these results suggest that certain aspects of name retrieval are already affected by midlife. Furthermore, we found statistically significant age-related differences in the integrity of white matter microstructure across a range of white matter tracts. Overall, the differences in DTI parameters between midlife and young adulthood observed across the major tracts supporting cognition Fig. (**[2](#f2){ref-type="fig"}**) are consistent with previous findings, indicating a negative effect of age on white matter microstructural integrity \[[@r30], [@r44]-[@r46]\]. Importantly, our data indicate that the age-related differences in microstructural integrity of the right SLF are associated with the ability of MA adults to retrieve proper names. More specifically, we found a significant association between the inability to retrieve proper names and the FA values in this tract in the MA group. However, we did not find a significant relation between the DTI parameters' values and TOT scores in MA. One could argue that testing more narrowly for an association between SLF III segment, which presumably connects BAs 44 and 40 \[[@r22]\], and TOT states could have resulted in different findings. While this objection is merited, one should keep in mind that multiple tracts run through the white matter underlying the supramarginal gyrus (SLF II, SLF III, middle longitudinal fasciculus, frontooccipital fasciculus) and that DTI data from this region "like gross dissection, cannot resolve the specific fiber bundles that mingle with each other" \[[@r47]\]. In fact, the difficulty of teasing apart fibers from different cerebral white matter fasciculi \[[@r48]\] and inaccuracies of DTI in mapping the fiber architecture of the areas with intersecting fibers' trajectories are so pronounced that they have apparently motivated a renewed interest in sophisticated fiber dissection techniques \[[@r49]\]. A related issue is whether the two dorsal tracts for language, the arcuate fasciculus and the superior longitudinal fasciculus, can be reliably separated \[[@r47], [@r50], [@r51]\]. Since axons from the arcuate fasciculus "mingle with those of SLF II and SLF III in the white matter of supramarginal gyrus", establishing their respective terminations using current DTI methodology would be challenging, even though evidence from the macaque monkey shows that SLF III terminates in BA 44 \[[@r21], p.[@r38]\]. This uncertainty is further exacerbated by lack of consensus on a model of dorsal language tracts. According to the traditional view, the left arcuate fasciculus is the classical language pathway, connecting Broca's and Wernicke's areas. On the other hand, according to one currently prominent view, the superior longitudinal fasciculus is a complex fiber system \[[@r52]\], consisting of four components in the human brain (see \[[@r43]\] for a review). In this multicomponent system view, the arcuate fasciculus is a part of 
the superior longitudinal fasciculus system \[[@r21], [@r22]\]. Thus, search for a possible role of a more narrowly defined portion of the SLF in name retrieval may be problematic for methodological and theoretical reasons. Finally, among frequently observed limitations of DTI \[[@r53]\] there is also the limitation that this technique allows only indirect measures of the white matter properties. We need to keep in mind this lack of direct mappings between the differences in signal intensities measured by this technique, on the one hand, and neurocognitive processes, on the other, when interpreting our findings. CONCLUSION ========== In conclusion, the present data indicate significant age-related group differences in proper name retrieval, with more pronounced naming difficulties in midlife relative to young adulthood, as also found in \[[@r19]\]. Furthermore, significant age-related differences were found in the microstructural integrity in a range of white matter tracts, consistent with previously observed effects of aging on the integrity of white matter microstructure \[[@r35], [@r29]\]. In addition, we have identified an association between the right SLF FA values and the retrieval of proper names in middle-aged subjects. Thus, although we did not observe specifically an association between white matter deterioration and TOT states in MA, the present data suggest the relevance of the anatomical connections between the right inferior frontal (BA 44) and inferior parietal areas (BA 40) to proper name retrieval in midlfie, as hypothesized by Kljajevic & Erramuzpe \[[@r19]\]. Declared none. ETHICS APPROVAL AND CONSENT TO PARTICIPATE ========================================== The protocol for the CamCan study has been approved by their Institution's Ethics Committee (Reference: 10/H0308/ 50), as stated in \[[@r39]\]. HUMAN AND ANIMAL RIGHTS ======================= Not applicable. CONSENT FOR PUBLICATION ======================= Not applicable. AVAILABILITY OF DATA AND MATERIALS ================================== Data collection and sharing for this project was provided by the Cambridge Centre for Ageing and Neuroscience (CamCAN). FUNDING ======= CamCAN funding was provided by the UK Biotechnology and Biological Sciences Research Council (grant number BB/H008217/1), together with support from the UK Medical Research Council and University of Cambridge, UK. CONFLICT OF INTEREST ==================== The authors declare no conflict of interest, financial or otherwise. {#f1} {#f2} ###### Demographics and behavioral scores of two groups. **MA (n = 72)** **YA (n = 59)** ***Test value*** ***p-*value** ---------------- ----------------- ----------------- -------------------- --------------- Age (Mean, SD) 49.7 ±3.3 25.5 ±3.4 *t* (129) = 40.882 \< .001 Age range 45-55 18-30 \- \- Gender (m/f) 36/36 24/36 χ^2^ (1) = 1.135 = .29, n.s. Know-correct 20.1 ±10.2 15.3 ±9.4 *U* = 1525.5 = .006 Know-incorrect 5.2 ±3.6 2.9 ±2.5 *U* = 1265.5 \< .001 Don't know 10.7 ±9.4 22 ±11.1 *U* = 930 \< .001 TOT 12.2 ±6.5 8.8 ±5.2 *U* = 1498.5 = .004

Introduction ============ Organized sports are a prominent achievement context for youths, and large numbers of children and youths participate in organized sports every year ([@B55]). Participation in a sport provides youths with the opportunity to engage in physical activity, which in turn results in a number of positive health effects, well-documented through extensive research ([@B60]; [@B31]; [@B17]). In addition, participation in sports has beneficial effects upon a variety of motor, mental, and social factors ([@B15]; [@B26]; [@B18]). Furthermore, a high level of physical activity in youth increases the likelihood of being physically active in adulthood ([@B56]; [@B24]). Additional effects can be obtained by participating in team sports, as compared with individual sports. For example, [@B48] found a reduction in social anxiety over time in children participating in team sport compared with those participating in an individual sport or no sport, and [@B7] found team sport involvement to be positively associated with social acceptance, while it partially mediated risks of depressive symptoms. In their comprehensive review, [@B18] concluded that team sport engagement seemed to be associated with improved health effects compared to engagement in individual sports. The coach is arguably a very important person influencing youths' general sport experience. The coach's organization, facilitation, and behavior in practice sessions and competition has been shown to influence athletes' motivation to participate ([@B20]; [@B47]; [@B49]). Coaches may positively affect individuals' abilities, beliefs, and enjoyment, and induce a desire for challenging and mastery experiences ([@B35]; [@B61]). It has even been suggested that the coach may enhance youths' personal development and life skills ([@B22]). On the other hand, coaches have the potential to induce anxiety and burnout in athletes, and ultimately dropout from the sport ([@B52]; [@B61]). The coaches' influence has been attributed, in part, to the motivational climate they create through the transfer of attitudes and values, as well as their recognition and evaluations, and is linked to the athletes' learning and performance ([@B27]). Therefore, coaches play a critical role in either hindering or strengthening an athlete's involvement in, and motivation for, sports ([@B57]; [@B1]). A commonly applied theoretical perspective when studying motivational climates in youth sport is the achievement goal theory (AGT) ([@B40]). AGT attempts to explain how an individual cognitively processes and develops his or her views about achievement under various social contexts and influences. The term motivational climate refers to the structure of the learning environment in achievement settings that affects an individual's participation, thoughts, feelings, and behavior, and reflects the actions of coaches and parents, such as their use of rewards, punishments, and feedback ([@B2]; [@B27]). A mastery-oriented climate is characterized by valuing the learning process, such as cooperating with others, and trying new solutions, whereas a performance-oriented climate values outperforming others, and is characterized using external rewards, and by discriminating in favor of the best athletes ([@B16]). Indeed, studies have shown that a coach-cultivated motivational climate centering on task goals (i.e., a mastery-oriented climate), fosters more positive cognitive and emotional responses and adaptive achievement patterns among athletes than does an ego-involving environment (i.e., a performance-oriented climate) ([@B3]; [@B35]). Athletes' perception of a mastery-oriented motivational climate in sports settings is associated with a variety of positive factors concerning thoughts and attitudes toward the activity and increased intrinsic motivation and social values related to the activity, which are of importance to persistence and continuation of a sport ([@B42]; [@B44]). More specifically, the perception of a mastery-oriented climate is associated with sports persistence, whereas the perception of a performance-oriented climate is associated with dropout ([@B6]; [@B25]). For example, [@B20] found a positive relationship between youth soccer players' perception of a caring, or mastery-oriented, climate and their intentions of engaging in soccer in the future, while [@B13] found that the motivational climate was, in fact, more important than the win-loss percentage of the team for young athletes' enjoyment and desire to continue playing for the coach. The coach may have every intention of creating a mastery-oriented climate, but may not be able to do so. This could be due to a lack of pedagogical or educational skills, which are not always possessed by coaches just because they have extensive sporting experience, or coaching badges ([@B62]). It is well known that coaches are more frequently chosen because of their sport-specific competence than their interpersonal skills (see [@B21]). Furthermore, as shown by [@B53], [@B54]), coaches' personal well- or ill-being affects their coaching behavior. Further, a coach may have the intention of creating a mastery-oriented motivational climate, and be capable of doing so in less competitive practice sessions, but may not be able to transfer this climate to a match situation, in which the pressure is greater ([@B49]). Despite extensive study of motivational climates, the topic of how coaches perceive motivational climates, and whether the coach's perception of the motivational climate within the team (created by himself or herself) is in concordance with the perceptions of the players has been under-researched. Divergences in perceptions of the motivational climate could potentially be a reason for dissatisfaction among players, and ultimately, why players choose to drop out from the team, and potentially, from the sport altogether. Among the relatively few studies that have focused on the coach's perspective, [@B53], [@B54]) studied the effect of coaches' psychological well- and ill being on their coaching behavior across a number of sports and found that coaches' well-being positively predicted their autonomy-supportive behavior toward their athletes, while ill-being was associated with a controlling coaching style. Stebbings et al.'s studies, however, included coaches only. In previous studies, when comparing players and coaches' perceptions of motivational climates, only correlations have been reported. Such studies have included findings of a moderate positive relationship between players' and coaches' perceptions of the motivational climates ([@B8]), or little such relationship at all ([@B14]). Neither the magnitude nor the direction of differences in perceptions of motivational climates or coach behavior was elaborated upon in those studies. Correlations between players' and coaches' perceptions of both climates (task-involving and ego-involving) in the research of Boyce et al. were only approximately 0.50, and Curtis et al. found generally low correlations on perceptions of coach behavior between coaches and their young baseball players. The results of both studies thus indicate that the convergence of perceptions may be less than complete. [@B50] argued that including coaches' perceptions of the motivational climate in research studies would provide a more comprehensive assessment of the environment. The results of their study, which included both players' and coaches' perceptions, compared with reports of independent observers, showed that there were moderate positive associations between players' and coaches' perceptions of the maladaptive and controlling behaviors of the coaches, but less agreement with respect to the more positive, empowering dimensions of the motivational climate. One possible (albeit speculative) reason for the overall lack of empirical studies on the coach's perspective on motivational climates may be that researchers fear that answers may be biased, as coaches are aware of the motivational climate desired by their players and how their behavior may negatively influence their players (see also above, [@B41]; [@B49]). This awareness might prime coaches into underestimating the performance-orientation of their approach and instead portray their team as being typified by the more socially desirable mastery-oriented climate, an effect known as social desirability bias, which is reported in self-report measures across all social sciences (see [@B19]). [@B53], [@B54]) attempted to control for such social desirability bias, in that they presented sport coaches in their study with a social desirability scale, assessing their tendency to respond in a socially desirable manner. Another potential strategy for securing more honest answers from coaches is that of [@B50], in which coaches and players responded to the same questionnaire, assessing the coaching environment within their teams. The present study applied a similar design to that of [@B50], having coaches and players answer the same questionnaire, the Perceived Motivational Climate in Sports Questionnaire-2 (PMCSQ-2) ([@B38]), and each group was aware of this fact. Thus, the purpose of the present study was to extend the previous literature on perceptions of the motivational climate in youth soccer, by examining both players' and coaches' perceptions of the motivational climate within their teams and comparing them. Unlike previous studies that reported correlations of perceptions ([@B14]; [@B8]), the present study directly compared each player's responses to those of his or her own coach and tested for differences across groups. Girls have been shown to be more in favor of a task-oriented motivational climate, compared with boys ([@B30]; [@B33]), and it has been argued that they may also be somewhat differently motivated compared with boys (see [@B15]). Furthermore, girls are more prone to dropping out from soccer ([@B36]). Therefore, analyses were separated by gender in order to investigate possible differences in girls' versus boys' convergence of perceptions of the motivational climate when compared with their coaches. In addition, demographic background variables were included in the questionnaire in order to investigate the possible effects of team or squad structure, as well as coaches' soccer-specific and formal education, and their coaching experience. Coaches' backgrounds may be important for both their perceptions of, and their knowledge about motivational climates, as well as their capacity to create a certain climate ([@B35]; [@B6]; [@B47]). Materials and Methods {#s1} ===================== Participants ------------ Players and coaches belonging to 17 different soccer teams were recruited from two regions in Norway through a mailed request. Inclusion criteria for both players and coaches were that they had to have completed at least one season with the current team in order for the motivational climate to be well established. As the literature does not give any formal guidelines on how long it takes for the motivational climate to be established ([@B46]), this was done as a precaution. Across the teams in the sample, 47.1% were male players with an average of 26 players (range 12--41) per team. Seventy percent of the squads were divided based upon skill level, and the coaches reported an average of eight players (range 0--31) dropping out in the past 3 years. ### Coaches The sample consisted of 29 coaches (one female) with a mean (range) age of 40.9 (21--57) years. They had been coaching the current team for a mean (range) of 4.8 (1--10) years, and 62% had also coached other teams before. The overall mean (range) of coaching experience was 8.9 (1--30) years. The overall education level was completed high school (52%) or college education. For coaching-specific education, 55% had a national level license, and 14% had a Union of European Football Association Level 3 (UEFA-B) coaching license. Thirty-one percent of the coaches received a salary from their club. ### Female Players One hundred twenty-eight girls with a mean (range) age of 15.7 (15--17) years participated in the study. They had been with their current team for a mean (range) of 5.6 (1--11) years and had been playing soccer for a mean (range) 7.3 (1--11) years. Forty-four percent reported that they were playing on their first team. ### Male Players One hundred twenty-eight boys with a mean (range) age of 15.6 (15--17) completed the questionnaire. They had been with their current team for a mean (range) of 5.9 (1--12) years, and their total soccer experience was a mean (range) 9.1 (3--15) years. Forty-six percent reported that they were playing on their first team. Procedure --------- For each team, all questionnaires were distributed within a single practice session, in which completion took approximately 20 min. All data were collected in the pre-season period between January and March. Questionnaires -------------- The first part of the questionnaire contained demographical questions concerning the respondents' age, gender, number of years playing soccer, and number of years on the current team, as well as whether the squad was divided into several teams (1st, 2nd, or 3rd), and whether this selection was based on players' performance levels. The coaches also gave information about their general educational level and their coach-specific education or training. The Perceived Motivational Climate in Sports Questionnaire-2 (PMCSQ-2) ---------------------------------------------------------------------- Perception of the motivational climate within the team was assessed by a Norwegian version of the PMCSQ-2. The PMCSQ-2 has been used in several studies related to the motivational climate in sports, and initial studies using the PMCSQ-2 have found it to have adequate internal reliability and factorial validity for youths as well ([@B38]; [@B39]). The PMCSQ-2 is a 33-item inventory, and consists of two higher-order factors measuring *performance*- (16 items) and *mastery*- (17 items) oriented motivational climates. Each of these two higher-order factors consists of three lower-order factors (the result being six lower-order factors). For the higher-order factor of mastery-oriented motivational climate, the lower-order factors are labeled *cooperative learning*, *effort*, *and improvement*, and *each player has an important role*. For the performance-oriented motivational climate, the lower-order factors are labeled *intra-team member rivalry*, *unequal recognition from the coach*, and *intolerance of mistakes*. The stem for each item is; "On this team...," and responses were indicated on a 5-point Likert scale anchored by strongly disagree (1) and strongly agree (5), giving data on an ordinal level. Translation Procedure --------------------- The PMCSQ-2 was translated into Norwegian and adapted to soccer. The translation was based on the original English version, as well as a version that was slightly modified for use on dancers ([@B9]) that had already been translated into Norwegian. Two graduate sociologists reviewed the translated version of the questionnaire along with the original version, and in this way, back-translation was secured, which is of importance for the construct validity ([@B11]). The aim of the present study, however, was to compare the players' perceptions of the motivational climate to that of their coaches. Therefore, it was necessary to reformulate the phrases in the questionnaire in order for them to refer to the coaches. The questions in the player's version of the questionnaire (the original one) were thus inverted so that they reflected the coach's perspective, much in the same way as done in the work of [@B53] and [@B50]. An illustration of this is the phrase, "On this team, the coach wants us to try new skills," which, after reformulating, would say, "On my team, I encourage the players to try new skills." The same was done for all items of the PMCSQ-2. Statistical Analyses -------------------- Kolmogorov--Smirnov tests, histograms, and Q-Q plots were applied to confirm normality assumptions of the distributions in the eight factor scores from the PMCSQ-2. Despite evidence of the factorial validity of the PMCSQ-2 (see e.g., [@B39]; [@B43]), the Norwegian version of the inventory was subjected to confirmatory factor analysis (CFA) in order to confirm the expected hierarchical, second order factor structure. This analysis was conducted on the players' data, as there were too few coaches in the sample to complete the CFA on this sub-sample. In this model, the mastery-oriented climate serves as the correlated higher-order factor of the subscales cooperative learning, effort and improvement, and each player has an important role. Furthermore, the higher-order factor performance-oriented climate captures shared variance between the lower-order factors intra-team member rivalry, unequal recognition, and intolerance of mistakes. The multi-dimensional hierarchical model was evaluated against various types of overall goodness-of-fit indices for the constructed model: chi-squared (χ^2^) ([@B4]), root mean square error of approximation (RMSEA) ([@B34]), and normed fit index (NFI) ([@B5]). The CFA were conducted with the IBM AMOS 23.0.0 software (IBM SPSS, US). The players' scores on the higher and lower-order factors obtained from the PMCSQ-2 were analyzed against demographical variables with a general linear model (GLM) MANOVA. For this full factorial model, the variables gender and whether they played on the 1st, 2nd, or 3rd team were designated as fixed factors, and variables of age, years of playing soccer, and years of playing on the team as covariates. Similarly, the coaches' perceptions of the motivational climate were subjected to a similar GLM MANOVA procedure with demographical variables of receiving a salary from the club (yes or no), general educational level, and coach-specific education as fixed factors, and variables of age, years of coaching the current team, and overall years of coaching experience as covariates. In all pairwise multiple comparisons, the alpha was Bonferroni corrected, and the partial eta squared ($\eta_{p}^{2}$) was applied as measure of effect size. As a first step, in order to examine the association between coach and athletes' perceptions of the motivational climate, bivariate correlations were used to examine the relationships between player- and coach-perceived higher- and lower-order environment dimensions and are reported as Pearson's product-moment correlation coefficients (*r*). Further statistical analyses incorporated the fact that the nature of the data, with players nested within their teams and respective coaches, requires multilevel analysis in order to examine the convergence between players' and coaches' perceptions of the motivational climate within their teams. The specified linear mixed model with the restricted maximum likelihood method included 29 coaches at level 2 and 256 athletes at level 1. The first step in the multilevel modeling involved running baseline component models to determine the amount of variance attributed to the grouping of athletes within teams for each of the two higher-order factors, and for each of the six lower-order factors. Intra-class correlation values (ICC) of 8.8 and 22.2% for mastery- and performance-oriented climate, respectively, 1.8% for cooperative learning, 11.1% for effort and improvement, 10.4% for each player has an important role, 19.2% for intra-team member rivalry, 13.8% for unequal recognition, and 27.3% for intolerance of mistakes, suggested that a significant amount of variance in the athletes' reports of the eight environment dimensions could be attributed to the grouping of athletes within teams (i.e., within coach). Thus, further examination of the convergence between the players' and coaches' responses were examined by specifying these as fixed effects in the model and conducting Bonferroni corrected pairwise comparisons with Cohen's *d* as the measure of effect size. MANOVAs and multilevel modeling were conducted using Predictive Analytics Software (PASW, IBM, US; previously SPSS) Version 23.0.0 with *p* \< 0.05 as the statistical significance criterion. Results ======= Descriptive statistics (mean, SD) for the players' and coaches' responses on the higher and lower-order factors from the PMCSQ-2 can be found in **Table [1](#T1){ref-type="table"}**. ###### Descriptive statistics \[Mean (SD)\] for players and coaches and internal consistency for higher- and lower-order factors from the Perceived Motivational Climate in Sports Questionnaire-2 (PMCSQ-2). Players ------------------------------ ------ ------------- ------------- ------------ *Higher-order factors* Mastery-oriented climate 0.88 72.3 (7.5) 70.5 (9.8) 79.1 (7.4) Performance-oriented climate 0.88 36.3 (10.3) 41.8 (11.5) 29.7 (9.1) *Lower-order factors* Cooperative Learning 0.75 12.3 (2.0) 12.05 (2.4) 13.8 (1.0) Effort and Development 0.76 35.2 (3.5) 34.3 (4.3) 37.6 (2.1) All have an Important Role 0.79 24.7 (3.6) 24.1 (4.3) 27.7 (2.4) Intra-Team Member Rivalry 0.61 10.8 (3.1) 11.8 (3.2) 8.3 (3.9) Uneven Recognition (coach) 0.86 14.5 (5.7) 17.5 (6.4) 11.4 (3.9) Intolerance of mistakes 0.74 10.9 (3.5) 12.5 (4.2) 9.9 (3.6) 1 Cronbach's coefficient alpha. Factor Structure of the PMCSQ-2 ------------------------------- The CFA of the expected hierarchical, second order factor structure resulted in acceptable goodness-of-fit indices (χ^2^ = 11.3, *df* = 5, *p* \< 0.05; RMSEA = 0.07, 90% CI Low/High = 0.009, 0.119; NFI = 0.98) ([@B5]; [@B34]; [@B4]). The covariance between the higher-order factors of mastery- and performance-involving climates was -0.59, and the loadings of the subscales onto the higher-order factor mastery-oriented climate ranged from 0.83 to 0.85, whereas the loadings of the designated subscales for the performance-oriented climate factor ranged from 0.74 to 0.86. All factor loadings were statistically significant. Cronbach's coefficient alpha for internal consistency was 0.88 for the higher-order factors and in the range of 0.61--0.86 for the lower-order factors. Although the lower end of the alpha values was somewhat below the suggested 0.70 criteria ([@B12]) for internal consistency (0.61 for the factor *Intra-team member rivalry*, comprising three of the items from the PMCSQ-2), all factors fitted the CFA model and the pattern of results regarding player/coach divergence was similar for each lower-order factor (outlined below). Players' Responses on the PMCSQ-2 --------------------------------- The GLM for the two higher-order factors indicated a significant difference (*F* = 4.32, *df* = 1, *p* \< 0.05, $\eta_{p}^{2}$ = 0.023) in boys' and girls' perceptions of the performance-oriented climate dimension, in which boys reported a mean score of 6.6 (95% CI: 0.35--12.97) higher compared to the girls. No such difference was obtained for the mastery-oriented climate factor (*F* = 0.82, *df* = 1, *p* \> 0.05, $\eta_{p}^{2}$ = 0.01). For the lower-order factors, boys reported significantly higher scores compared to girls on the subscale unequal recognition (*F* = 5.65, *df* = 1, *p* \< 0.05, $\eta_{p}^{2}$ = 0.03), with a mean difference of 4.2 (95% CI: 0.71--7.65), and a significant mean difference of 2.3 (95% CI: 0.03--4.57) on the subscale intolerance of mistakes (*F* = 3.90, *df* = 1, *p* \< 0.05, $\eta_{p}^{2}$ = 0.02). There were no significant differences in girls' vs. boys' scores on the other subscales. Furthermore, there were no significant differences in players scores in relation to whether they played on the 1st, 2nd, or 3rd team, or related to how many years they had been playing soccer. Significant effects of players age (15, 16, or 17 years old) was found (in which the younger players reported lower scores) on the higher-order factor performance-oriented climate (*F* = 5.36, *df* = 1, *p* \< 0.05, $\eta_{p}^{2}$ = 0.03) and the lower-order factors effort and improvement (*F* = 6.82, *df* = 1, *p* \< 0.05, $\eta_{p}^{2}$ = 0.04) as well as unequal recognition (*F* = 4.75, *df* = 1, *p* \< 0.05, $\eta_{p}^{2}$ = 0.03). Lastly, the number of years they had been playing on their current team was significantly associated with the higher-order dimension mastery-oriented climate factor (*F* = 4.63, *df* = 1, *p* \< 0.05, $\eta_{p}^{2}$ = 0.02) and the lower-order factor effort and improvement (*F* = 5.49, *df* = 1, *p* \< 0.05, $\eta_{p}^{2}$ = 0.03). Coaches' Responses on the PMCSQ-2 --------------------------------- Analysis of the coaches' perceptions of the motivational climate with GLM MANOVA indicated that there were no significant differences (*p* \> 0.05) in either higher- or lower-order factors between coaches with different general education levels (secondary school up to more than 4 years in university) or between coaches with or without soccer-specific coaching education (grassroot or UEFA-licenses). Furthermore, pairwise comparisons demonstrated that none of the variables *receiving a salary or not*, *age*, *years of coaching the current team*, and *years of overall coaching experience* induced significant differences (*p* \> 0.05) on any of the eight motivational climate factor scores. Coaches' vs. Players' Responses on the PMCSQ-2 ---------------------------------------------- Pearson's *r* for the association between coaches' and players' perceptions of the motivational climate, depicted in **Table [2](#T2){ref-type="table"}**, indicated moderate significant correlation coefficients for the higher-order factor mastery-oriented climate and the lower-order factors effort and development and intra-team member rivalry. ###### Inter-correlations between players' and coaches' scores on higher- and lower-order factors from the PMCSQ-2. Pearson product-moment correlations (*r*) ------------------------------ ------------------------------------------- *Higher-order factors* Mastery-oriented climate **0.39**^∗^ Performance-oriented climate 0.28 *Lower-order factors* Cooperative Learning 0.34 Effort and Development **0.40**^∗^ All have an Important Role 0.14 Intra-Team Member Rivalry **0.46**^∗∗^ Uneven Recognition (coach) 0.15 Intolerance of mistakes 0.25 Significant correlations ( ∗ p \< 0.05, ∗∗ p \< 0.01) in bold. Further analysis, in which players' and coaches' responses were specified as fixed effects in nested multilevel models, indicated a significant overall effect on the responses on PMCSQ-2 that was dependent upon whether a player or coach answered the questionnaire. For the higher-order factor mastery-oriented climate, the players scored statistically significantly lower, on average 7.5 (95% CI for difference = 4.4--10.9), compared to the coaches (*t* = 4.3, *df* = 283, *p* \< 0.001, *d* = 0.55) and significantly higher on the higher-order factor performance-oriented climate (*t* = 4.6, *df* = 283, *p* \< 0.001, *d* = 0.51), on average 9.4 (95% CI for difference = 5.1--13.7). Multilevel analysis of the six lower-order factors indicated a significantly lower score (mean difference 1.5, 95% CI = 0.7--2.3) for the players compared to the coaches on the mastery-oriented climate subscales cooperative learning (*t* = 3.7, *df* = 283, *p* \< 0.001, *d* = 0.44), effort and development (mean difference 2.8, 95% CI = 1.4--4.3, *t* = 3.8, *df* = 283, *p* \< 0.001, *d* = 0.45) and all have an important role (mean difference 3.3, 95% CI = 1.8--4.6, *t* = 4.4, *df* = 283, *p* \< 0.001, *d* = 0.52). Furthermore, statistically significant higher scores amongst the players compared to the coaches were observed on the performance-oriented climate subscales intra-team member rivalry (mean difference 3.0, 95% CI = 1.8--4.2, *t* = 4.9, *df* = 283, *p* \< 0.001, *d* = 0.58), uneven recognition (mean difference 4.6, 95% CI = 2.3--6.9, *t* = 3.9, *df* = 283, *p* \< 0.001, *d* = 0.46), and intolerance of mistakes (mean difference 1.9, 95% CI = 0.4--3.4, *t* = 2.5, *df* = 283, *p* \< 0.05, *d* = 0.30). Discussion ========== The purpose of the present study was to directly compare players' and coaches' perceptions of the motivational climate within their youth soccer teams. Furthermore, possible effects of demographic variables upon these relationships were investigated within both groups. The study applied a Norwegian version of the commonly applied PMCSQ-2 questionnaire for assessing the motivational climate, in which CFA of the players' data suggested the expected two-level factorial structure. The results showed that there was a divergence between coaches' and players' perceptions in that coaches perceived the motivational climates as significantly higher in mastery-orientation, and significantly lower in performance-orientation, compared with their players. Aside from a gender difference, in which boys reported higher scores on factors associated with a performance-oriented climate, other demographic variables from players (age, division of the squad, which team the player belongs to) or coaches (formal and sport-specific education) did not affect the overall pattern of results. Even though previous studies with similar designs have found correlations between players' and coaches' perceptions of the motivational climate to be rather low ([@B14]; [@B8]; [@B50]), such divergences of perceptions on direct comparison, as shown in the present study, have not been previously reported. The results may be accurate, in that there is a divergence of perceptions between players and coaches about the motivational climate within the team, or they might signal biased reporting from coaches, players, or both. If the results are accurate, coaches may wrongly perceive that they create a mastery-oriented climate while creating a climate that is more performance-oriented, which the players correctly perceive. On the other hand, coaches may create a mastery-oriented climate, as they report, but this is wrongly perceived as more performance-oriented by the players. Such misperceptions may be due to unintended behaviors by coaches because of lack of pedagogical or educational skills or experience in otherwise soccer-competent individuals ([@B62]; [@B21]), or it may be due to coaches' psychological ill-being, which has been shown by [@B53], [@B54]) to predict controlling behaviors toward their players. The present results thus support the assumption set out by [@B45], that the coach may have the best intentions of focusing on a mastery-oriented climate, but still the athlete perceives the climate to be performance-oriented perhaps due to unintended behaviors of the coach. If correct, our results indicate that this assumption also for youth soccer players of both genders. Coaches may also, seek to create a motivational climate for the team that is in accordance with their personal ambitions and views about coaching, and less in accordance with players' wishes or official goals set by clubs or associations, as was suggested by [@B28]. In such cases, it is possible that coaches' responses are biased, and that they are underreporting the performance-orientation within their team. Coaches may seek to portray the motivational climate as being more mastery-oriented, as they know that this would be more desirable and socially acceptable. On the other hand, it is also possible that players are biased and portray the performance orientation of the motivational climate more extremely, perhaps as a signal of their disagreement and/or dissatisfaction with the motivational climates, and/or the coach's (or coaches') behaviors. From the present results, it is not possible to reach a conclusion about such possible biases. However, evidence supports that if there were any bias or misperceptions regarding the actual climate, it would be among the coaches, as [@B51] found scores on an objective measure of the motivational climate (the Multidimensional Motivational Climate Observation System, MMCOS) to be more consistent with athletes' reports than with those of their coaches. Whatever the cause, however, such biased reporting would seem to support the fact that coaches create a more performance-oriented motivational climate than what is desired by the players, and since it is the players who are motivated, or not, by the climate, their perceptions would be the ones that matter the most. Thus, if coaches wish to succeed with their teams, they should try to create a motivational climate that is more in accordance with the players' preferences and wishes. Players who are dissatisfied with the motivational climate of the team, or with coaching behaviors associated with exaggerated performance orientation, may choose to leave the team, and even the sport altogether ([@B20]). The boys who participated in this study tended toward perceiving a higher performance-orientation than the girls. This is in line with previous research that has repeatedly found female athletes to perceive the motivational climate created by the coach as being more task-involving or mastery-oriented compared with their male counterparts ([@B29]; [@B63]; [@B10]; [@B59]), and probably also reflects an actual difference in motivational climates within girls' and boys' teams. In a similar vein, studies have reported higher ego-orientation and lower task-orientation in male players than in female players across a variety of team sports ([@B30]; [@B33]). These gender differences, associated with players' perceptions of the motivational climate, can be explained by a general trend of a more competitive environment that emphasizes winning and outperforming others in male teams, which might expose players to performance-oriented practices ([@B37]). Another postulated explanation is that the sports arenas provide boys with an opportunity to show their masculinity and gain popularity by peers, by demonstrating their strength and athletic skill ([@B32]). A third possibility concerns gender differences in competence-judging criteria, as boys tend to evaluate their competence in line with the characteristics of a performance-oriented climate, whereas girls are known to use more self-referenced criteria, e.g., skill improvement, in line with the characteristics of a mastery-oriented climate ([@B23]). Clearly, establishing the specific nature of causes and effects of these gender differences in team sports, especially in terms of dropout, is an important area for further research. The overall perceptions of the motivational climate drawn from the present sample of coaches did not systematically vary as a function of completed formal soccer coaching training or general education level. Although this finding might indicate that the coaching style is unaffected by these variables, it must be considered that a relatively low proportion of coaches were categorized as *without formal coaching training* or of *lower general educational level*. Previous research has shown that interventions aimed at getting coaches to facilitate a mastery-oriented climate in sports are effective without being very costly or time consuming. These interventions are associated with changes in athletes' anxiety and perceptions of the coaches being more mastery-oriented ([@B52]). Furthermore, dropout rates have been found to be higher for non-trained coaches compared to coaches with formal sport-specific education ([@B47]). As we did not include any specific information about the content of the coaches' formal training, we can only speculate whether the motivational climate was part of the curriculum. [@B58] demonstrated that a coach's gender may have an effect upon players' perceptions of the motivational climate. Vazou's results indicated that athletes perceived female coaches to promote a more task-involving, that is, mastery-oriented climate, and less of an ego-involving or performance-oriented climate compared to male coaches. Unfortunately, because only one female coach participated in the present study, we were unable to examine this potential impact of gender. An open question for further study, therefore, is whether there also are gender differences in perceptions of motivational climates among coaches, and whether female coaches' perceptions of the motivational climate within their teams are more in accordance with those of their players. Implications ------------ A divergence of players' and coaches' perceptions of the motivational climate within their youth soccer team may indicate that motivational climates within teams are more performance-oriented and less mastery-oriented than the coaches report. As players' motivations are dependent on the motivational climate that the coaches create, their perceptions would seem to matter more, and thus, the motivational climates should be adjusted so that they are more in accordance with players' preferences. Players, and especially girls, tend to favor more mastery-oriented climates, thus the climates within the teams included in the present study may in fact be a cause of dissatisfaction among players, which may help in explaining why so many players, especially girls, drop out from the sport. From the present findings, it can be argued that coaching education should emphasize the importance of the motivational climate within a team, and should encourage the creation of a more mastery-oriented motivational climate within their teams. Limitations and Future Research Directions ------------------------------------------ The present study did not include any objective measure that would have indicated which of the groups' (players or coaches) perceptions of the motivational climates were correct, and did not control for social desirability bias. However, as argued above, any divergence would signal some issues within the team, and therefore would bias reporting from any group. The present study applied the PMCSQ-2 with slight modifications (inverting the questions to fit the coaches' perspective), similar to previous studies ([@B14]; [@B53], [@B54]; [@B50]). However, in the present study, the direction and magnitude of the divergence was tested. Thus, the present design could motivate further studies targeting the generalizability of the current findings. In particular, a possible line of research would be to examine the perceived motivational climate reported by players no longer playing or those considering dropping out, and compare their perceptions with those of their current or past coaches. In the current study, the CFA was only applied to the players' data due to a limited *n* in the sub-sample of coaches. Thus, evaluating the psychometrical properties of the Norwegian version of the PMCSQ-2 awaits further study. Substantial and potentially significant differences in the factor structure of players and coach's data could also shed further light on the divergence in perception of the motivational climate. Conclusion ========== In the present study, coaches and their players perceived the motivational climate within their teams differently in that the coaches reported a more mastery-oriented and less performance-oriented motivational climate than did their players. It is suggested that coaches, inadvertently or on purpose, may create a climate that is more in accordance with their personal preferences, and not necessarily in accordance with the preferences and wishes of their players. Players, and especially girls, tend to favor more mastery-oriented climates. Thus, the climates within the teams included in the present study may in fact be a cause of dissatisfaction among players, and may help in explaining why so many players, especially girls, drop out from the sport. Coaching education should therefore emphasize the importance of the motivational climate within a team, and should encourage coaches to create more mastery-oriented motivational climates within their teams. Ethics Statement ================ The study was approved by The Norwegian National Research Ethics Committee. Information letters about the study, along with a request for respondents, were mailed to each team's contact person. When agreeing to participate in the study, the teams were contacted again and given further information about the study in the guise of a written information sheet. Completed questionnaires were put in closed envelopes that were each given a code to ensure the respondents' anonymity. The participants were above the age of 15 which, in accordance with The Norwegian National Research Ethics Committee, does not introduce a requirement for parental consent. Author Contributions ==================== Conception and design of study: NM, AP, and HL; acquisition of data: NM; analysis and/or interpretation of data: NM, AP, and HL; drafting the manuscript: NM, AP, and HL; revising the manuscript critically for important intellectual content: NM, AP, and HL; approval of the version of the manuscript to be published: NM, AP, and HL. Conflict of Interest Statement ============================== The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors would like to thank the players and the coaches for participating in the study. [^1]: Edited by: *Adam Robert Nicholls, University of Hull, UK* [^2]: Reviewed by: *Paul Appleton, University of Birmingham, UK; Juliette Stebbings, University of Birmingham, UK* [^3]: This article was submitted to Movement Science and Sport Psychology, a section of the journal Frontiers in Psychology

Introduction ============ The burden of hepatocellular carcinoma (HCC), accounting for the majority of primary liver cancers and a major complication of liver cirrhosis, is substantial -- ranking third in cancer-related deaths worldwide.[@b1-jhc-6-023] In the UK, the incidence of liver cancer has risen considerably over the last decade, with 5,736 new cases in 2015.[@b2-jhc-6-023] Mortality rates have also increased, witĥ5,417 deaths reported in 2016. This is particularly poignant given that up to 49% of UK cases of HCC are considered preventable ones.[@b3-jhc-6-023] Many of these patients present with advanced stage disease, where a lack of effective therapies contributes frequently to death within a year.[@b4-jhc-6-023] The term "surveillance" can be defined as the regular implementation of a diagnostic test to individuals at risk of developing a given disease. The principal aim of surveillance programs is to reduce disease-related mortality, the success of which can be influenced by the incidence of the disease in question in the individuals deemed to be at risk -- the "target population," the availability of a cheap and efficient test that is acceptable to the target population, and the availability of effective treatments if the disease is discovered. For patients with HCC, where the majority arise in the presence of cirrhosis, there are curative or effective therapies for tumors detected at an earlier stage. Detecting early cancers in patients fit enough to have these therapies can have a major impact on mortality. This review summarizes the changing trends in the epidemiology of liver diseases underlying the development of HCC and focuses on the role of surveillance in patients at risk, detailing the benefits, but also the limitations in different groups of patients. The changing epidemiology of HCC -------------------------------- There are several risk factors for liver cirrhosis, including chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, alcohol-related liver disease (ARLD), and obesity-related non-alcoholic fatty liver disease (NAFLD). Other causes include cholestatic and autoimmune liver diseases, metabolic liver diseases, and cryptogenic cirrhosis. In Asian and African nations, viral etiologies are more common. Their prevalence varies in European countries and in Italy and Spain, where there is a relatively higher HCC incidence and mortality, the differences have been attributed to the prevalence of HCV.[@b5-jhc-6-023] In the UK, where the prevalence of viral hepatitis is lower, ARLD and NAFLD cirrhosis pose a bigger challenge and are responsible for the dramatic increases in both incidence of HCC and mortality attributed to HCC over the last 10 years.[@b4-jhc-6-023]--[@b6-jhc-6-023] Furthermore, data generated by Cancer Research UK suggest that the incidence of primary liver cancer is likely to continue increasing in the UK -- at least for the next 20 years, as a result of widespread obesity.[@b7-jhc-6-023] Risk of HCC in cirrhosis ------------------------ The pathophysiology of HCC is a multifactorial event. Hepatic inflammation, necrosis, fibrosis, and ongoing regeneration characterize the cirrhotic liver and contribute to HCC development. Approximately 2%--7% patients with cirrhosis are reported to develop HCC annually, although the risk attached to different etiologies varies.[@b8-jhc-6-023],[@b9-jhc-6-023] When considering the effectiveness and costs of a surveillance strategy, it is worth considering this variation. Worldwide, \~80% of HCC cases are attributable to chronic HBV and/or HCV infection, especially in the setting of advanced fibrosis and established cirrhosis.[@b10-jhc-6-023] In China, several meta-analyses have demonstrated a 15--20 times greater risk of HCC among HBV-infected individuals compared with the uninfected population.[@b11-jhc-6-023] Moreover, increased incidence and mortality rates of HCC have been reported in countries with chronic HBV infection prevalence \>2%.[@b12-jhc-6-023] The lifetime risk of HCC among chronic HBV patients is estimated to be 10%--25%. Interestingly, HBV cirrhosis is more strongly associated with HCC in Asia and Africa compared to Western Europe and America, with 5%--50% developing HCC in Asia and Africa compared to 3%--10% developing HCC in Western Europe and America.[@b12-jhc-6-023] The risk of HCC in treated patients with HBV E-antigen seroconversion or suppressed HBV replication is lower, but not eliminated.[@b13-jhc-6-023] Prospective studies have also shown a markedly increased risk of HCC in HCV-infected patients. In Japan, \~70% of cases diagnosed with HCC over the last 10 years were HCV antibody (HCV Ab)-positive.[@b14-jhc-6-023] In Korea, 10%--20% of HCC patients are positive for HCV Ab. A meta-analysis of case--control studies suggested that individuals positive for HCV Ab have a 17-fold increased risk of developing HCC compared with those negative for HCV Ab.[@b15-jhc-6-023] Similar to patients with HBV, patients treated successfully for HCV are also growing in number and while viral clearance markedly reduces their risk, again, it is not eliminated.[@b16-jhc-6-023] The 5-year cumulative risk of HCC in an individual with ARLD cirrhosis is reported to be \~8%.[@b17-jhc-6-023] While this is less than that for patients with viral hepatitis, it is an important cause of HCC in regions where alcohol excess is common.[@b4-jhc-6-023] Consequent to a global epidemic of obesity, NAFLD now affects a large proportion of the world population and both its incidence and prevalence are increasing.[@b6-jhc-6-023] Although only a minority of patients with NAFLD progress to cirrhosis, NAFLD is now so widespread that it has become the commonest cause of cirrhosis in western nations.[@b18-jhc-6-023]--[@b20-jhc-6-023] The risk of HCC in NAFLD cirrhosis is estimated at 2.6% and NAFLD cirrhosis now typically accounts for 10%--14% of HCC cases in Europe or North America.[@b21-jhc-6-023]--[@b23-jhc-6-023] In Northern England, the number of HCC cases referred to the tertiary center in Newcastle upon Tyne has increased over tenfold with NAFLD accounting for 35% of cases.[@b4-jhc-6-023] Cumulative evidence on which the recommendations in the recently published European Association for Study of the Liver (EASL) guidelines were based suggests cost-effectiveness where the incidence of HCC in cirrhotic patients exceeds 1.5% per year -- which includes all cirrhotic patients regardless of their underlying etiology.[@b24-jhc-6-023] Risk of HCC in non-cirrhotic patients ------------------------------------- A large cohort study published by Giannini et al in 2013 reported that 52 of 3,027 cases (1.7%) of HCC were found in non-cirrhotic livers.[@b25-jhc-6-023] Notably, these patients were more likely to present at a more advanced tumor stage, associated with a poorer prognosis. Therefore, while cirrhosis is clearly the most important risk factor for HCC development, there are some identifiable groups of patients without cirrhotic liver disease who have an elevated risk of developing HCC. As these patients may have better preserved liver function with more opportunities for curative interventions for early stage disease, cost-effectiveness may justify surveillance in some of these groups even if the incidence is \<1.5%. Hence there is a need to consider these groups carefully. HBV can have oncogenic effects as a consequence of its integration into the human genome, and therefore, patients with chronic infection have an elevated risk of developing HCC even in the absence of cirrhosis. The risk is reportedly elevated in the presence of higher levels of HBV replication and is higher in males and also in Asian and African countries, as compared to western countries.[@b26-jhc-6-023] For patients with chronic HCV, those with more advanced or bridging fibrosis falling short of cirrhosis have a higher risk of HCC.[@b27-jhc-6-023] For these groups of patients with viral hepatitis without cirrhosis, surveillance is justifiable. The epidemiology and natural history of NAFLD-HCC are presently unfolding, with the numbers of patients with NAFLD-HCC without cirrhosis steadily increasing and reported to be as high as 25%--45% in some series.[@b4-jhc-6-023],[@b28-jhc-6-023] While these numbers are substantial, the population at risk is very large and an individual patients' risk of developing HCC in the presence of non-cirrhotic NAFLD is actually very small.[@b29-jhc-6-023] In patients with type 2 diabetes, who have an elevated risk compared to the general population, the risk is still smaller -- estimated to be in the region of 0.8/1,000 patient-years.[@b30-jhc-6-023] Consequently, surveillance in NAFLD patients without cirrhosis, or patients with obesity or type 2 diabetes in the absence of cirrhosis, is not advocated. Benefits of surveillance ------------------------ As outlined above, HCC is a substantial cause of death worldwide, with the majority of cases presenting at advanced and incurable stages. As there are effective curative therapies for early stage cases, and the majority of cases arise in individuals with a known predisposing condition, an acceptable test, applied regularly to a fit target population known to have an elevated risk could potentially have a major positive impact on mortality. The tests most commonly used for HCC surveillance include a serum measurement of tumor marker alpha fetoprotein (AFP) and an abdominal ultrasonography (US).[@b24-jhc-6-023] In a population-based Chinese study targeting a region with a high prevalence of HBV, all the villages in the region were randomized either to receiving US and AFP measurements every 6 months or to "no surveillance." Despite a compliance rate of only 55% in the surveillance arm, HCC mortality was reduced by 37% -- as a result of a greater detection of earlier stage lesions and a higher rate of curative resection.[@b31-jhc-6-023] This was a randomized controlled trial, providing the strongest level of evidence in favor of a benefit for HCC surveillance. While highly encouraging, the study by Zhang et al described above is the only published randomized controlled trial addressing the benefit of surveillance vs no surveillance. It was population based in a region with a significant mortality attributed to HBV-HCC, and its positive outcome should not necessarily be assumed to be representative of other target populations -- perhaps where the relative risk in the target population, the fitness to undergo curative treatments, and the sensitivity of US and AFP as diagnostic tests may be lower. Performing additional RCTs may well have been worthwhile from a cost-effective viewpoint, but the ethical dilemma of randomizing patients at risk to a non-surveillance arm has been a major hindrance. Instead, there have been other types of studies, including cohort studies exploring the impact of surveillance in individuals with cirrhosis.[@b32-jhc-6-023],[@b33-jhc-6-023] In addition, there have been high-quality studies in cirrhotic patients, comparing 3-, 6-, and 12-month surveillance intervals.[@b34-jhc-6-023],[@b35-jhc-6-023] There have also been cost utility analyses. A UK-based study using a decision analytic model suggested that 6-monthly US had an incremental cost-effectiveness ratio of around £30,000 per quality-adjusted life year (QALY).[@b36-jhc-6-023] Adding AFP to 6-monthly US reportedly increased the cost to £60,100 per QALY, as the additional pick up rate of 6%--8% was thought not to counteract the increased rate of false-positive results and additional diagnostic testing.[@b37-jhc-6-023] North American studies have modeled US with or without AFP to cost around \$30,000 per QALY.[@b38-jhc-6-023],[@b39-jhc-6-023] Annual computed tomography (CT) and magnetic resonance image (MRI) surveillance were estimated to cost between \$25,323 and \$50,000 and \$118,000 per QALY, respectively.[@b38-jhc-6-023],[@b39-jhc-6-023] In combination, these studies have largely reinforced the benefit of surveillance in cirrhotic patients, and the 6-month interval with US, possibly in conjunction with AFP, is widely promoted.[@b40-jhc-6-023]--[@b42-jhc-6-023] International guidelines on the surveillance of HCC are summarized in [Table 1](#t1-jhc-6-023){ref-type="table"}. The benefits of surveillance are summarized in [Table 2](#t2-jhc-6-023){ref-type="table"}. The limitations of surveillance and its potential harms ------------------------------------------------------- Much of the criticism of surveillance programs stem from the lack of evidence underpinning its widespread application in individuals where the true efficacy and cost-effectiveness are unknown. There are numerous supportive studies, but a well-recognized weakness of cohort studies or case series is that they often fail to take into account the impact of lead time and length time bias. Lead time bias refers to an apparent improvement of survival simply because the diagnosis of cancer is made earlier. Length time bias can occur if there is an over-representation of patients with slower-growing tumors, if those with the most aggressive and rapidly growing tumors present and die within the surveillance interval and their data are not captured. While these are valid criticisms, correction formulas can be applied and a retrospective case-- control study from Italy recently reported that survival benefit beyond 3 years could confidently be attributed to successful surveillance rather than lead time bias.[@b43-jhc-6-023] The other factor not always appreciated is the impact of being in a surveillance program on individual patients. There are cost implication for patients, not just health care bodies, in terms of time, finances for travel or parking, as well as hospital visit-related stress. Those in whom HCCs are detected at an early stage who then undergo curative or life-prolonging therapies do benefit overall, but this needs to be interpreted alongside an appreciation of the negative impact and burden of tests in individuals in whom cancers are not detected, or in fact where cancers are detected, but at a stage too late to be of benefit. For those undergoing surveillance, abdominal US is a safe and noninvasive test that is acceptable to patients. However, it is widely appreciated that HCC can be difficult to detect in cirrhotic livers and its usefulness is operator dependent.[@b44-jhc-6-023],[@b45-jhc-6-023] Detection in centrally obese individuals is also more challenging, and the test is not as sensitive in these individuals.[@b46-jhc-6-023] However, its cost is relatively modest, with an overall sensitivity that ranges from 58% to 89% and a specificity \>90% -- which is deemed acceptable for a surveillance test.[@b47-jhc-6-023] US also has the capacity to detect other complications of cirrhosis that may benefit from earlier intervention -- such as subclinical ascites or a portal vein thrombosis. Serum AFP has been used for decades but has poorer sensitivity for the detection of early lesions and is in fact no longer recommended by the EASL guidelines as a surveillance tool.[@b24-jhc-6-023] The recently revised AASLD and Asian Pacific Association for the Study of the Liver guidelines suggest that it may have a role alongside US, although advises noting the rate of increase rather than just the absolute level, as the latter can be raised in some individuals with chronic liver disease who do not have HCC, contributing to the test's poor specificity.[@b40-jhc-6-023],[@b41-jhc-6-023] Despite these negative attributes, the test is cheap and its addition to US reportedly facilitates the detection of an additional 6%--8% of cases overall.[@b48-jhc-6-023] A recent meta-analysis including 32 studies reported US sensitivity of 84% for all stages of HCC, although this percentage was notably higher for larger cancers and significantly lower for early stage cases.[@b49-jhc-6-023] This meta-analysis reported that US with and without AFP detected early stage HCC with 63% and 45% sensitivity, respectively. Thus, the combination is most likely superior to US alone and remains standard practice in many centers.[@b49-jhc-6-023] In addition to patient inconvenience and cost, the added harms of false-positive tests should also be appreciated. An US scan detecting a small suspicious nodule, or a slightly raised AFP, will result in further investigations. A CT scan to visualize and further characterize a suspected lesion leads to radiation exposure and requires the use of intravenous contrast, with an injection and potential renal toxicity. An MRI scan may be considered as an alternative or in addition and has no irradiation exposure. However, a contrast injection is still required, and there is still a small risk to renal function. In addition, many patients find MRI scans distressing as they take more time to complete, and the "tunnel-like" environment in which the patient is placed often causes distress and anxiety. Patients may also be subjected to invasive procedures such as liver biopsy in order to sample a suspicious lesion, if scans are insufficient to characterize it confidently. Having the reassurance that a nodule is regenerative, or dysplastic requiring heightened observation rather than an HCC requiring intervention, can be very informative. Thus, while some may class an additional scan or biopsy that confirms a non-cancerous lesion as a "surveillance harm event," both the clinician and the patient may well value that information, rather than regard it as resulting from an "unnecessary" investigation. Not to be forgotten though is that there are also risks -- albeit small -- associated with liver biopsy. These include bleeding, which very occasionally can be life threatening.[@b44-jhc-6-023],[@b45-jhc-6-023] A decision to biopsy a lesion in a cirrhotic liver should be carefully considered within a multidisciplinary team.[@b24-jhc-6-023] While we generally accept, based on guidelines, that surveillance is recommended and that false-positive events will trigger additional investigations, studies over the last decade have estimated that every year up to 5% of patients undergoing HCC surveillance will have a false-positive test warranting further investigations.[@b34-jhc-6-023],[@b50-jhc-6-023] Therefore, the number and associated costs are high. A recent report by Atiq et al suggests that false-positive harm events are rising rather than falling.[@b51-jhc-6-023] The study included 680 cirrhotic patients of which 11.5% (78) developed HCC over a 3-year period. Forty-eight (61.5%) of the HCCs were identified by using surveillance, including 43.8% by US, 31.2% with AFP and the remainder (25%) by a combination of the two. Surveillance harm events over the same period, defined largely as unnecessary testing, were identified in 187 cohort patients, equating to \~9% per year and adding significantly to the cost of each true HCC detected. Of note, US-related harms were more frequent than AFP-related harms.[@b51-jhc-6-023] As the prevalence of obesity and poor sonar subjects increases, the rising costs associated with false-positive tests may have a negative impact on cost--benefit analyses and ultimately result in the practice being simply too expensive unless surveillance tools for the at-risk populations improve. Another surveillance harm is that of false-negative imaging. A recent study evaluated 352 patients who were being assessed for liver transplant for indications other than HCC. Individuals underwent US, and this was compared with CT or MRI. Patients with body mass index (BMI) ≥30 kg/m^2^ had an US sensitivity for HCC detection of 0.76 vs 0.87 for BMI \<30 kg/m^2^ (*P*=0.01). US sensitivity was further decreased in patients with NAFLD-cirrhosis vs other etiologies (0.59 vs 0.84, *P*=0.003).[@b52-jhc-6-023] This study supports the deteriorating sensitivity of the tools we use in the fastest growing at-risk population (obese individuals with NAFLD), and as a consequence, inadequate or false-negative imaging is likely to increase. A cancer diagnosis is very commonly associated with fear and anxiety, and a late or missed diagnosis in a patient entered into a surveillance program adds anger and disappointment to the psychosocial harm, for both the patient and their families. The limitations of surveillance are summarized in [Table 2](#t2-jhc-6-023){ref-type="table"}. HCC surveillance in developing countries ---------------------------------------- Around 80% of HCC occurs in developing countries due to the high prevalence of HBV infection and aflatoxin B1 exposure.[@b53-jhc-6-023],[@b54-jhc-6-023] The prognosis of HCC is significantly worse in these countries compared to developed ones, owing to lack of detection of HBV, lack of HCC surveillance programs, late diagnosis of cancers, and limited access to treatments.[@b55-jhc-6-023],[@b56-jhc-6-023] Recognizing the risk to HBV-infected individuals, but without the resource to implement more costly surveillance initiatives, AFP measurement alone has been recommended for HCC surveillance in Sub-Saharan Africa.[@b57-jhc-6-023] The success of strategies such as these is not easy to evaluate, but in resource-limited counties with a high incidence of HCC, this approach may well extend survival if affected individuals are offered treatments. Summary and recommendations =========================== The evidence available supports the use of HCC surveillance with a combination of 6-monthly US scan and serum AFP in individuals at higher risk of developing HCC. The measure of success in terms of reduced mortality and life years saved varies in different target populations, reflecting the level of risk, the fitness of the individuals, and the effectiveness of the tools used -- each of which vary in the context of the different etiologies predisposing to HCC. The following factors should be considered by physicians directing surveillance: weighing up for each individual his or her risk of developing HCC, the likelihood of being able to offer him or her life-prolonging therapy, alongside the burden of surveillance for both the patient and the health care provider. If there is a good case for offering surveillance, this -- as well as the limitations -- should be explained to the individuals and their consent to proceed obtained. The recent EASL guideline recommends surveillance in all cirrhotic patients with preserved liver function graded as Child--Pugh stage A or B, as well as in patients of Child--Pugh stage C awaiting liver transplantation.[@b24-jhc-6-023] For patients with HBV without cirrhosis, assessing risk with the PAGE-B score is advised, offering surveillance to those with a score \>9.[@b24-jhc-6-023] These tend to be older, male patients with reduced platelet number -- age, sex, and platelet count being the three factors on which the score is based. Surveillance is also recommended for individuals with advanced fibrosis (Metavir F3) falling short of cirrhosis regardless of etiology. In each of these patient groups, there is no evidence to support withdrawal of surveillance beyond a certain age, although withdrawal based on futility should be explained to patients developing comorbidities or frailty that would limit the application of life-prolonging therapies. Although one of the most rapidly growing groups of patients with HCC are those with non-cirrhotic NAFLD, cost-effective analyses do not support surveillance in those with NAFLD who do not have cirrhosis or advanced fibrosis. Continued efforts to stratify risk in these individuals -- perhaps including factors such as age and sex in conjunction with NAFLD-HCC risks such as PNPLA3 genotype -- alongside efforts to identify more sensitive and specific tools relevant to these patients may change this recommendation in future years.[@b58-jhc-6-023],[@b59-jhc-6-023] **Disclosure** The authors report no conflicts of interest in this work. ###### Summary of current international guidelines on HCC surveillance ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Guidelines Surveillance population Surveillance modality Surveillance interval ---------------------------- ----------------------------------------------------------------------------------------------------------------- ----------------------- ----------------------- AASLD 2018[@b40-jhc-6-023] All patients with liver cirrhosis except patients with Child--Pugh stage\ US ± AFP 6 months C cirrhosis unless on transplant waiting list APASL 2017[@b41-jhc-6-023] All patients with cirrhosis\ US + AFP 6 months Chronic HBV carriers without cirrhosis\ • Asian females \>50 years\ • Asian males \>40 years\ • Africans \>20 years\ • Family history of HCC EASL 2018[@b24-jhc-6-023] Cirrhosis Child--Pugh stage A and B\ US 6 months Cirrhosis Child--Pugh stage C awaiting liver transplant\ Chronic HBV without cirrhosis at intermediate (10--17) or high risk (≥18) of HCC according to PAGE-B score\ Non-cirrhotic patients with Metavir F3 fibrosis regardless of etiology ESMO 2018[@b42-jhc-6-023] All patients with cirrhosis as long as liver function and comorbidities allow curative or palliative treatment\ US ± AFP 6 months Chronic HBV and HCV carriers with Metavir F3 fibrosis\ Asian chronic HBV carriers with serum HBV-DNA above 10,000 copies/mL ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Abbreviations:** AASLD, American Association for the Study of Liver Disease; AFP, alpha fetoprotein; APASL, Asian Pacific Association for the Study of the Liver; EASL, European Association for the Study of the Liver; ESMO, European Society for Medical Oncology; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; PAGE-B score, Platelets, Age, Gender, Hepatitis B; US, ultrasound. ###### The benefits vs limitations and risks of HCC surveillance ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Benefits of surveillance Limitations and potential harms of surveillance ------------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Allows diagnosis of HCC at an earlier stage Limitations in study design of current evidence such as lack of randomized controlled trials and lead time bias Reduces HCC mortality Financial cost\ • Questionable cost-effectiveness\ • Cost to health care system\ • Cost to patients Increases the rate of curative resection Limitations of surveillance tests\ • US -- operator dependent and sensitivity reduces in patients with central obesity\ • AFP -- poor sensitivity and poor specificity leading to false-positive and false-negative results US allows detection of other complications of cirrhosis such as ascites and portal vein thrombosis Up to 5% of patients have false-positive results\ Harms of false-positive results\ • Radiation exposure with cross-sectional imaging\ • Contrast risk\ • Risk of invasive procedures such as liver biopsy\ • Psychosocial harm to patient and family Unarguable value in well-defined target populations who have a high incidence of HCC and are fit for intervention Although HCC in NAFLD without cirrhosis is increasingly common, the target population is so large, and the incidence within the population is so small, surveillance in NAFLD patients without cirrhosis is not cost-effective and is not advocated ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Abbreviations:** AFP, alpha fetoprotein; HCC, hepatocellular carcinoma; NAFLD, non-alcoholic fatty liver disease; US, abdominal ultrasound. [^1]: These authors contributed equally to this work

The value of teaching preK-12 students the knowledge and skills needed for healthy living has long been recognized ([@bibr7-1524839919870196]; [@bibr33-1524839919870196]), and leaders in both public and school health education have long emphasized the role school health education should play in ensuring a healthy and health literate population ([@bibr7-1524839919870196]). In 2014, the Centers for Disease Control and Prevention (CDC) and ASCD, an education professional organization, collaboratively developed the Whole School, Whole Community, Whole Child (WSCC) framework for promoting health and academic success for school-age students. The WSCC framework includes 10 essential components, one of which is school-based health education ([@bibr9-1524839919870196]). In the years since its launch, the CDC, ASCD, and other supporting organizations have promoted the widespread adoption of the WSCC framework in schools, including strengthening health education. Distressingly, driven by controversial federal and state priorities, laws, and policies associated with high-stakes testing during the preceding years ([@bibr30-1524839919870196]), instruction in untested subjects, including health education, has been reduced or entirely eliminated in many schools in order to devote more attention to tested subjects, primarily reading, math, writing, and science ([@bibr37-1524839919870196]; [@bibr43-1524839919870196]). As a result, teaching and learning about health has become a low priority in many schools. For example, with the exception of violence prevention, requirements for teaching about priority health topics trended down in elementary and middle schools from 2000 to 2016 ([@bibr15-1524839919870196]). Eight in 10 U.S. school districts only required teaching about violence prevention in elementary schools and violence prevention plus tobacco use prevention in middle schools, while instruction in only seven health topics was required in most high schools ([@bibr15-1524839919870196]). Although 8 of every 10 districts required schools to follow either national, state, or district health education standards, just over a third assessed attainment of health standards at the elementary level while only half did so at the middle and high school levels. ([@bibr15-1524839919870196]). This erosion of the education system's commitment to health education is particularly troubling within the broader societal context. Public interest in health has become culturally pervasive as health information is more available, more sought out, and more accessed than ever before ([@bibr4-1524839919870196]). Technology has exponentially increased access to health misinformation and the sophisticated, dynamic, and rapid evolution of the health sciences requires more qualified and competent guidance from all types of health education professionals ([@bibr11-1524839919870196]). However, recent efforts to reestablish the importance of school health education have been met with slow progress, if not resistance. Initiatives designed to better implement school health education are intertwined with complex and sometimes combative education and political systems that may resist reform, misdiagnose challenges to service delivery, or blame individual teachers and schools for system-level problems ([@bibr28-1524839919870196]; [@bibr40-1524839919870196]; [@bibr39-1524839919870196]). Addressing these and other implementation challenges associated with our current education system demand new types of professional thinking about how to ensure the reliable, large-scale delivery of effective school health education. Although understanding how teachers can best deliver health instruction in their classrooms will always be important, to be truly effective, school health educators must also learn to (1) develop effective large-scale implementation capacities not typically emphasized in traditional health education teacher preparation programs and (2) enlist the support and assistance of all professional health educators as well as other advocates and partners. Therefore, the goal of this article is to propose a reinvigorated national pathway to ensure that every child in every school learns the functional health knowledge and achieves the level of health literacy necessary to live healthy, happy, and successful lives. To this end, four challenges to the reliable, large-scale implementation of effective school health education have been identified, along with corresponding proposals regarding the professional activities and growth necessary to overcome each challenge. These challenges were identified by the Society for Public Health Education (SOPHE) National Committee on the Future of School Health Education in the 21st Century. This committee includes a select group of 10 recognized leaders in school health education who have extensive experience as policy makers, practitioners, administrators, and scholars in school health, some of whom have maintained careers in the field that include 40 or more years of experience. This list of challenges was developed through a series of structured group discussions held by telephone conference over a 7-month period (February 2017--September 2017) through which common themes were identified, refined, and confirmed by group consensus. These four challenges are also part of a larger list of challenges identified by the committee, the rest of which are presented in two companion articles and a commentary that are included in this focus issue dedicated to the Future of School Health Education in the 21st Century. Theoretical and Historical Background {#section1-1524839919870196} ===================================== The challenges identified by the committee may best be considered through a historical lens informed by diffusion of innovations theory constructs ([@bibr38-1524839919870196]). Diffusion of innovations theory describes a series of stages through which an innovation may be developed, disseminated, adopted, implemented, and maintained in a sustained, routine manner over time. Clearly, not all innovations are adopted, implemented, or made a routine part of any given profession. Diffusion of innovations theory provides critical insights designed to identify challenges to the full diffusion of innovations as well as strategies meant to help various types of adopters successfully incorporate a given innovation into their work or lives (e.g., early adopters vs. late adopters). Comprehensive school health education became a recognized innovation designed to ensure the effectiveness of school health education following the completion of the School Health Education Study in 1972 ([@bibr24-1524839919870196]). It consisted of an approach to school health education that was planned across all grade levels, purposefully sequenced, emphasized skill building, and addressed multiple dimensions of health through 10 core content areas. In 1985, the School Health Education Evaluation funded by the U.S. Department of Health & Human Services, Office of Disease Prevention and Health Promotion, established that health instruction of 50 or more hours per year was effective for influencing knowledge, attitudes, and practices. In the early days of this innovation, increased opportunities for teacher professional development and access to resources played a critical role in the rapid expansion of comprehensive school health education curriculum and implementation ([@bibr18-1524839919870196]). Quickly, barriers to the successful adoption of comprehensive school health education began to emerge. For instance, single-issue topical health units for elementary, middle, and high schools began to be developed, evaluated, and disseminated ([@bibr23-1524839919870196]; [@bibr24-1524839919870196]; [@bibr35-1524839919870196]). Competition from these single-issue curricular units, often promoted by influential national organizations, began to emerge as competing innovations and barriers to the successful diffusion of comprehensive school health education ([@bibr43-1524839919870196]). These single issue units appear to have unintentionally distracted adopters and created confusion about the true nature and requirements of effective comprehensive school health education, as well as adding perceived pressures on school administrators to respond to multiple and competing demands (e.g., responding to multiple stake holders, competition for time in the overall health curriculum, redundancy in the health curriculum, unnecessary complexity). In essence, while most school health professionals advocated for comprehensive school health education, competition arising from the uncoordinated activities of other innovators in the public, nonprofit, and for-profit domains to disseminate single-issue units may have inadvertently derailed or slowed the adoption of comprehensive school health education. Early advocates for comprehensive school health education also faced two systemic barriers to adoption. These included (1) confusion about the change agent and (2) inability to achieve the final state in the innovation adoption process---routinizing ([@bibr38-1524839919870196]). Change agents influence innovation adoption decisions. In this case, the change agents were primarily federal governmental entities that also experienced varying degrees of change over time. Diffusion of innovations theory suggests that disruptions in the consistency or strength of opinion and implementation leadership may slow the adoption of innovations and impede efforts to routinize the innovation. Historical federal responses to the drug, HIV/AIDS, and obesity epidemics may serve as examples of the disruptive influence of shifting change agents. First, in response to the adolescent drug abuse epidemic in the 1980s, the U.S. Department of Education, Office of Safe and Drug-Free Schools (OSDFS) was identified as the primary change agency and provided pass through funds to school systems via state agencies to support personnel, policy development, teacher training, curriculum implementation, and surveillance surveys ([@bibr24-1524839919870196]). Having lost status over a number of years, OSDFS was eliminated by Congress in 2011 with some initiatives assumed by a new Office of Safe and Healthy Students ([@bibr48-1524839919870196]). Additionally, during the HIV/AIDS epidemic, the CDC Division of Adolescent and School Health (DASH) within the National Center for Chronic Disease Prevention and Health Promotion (NCCDPHP) emerged as another change agent in 1988 ([@bibr24-1524839919870196]). Uniquely, DASH funded HIV coordinators in all state and territorial education agencies (not state health agencies) and 15 to 20 large urban school districts to disseminate HIV/AIDS policies, teacher training, and curriculum development. DASH also developed and fielded the Youth Risk Behavior Survey and the School Health Profiles Survey at the national, state, and local levels ([@bibr16-1524839919870196]). Furthermore, DASH funded personnel in highly influential national education organizations such as the National School Boards Association, the National Association of State Boards of Education, and many others to support HIV/AIDS prevention in schools ([@bibr24-1524839919870196]). Beginning in the mid-1990s, DASH supported over 15 state education agencies annually to develop comprehensive school health infrastructure with funding for two school health directors (one in the public health agency), a health education coordinator, and a HIV coordinator. By the late 1990s, these efforts expanded to include a focus on physical activity, nutrition, and tobacco use prevention with added Congressional funding ([@bibr36-1524839919870196]). In 2011, however, DASH was relocated to the National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention. Today DASH has reduced its reach to state-level agencies and shifted its remaining resources to focus on HIV/AIDS prevention in 28 large urban school districts and six national organizations, none of which focuses primarily on education ([@bibr17-1524839919870196]). These funds are now used to support work in HIV/AIDS prevention, sexually transmitted disease, and pregnancy among teens. DASH also continues to support the implementation of the Youth Risk Behavior Survey and, until recently, the School Health Policies and Practices Study. CDC's NCCDPHP School Health Branch funds state education agencies, local education agencies, and national organizations to increase the quantity and quality of physical education, health education, and physical activity; improve the nutritional quality of foods provided in schools; and improve the capacity of schools to manage chronic conditions. Finally, the obesity epidemic is currently a high-profile child and adolescent health issue being addressed in schools. Due to the Local Wellness Policy mandate (unfunded) in the Healthy Hunger-Free Kids Act, the school health education leadership void was somewhat filled by state education agency National School Lunch Program personnel funded by the [@bibr46-1524839919870196]. Local school districts who participate in the National School Lunch Program are required to adopt a local wellness policy that includes goals for nutrition education and physical activity. USDA funding is provided to local school food service personnel who are also responsible for compliance but typically have no role in curriculum and instruction. With regard to obesity prevention, new, independent national organizations such as [@bibr1-1524839919870196] and the [@bibr2-1524839919870196] also emerged to engage schools; however, the impact on nutrition education seems minimal ([@bibr15-1524839919870196]). Together, the closure and federal reorganization of OSDFS and changes in funding to CDC may have contributed to a reduction in the overall number of school health education change agents and advocates. Likewise, as state education agencies were increasingly pressed for funding and focused on academic metrics related to reading, writing, math, and science, many school positions not explicitly focused those metrics were eliminated at both the state and local levels, including school health education ([@bibr39-1524839919870196]; [@bibr45-1524839919870196]). Diffusion of innovations theory suggests the importance of supporting and maintaining the strength and consistency of relevant change agents, as well as a degree of coordination and clarity of roles across agencies and systems, until full adoption and routinization of the innovation has occurred ([@bibr38-1524839919870196]). Although national organizations have emerged to provide leadership on specific issues (e.g., childhood obesity), the most recent results from the School Health Policies and Practices Study ([@bibr13-1524839919870196], [@bibr14-1524839919870196], [@bibr15-1524839919870196]) suggest a continued need (1) to marshal increased support for comprehensive school health education, (2) to strengthen change agents devoted to leading efforts to increase the adoption of comprehensive school health education, and (3) to better coordinate the activities of relevant change agents, especially efforts to align agencies and organizations devoted to single health issues with the comprehensive school health education approach. In summary, over the past 40 years, schools have been tasked with providing health education related to discrete health issues rather than comprehensive school health education. When new child and adolescent health problems emerged, state, and local school systems adopted policies, training, and instruction specific to each. Attention and resourcing were initially robust but, diminished over time as health priorities shifted and national-level change agents, advocates, and support systems experienced their own fluctuations in resources and responsibilities. Hence, the place of health education in the overall school curriculum remains tenuous. Furthermore, when reviewing recent historical factors influencing the adoption of comprehensive school health education through the lens of diffusion of innovations theory, it is clear that efforts to increase collaboration and coordination, decrease competition, and strengthen key change agents may enhance the adoption and routinization of comprehensive school health education. Therefore, by addressing the four challenges presented below, health educators working in schools, with the support and assistance of community health educators and other advocates, can promote the social and system-level conditions required to support, elevate, and ensure the reliable delivery of effective health education to every student in every school every year. Challenge 1: Establish School Health Education as an Undeniable Social and Cultural Priority through Improved Advocacy {#section2-1524839919870196} ====================================================================================================================== Explicit social values and priorities drive institutional commitments to building organizational capacity and distributing resources ([@bibr34-1524839919870196]). In this way, public institutions, including school systems, respond to perceived public imperatives with time, attention, and funding ([@bibr5-1524839919870196]). For example, the recent prominence and funding of Science, Technology, Engineering, and Math (STEM) subjects exemplifies the education system's response to strongly stated priorities with clear and compelling rationales and public support. The importance of containing health care costs ([@bibr26-1524839919870196]), the public's increasing interest in health information ([@bibr4-1524839919870196]), and historically high levels of parental support for school-based health education ([@bibr7-1524839919870196]) provide compelling confirmation of the importance of resourcing school health education. As evidenced by health education being legitimized for the first time in federal law through the Every Student Succeeds Act (2015), this narrative is gaining traction. Nevertheless, school health education must become an even higher priority within society-at-large and the education system. To this end, the health education profession may benefit from stronger advocacy at federal, state, and local levels. 1. **Recommended action:** Assign key national organizations the primary responsibility of advocating for delivery of effective school health education, along with coordinating and supporting national, state, and local advocacy work across multiple organizations. In many professional disciplines, there is one clear leading professional organization that serves as the "home base" for coordinating efforts to advocate for their given field. Currently, the field of school health education does not have a clear "home base" professional organization responsible for advocating for school health education as a whole. Recent history has led to multiple organizations competing for recognition as the leading professional advocacy and professional development organization within the field. Although having one clear "home base" organization may not be required to successfully advocate for school health education, the field would certainly benefit from---at a minimum---strong partnerships, open communications, and clear coordination of roles among all relevant organizations. To this end, either (1) identifying and supporting one lead "home base" organization for school health education advocacy or (2) developing and coordinating a coalition of relevant organizations able to work in a strong partnership, seems essential to maximizing our efforts to establish school health education as an undeniable social and cultural priority. As important as professional organizations can be when advocating for their profession, professional organizations devoted to school health education tend to have limited reach and may benefit from collaborating with better resourced and more powerful organizations. For example, SOPHE and other national professional organizations related to school health education were finally successful at advocating for the inclusion of health education in federal education law ([@bibr41-1524839919870196].) but this occurred 30 years after other subjects had been supported via Education Goals 2000 and so far no health education-specific funding has been allocated. Additionally, professionals working within public sector systems commonly face constraints and both can be viewed by policy makers as pursuing self-interests. Thus, an important task for a professional organization is to engage one or more highly influential, health-related organizations with clear vested interests in children's health, such as the American Academy of Pediatrics, the American Medical Association, or America's Health Insurance Plans, in funding and spearheading an advocacy and communications campaign promoting school health education (i.e., "health for every kid, every school, every day"). Involving such organizations can help exert political influences and generate other resources required to make school health education a clear public imperative dictated by explicit social values. Furthermore, non--health-related organizations might also play critical roles as partners committed to better establishing the importance of promoting children's health in schools as an undeniable cultural and social priority. Adding the supportive voices of educators more broadly---perhaps by more explicitly or more effectively partnering with education-focused professional development organizations serving teachers, school administrators, school board members, and other relevant school personnel---may add weight to arguments to expand health education in schools. Likewise, enhancing partnerships with parent-focused educational organizations may also provide similar opportunities and benefits. Finally, many academic subject areas also benefit from the coordinated efforts of national professional, philanthropic, and business advocacy groups that assume primary responsibility for generating, sustaining, and leveraging public support for specific school subjects; for example, the Arts or STEM education. Successful national advocacy organizations can channel the voices, energy, support of professionals, parents, community members, and highly credible and influential nongovernmental organizations ([@bibr7-1524839919870196]; [@bibr8-1524839919870196]; [@bibr44-1524839919870196]). Importantly, these types of supporters can join and reinforce our core national advocacy organization(s)' pursuit of clear, unified message delivery and system-level accountability. Challenge 2: Reform Educational Institutions to Strengthen their Capacity for Reliably Delivering Large-Scale, High-Quality, School-Based Health Education {#section3-1524839919870196} ========================================================================================================================================================== Most schools and school districts have met few of the Healthy People 2020 targets for health education ([@bibr13-1524839919870196], [@bibr14-1524839919870196], [@bibr15-1524839919870196]). Although the format of the 2016 School Health Policies and Practice Study (SHPPS) changed from previous years and did not directly report on schools' overall progress toward achieving the Healthy People 2020 targets, a range of trends suggested continued insufficient progress toward ensuring delivery of high-quality health education in every school ([@bibr15-1524839919870196]). Nevertheless, although the three most recent SHPPS reports indicate some progress in a few areas, such as improvement in rates of teaching violence and suicide prevention, overall findings from all three SHPPS reports suggest that the institutional capacity for ensuring the reliable and effective delivery of comprehensive school health education is less than desirable. Several historic policy events may have contributed to a lack of institutional capacity. The National Academy of Sciences 1997 report, *Schools and Health: Our Nation's Investment* ([@bibr24-1524839919870196]) recommended wide-ranging education system infrastructure reforms required to better support comprehensive school health education, highlighted needs at the federal, state, and local levels, and suggested a number of coordinating councils and partnerships at each level. However, the No Child Left Behind Act (PL107-110, [@bibr31-1524839919870196]) may have diverted attention from these types of partnerships to more institutionally affirmed priorities related to reading, writing, and mathematics test scores (Stanick, 2007). Subsequently, these still relevant recommendations appear to have been only partially implemented and/or inadequately resourced. Meanwhile, the capacities or foci of traditional governmental champions, including the Federal Interagency Committee on School Health, the National Coordinating Committee on School Health and Safety, and the CDC DASH, shifted. The 2015 Every Student Succeeds Act ([@bibr41-1524839919870196].) offers new possibilities for schools ([@bibr22-1524839919870196]) by recognizing a fuller range of previously ignored academic subjects, including delivery of health education. Nevertheless, students and schools will fully benefit only when such opportunities are driven by comprehensive systemic changes that reinforce the importance of school health education, along with the adoption of structural reforms within educational institutions. One obstacle to systems change may be educational leaders' limited understanding of the value of health education. School health education is typically portrayed as critical for addressing public health problems. Educational leaders are more likely to support comprehensive health education if aware of immediate benefits related to student learning and maintaining safe social--emotional school climates. Specific academic benefits of school health education include less disruptive student behavior, improved attention, and decreased absenteeism ([@bibr23-1524839919870196]; [@bibr32-1524839919870196]). Students who learn skills such as identifying and expressing emotions effectively, preventing and resolving conflict, communicating assertively, managing stress, and refusing to engage in inappropriate behavior are better prepared to function in today's collaborative classrooms ([@bibr19-1524839919870196]). Thus, school health educators' goal should be to forward arguments and support actions that correctly position school health education as central and essential to the educational enterprise, and to do so in a manner that engages all educators and administrators that care about the health, well-being, and growth of their students. 1. **Recommended action:** Align academic missions, structures, and systems to support the reliable delivery of effective school health education in a manner that positively affects student health and well-being. Systems theory suggests that institutional and organizational design matters ([@bibr42-1524839919870196]). Ambiguous priorities, noninstitutionalized goals, or underresourced initiatives are unlikely to be achieved ([@bibr5-1524839919870196]). Only by clearly aligning institutional and organizational missions, structure, and systems can the highest goals of schools and school health education be realized ([@bibr28-1524839919870196]). In *From Tactics to Strategy: Creating and Sustaining Social Conditions That Demand and Deliver Effective School Health Programs*, [@bibr28-1524839919870196] proposed *26 Indicators of Institutionally and Organizationally Resilient School Health Environments*. These indicators were meant to provide examples of how to operationalize a strategic approach to creating a professional environment in which successful implementation of quality school health programs would be likely, if not inevitable. Although the full list of indicators is too long to include here, some examples of efforts aimed at reforming institutional capacity to reliably deliver effective school health education include: - Aligning the federal Department of Education, state departments of education, school districts, and school missions to explicitly include promoting the health and well-being of students, including the effective delivery of health education. - Strengthening department of education, CDC, and other public/community health partnerships at federal, state, and local levels. - Delivering health education in the context of an array of initiatives designed to promote student health, such as the CDC/ASCD's WSCC framework ([@bibr9-1524839919870196]; [@bibr20-1524839919870196]). - Aligning all health education curricula with the National Health Education Standards ([@bibr12-1524839919870196]). - Developing or strengthening federal, state, district, and school professional development, educational program implementation, and evaluation capacities by (1) providing sustained direction and technical assistance and (2) requiring all academic leaders to develop a minimum level of expertise in student health promotion, evaluating student health and maintaining effective cross-organization partnerships with governmental and nongovernmental public health agencies and organizations ([@bibr25-1524839919870196]). - Establishing Director of School Health Education positions in all state and territory education agencies who are tasked with championing health education best practices and holding schools accountable for improving student health and well-being. - Adequately resourcing each of the efforts listed above, including using innovative models of cross-agency collaborative funding and resource sharing where possible. Although the selection of strategies briefly outlined above do not represent a comprehensive list of ways to strengthen the broader education system's capacity to reliably deliver large-scale, high-quality school-based health education, they might represent a feasible start. Regardless, they represent an important path forward---moving the role of school health and school health education from the periphery of the educational enterprise to its center. Developing and acting collectively on a unified set of targets for action represents a critical step in the reform process. Challenge 3: Create Formal Collaboration Mechanisms for Coordinating Efforts of Governmental and Nongovernmental Organizations Tasked with Promoting Health and Providing thought Leadership to the School Health Education Profession {#section4-1524839919870196} ====================================================================================================================================================================================================================================== During this century, various governmental and nongovernmental organizations have built national mechanisms that could assist with the implementation of several components of the WSCC framework. For example, the American Academy of Pediatrics Council on School Health collaborates with other organizations to continuously implement a wide range of priority actions for improving school-based clinical health services. With initial support from the CDC, an alliance of national organizations was established for building and ensuring long-term success of a National Physical Activity Plan. The Healthy, Hunger-Free Kids Act of 2010 enabled the USDA to drive school food service reforms for improving student health and educational achievement ([@bibr47-1524839919870196]). Unfortunately, the last national effort to improve comprehensive school health education occurred long ago. In 1992, the American Cancer Society enabled 125 experts from 40 national education, health, and social service organizations to produce a National Action Plan for Comprehensive School Health Education ([@bibr3-1524839919870196]). As a result, National Health Education Standards were developed, with performance indicators ([@bibr12-1524839919870196]). However, these standards have not been updated in more than a decade. Currently, no mechanism exists for enabling interested national or state-level organizations to collaboratively help implement effective school health education programs, although several organizations may have interest in so doing. For example, the SOPHE, whose members include school health education experts from universities and educational organizations nationwide, established a National Committee on the Future of School Health Education in the 21st Century; which spearheaded development of this multiarticle series. The American School Health Association, Society of State Leaders of Health and Physical Education, ASCD, and the Student Health Advocacy Coalition provide continued leadership, especially in terms of professional development and support for state and local education agencies. The CDC DASH and CDC School Health Branch provide critical federal leadership for school health education, as to a lesser extent do the Health Resources and Services Administration, the U.S. Department of Education, and the USDA. Furthermore, the Robert Wood Johnson Foundation has provided important leadership for school health education, especially in the context of the WSCC model. Despite this widespread interest, a critical need remains for establishing mechanisms to coordinate the efforts of groups interested in supporting the large-scale delivery of high-quality school health education. 1. **Recommended action:** National organizations listed above, and possibly others, collaboratively develop a sustainable national mechanism to progressively: (1) identify challenges that most impede the implementation of large-scale effective school health education nationwide, (2) take action to address a manageable number of high-leverage challenges, and (3) periodically report the results of each action over time. The organizations and agencies listed above, along with other interested organizations, can convene meetings and begin a dialogue focused on how to create a mechanism for providing thought leadership to the field, especially as related to overcoming implementation challenges. The national challenges and respective recommendations for action listed within this and the other companion articles in this series can provide a focus, rationale, and initial action plan for this group. Additionally, similar to the recommendations found in Challenge 1 and the model provided by the American Cancer Association's 1992 sponsorship of the National Action Plan for Comprehensive School Health Education, it may be helpful to pursue support and assistance from partner organizations able to invest the resources needed to initiate and maintain this mechanism. This newly established mechanism could also be used to review the National Health Education Standards and update them if deemed necessary. Challenge 4: Build Multidisciplinary Research Capacities Necessary to Solve Problems Associated with Ensuring the Reliable, Large-Scale Implementation of Effective School Health Education {#section5-1524839919870196} =========================================================================================================================================================================================== From the 1960s to today, studies have repeatedly found similar supports and barriers to effective health education implementation ([@bibr29-1524839919870196]). Three recurring barriers include lack of administrative support, inadequate teacher preparation, and chronically low teacher pay. Yet, these barriers persist ([@bibr10-1524839919870196]), suggesting the need to identify and adopt effective strategies for successfully addressing these and other system-level barriers. Nevertheless, recent database searches indicate that only a small number of research studies focused on addressing school health education implementation ([@bibr10-1524839919870196]). It is also particularly important to recognize that although numerous researchers and practitioners have identified a range of approaches, strategies, and programs that the scientific evidence suggests would benefit students in schools, much of this work goes unimplemented. Therefore, building the multidisciplinary research capacities and conducting the types of research necessary to illuminate the pathways between what is known to be effective and what becomes standard practice seems critically important. 1. **Recommended action:** Conduct new research that identifies and addresses persistent barriers to large-scale school health education implementation and sustainability ([@bibr7-1524839919870196]; [@bibr29-1524839919870196]). A research agenda for school health education, and perhaps school health more broadly, could be developed with the goal of directing future research toward agreed on professional priorities meant to move the field forward ([@bibr6-1524839919870196]). Similar research agendas have been developed by other groups of professional educators and researchers (e.g., the Association of Middle Level Educators), and these efforts have successfully guided research into much needed, but previously neglected areas. Established research agendas also provide guidance to senior researchers and university faculty members about the types of research they need to prepare emerging and developing researchers to conduct. An essential area of inquiry within this broader research agenda could be devoted to school health education-focused implementation science, systems analysis, and the promotion of widespread changes in social values and support for children's health and health education. Some specific examples of the types of research likely to promote more effective implementation may include (1) identifying messages, messengers, and incentives most likely to influence policy makers and educational administrators at all levels; (2) examining the effects of infusing education leadership/administration graduate programs with content related to the benefits of and skills required to implement the WSCC framework and coordinated school health approach, with emphasis on health education ([@bibr10-1524839919870196]); (3) evaluating the impact of programs that prepare principals to identify and hire teachers who are well prepared to deliver health education; or (4) comparing the relative performance of school districts that provide varying levels of priority, support, and resources to school health education. In each of these examples, and perhaps in implementation science more broadly, the need for school health education researchers to be prepared to work in multidisciplinary teams seems obvious. Therefore, enhancing capacity and eagerness to engage other researchers from teacher preparation, school administration, sociology, economics, political science, and public administration---as well as our counterparts in public and community health education---represents an essential step in completing the type of research needed to more consistently implement comprehensive school health education. Other factors, such as who conducts research ([@bibr21-1524839919870196]), how findings are disseminated, and who funds such research, may also need to be considered. Since most preservice programs for elementary and secondary teachers and the graduate preparation of principals are not conducted by health education faculty, partnerships between health education researchers and researchers with expertise in other education disciplines may prove fruitful. Research findings may accrue greater acceptance if presented at conferences tailored to education professionals, more generally, and teacher education/preparation faculty members. Likewise, research findings may be more appealing if published in both practitioner and research journals devoted to the broader field of education or tailored to teacher education/preparation professionals. Additionally, having research support from a private-sector funder(s), such as a not-for-profit entity or philanthropic foundation that is capable of supporting multiyear studies, would provide the sustainability required for producing actionable research findings. Conclusion {#section6-1524839919870196} ========== Recent explosions of scientific knowledge and technological advancement have dynamically transformed health knowledge. These transformations regarding what we know and how we learn about health, likewise demand transformations in how we teach future generations about health. More than ever, students' health knowledge, skills, and intentions to behave in healthy ways must be enabled and supported by their schools as well as their families, communities, and the greater culture ([@bibr27-1524839919870196]). Clearly, one transformation must include ensuring a basic level of health and health literacy by reliably providing high-quality, comprehensive health education through our national public school system. This outcome has long been a goal and finally achieving it will require addressing the social and system-level conditions and challenges that have stood in the way of doing so. In this regard, an underlying theme of this article has been to emphasize the need for health educators to command the broad implementation skills required to bolster social and cultural values related to ensuring children's health, strengthen and connect institutions, and lead systems change. In order to best support this transformation, school health educators will benefit from sharply focusing on and further developing a specified set of "next generation" implementation skills. To this end, a brief list of *Next Generation Implementation Skills for School-Based Health Promotion* is proposed. This list is meant to encapsulate the core skills necessary to build the capacity needed to respond to the four challenges described above and other implementation challenges yet to be identified. Each skill is essential for creating an environment more conducive to the large-scale implementation of effective school health as embodied by the WSCC framework, including comprehensive school health education. Furthermore, the collective development and deployment of these skills is likely to exert an increasingly powerful influence as they are collaboratively employed among health education professionals in all settings. These *Next Generation Implementation Skills* include 1. Accurately diagnosing root causes of challenges to implementation as they occur within the broader social context and the unique education system ecology ([@bibr27-1524839919870196]; [@bibr28-1524839919870196]). 2. Proposing social and system-level solutions, policies, and practices that support and elevate the accomplishments of individual schools and teachers and eliminate barriers to professional success ([@bibr7-1524839919870196]; [@bibr27-1524839919870196]; [@bibr28-1524839919870196]). 3. Ensuring proper accountability at all levels by evaluating and acting on data related to the effectiveness of policy makers, public institutions, and system-level leaders, in addition to individual teachers and local school administrators ([@bibr28-1524839919870196]; [@bibr39-1524839919870196]). 4. Acting collectively to recognize, collaborate, and coordinate work on issues too large and complex for change through isolated, individual efforts ([@bibr7-1524839919870196]; [@bibr25-1524839919870196]; [@bibr28-1524839919870196]). Garnering the capacity to address the four challenges presented above using these four *Next Generation Implementation Skills* as a foundation for action represents a new way forward for school health educators. Taken together, they detail clear and concrete professional actions and skills that, when activated, will enhance the implementation of reliable, large-scale, and effective school health education. Since these skills have not typically been included in professional preparation programs for entry-level school health educators, they may need to be obtained via professional development programming provided by professional organizations. These skills are certainly in alignment with and well-supported by the Certified Health Education Specialist or Master Certified Health Education Specialist Health Education Specialist Practice Analysis competencies related to communications and advocacy. Therefore, in order to ensure opportunities to develop and deepen these skills, it may be important to work closely with the National Commission for Health Education Credentialing and the designated providers they partner with to provide new professional development for school health educators focused on these skills. Ensuring high-quality, school-based health education for every child is unlikely to be fully achieved within our education system as it is today. Rather, for this goal to be achieved, school health educators and counterpart health educators working in public health, community health, higher education, and other health promotion-related settings must lead and champion newly proposed, constructive social and system-level solutions to large-scale implementation problems. Rising to meet these challenges will be professionally demanding, but doing so is possible and represents the most promising means of extending the full benefits of high-quality health education to all children in every school during our lifetimes. **Authors' Note:**This article is part of a special School Health Collection developed under the guidance of the Society for Public Health Education (SOPHE). SOPHE received funding from the CDC School Health Branch in the National Center for Chronic Disease Prevention and Health Promotion (Grant number 6 NU38OT000315-01-01) to support printing and open access dissemination. No federal funds were used in the development of these manuscripts and the views and findings expressed in them are those of the authors and are not meant to imply endorsement or reflect the views and policies of the U.S. Government.

KSHV encodes 17 miRs, derived from 12 pre-miRs, with largely unknown functions[@R2]--[@R5]. These miRs are located in the latent genomic locus and expressed during viral latency and in KS tumors[@R2]--[@R6]. Several KSHV miRs target cellular genes, and thus might play roles in the pathogenesis of KSHV-induced malignancies[@R7]--[@R12]. However, their roles in viral infection and replication remain unclear. To determine the function of KSHV miRs in viral lifecycle, we generated a viral mutant, ΔmiRs, with a cluster of 14 miRs (except miR-K10a/b and -K12) deleted and its revertant ΔmiRs_rt ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S1](#SD2){ref-type="supplementary-material"}). The recombinant viruses were reconstituted in 293T cells, which can be efficiently transfected with large viral DNA genomes, support KSHV persistent infection and replication, and are the only cell type used successfully in conjunction with the KSHV genetic system so far[@R13],[@R14]. ΔmiRs cells had no difference in morphology, latent nuclear antigen (LANA) staining pattern, and genome copy number per cell from wild-type virus (WT) cells ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S2a--c](#SD3){ref-type="supplementary-material"}). Quantitative real-time reverse transcription PCR (RT-qPCR) confirmed the deletion of the miR cluster ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S2d](#SD3){ref-type="supplementary-material"}). WT and ΔmiRs cells had similar expression levels of latent genes vFLIP and vCyclin in uninduced cells and cells induced with 12-*O*-tetradecanoyl-phorbol-13-acetate and sodium butyrate (T/B) for viral lytic replication ([Fig. 1a](#F1){ref-type="fig"}, first panel). The expression of latent gene LANA was also unchanged in uninduced cells following the miR cluster deletion but was marginally higher in WT than ΔmiRs cells following lytic induction ([Fig. 1a](#F1){ref-type="fig"}, first panel). Immunofluorescence staining showed that WT and ΔmiRs cells had similar expression levels of LANA protein ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S2b](#SD3){ref-type="supplementary-material"}). In contrast, the expression level of replication and transcription activator (RTA), which activates viral lytic replication, was 4.2-fold higher in ΔmiRs cells than WT cells ([Fig. 1a](#F1){ref-type="fig"}, second panel). While RTA level in WT cells was increased by 3.2-fold following lytic induction, it remained higher in ΔmiRs cells by 2.3-fold. Similar results were observed with KSHV late lytic protein major capsid protein (MCP) ([Fig. 1a](#F1){ref-type="fig"}, third panel). Examination of expression kinetics showed that ΔmiRs cells had significantly higher expression levels of both RTA and MCP throughout the entire lytic induction period ([Fig. 1b, c](#F1){ref-type="fig"}). In addition, the RTA promoter activity was 1.6- and 2.2-fold higher in uninduced and T/B-induced ΔmiRs cells than those of WT cells, respectively ([Fig. 1d](#F1){ref-type="fig"}). Uninduced and T/B-induced ΔmiRs cells also had 3.1- and 4.6-fold more cells stained positive for early viral lytic protein ORF59 than WT cells had, respectively ([Fig. 1e](#F1){ref-type="fig"}). Consistent with these results, ΔmiRs cells produced 2.4-fold more viral particles than WT cells did following lytic induction ([Fig. 1f](#F1){ref-type="fig"}). This enhanced effect on viral lytic replication following the deletion of miR cluster was specific because viral replication in ΔmiRs_rt cells was reverted to WT cells as reflected by the RTA and MCP expression levels ([Fig. 1g](#F1){ref-type="fig"}). Furthermore, stable expression of the miR cluster in ΔmiRs cells, which resulted in the expression of miRs at levels similar to those of WT cells ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S3a](#SD4){ref-type="supplementary-material"}), reduced the RTA level by 50% and 41% in uninduced and T/B-induced cells, respectively ([Fig. 1h](#F1){ref-type="fig"}), indicating that the cluster could function in trans. Similar results were observed with MCP. Together, these results indicated that the miR cluster inhibited the expression of viral lytic genes in latent cells, and in cells induced for viral lytic replication. In a previous study[@R15], computational analysis has predicted several putative targeting sites for KSHV miR-K6 in the 3'UTRs of RTA and ZTA, which shared multiple polycistronic transcripts with the same 3'UTR sequences[@R16]. However, we failed to detect any effects of KSHV miRs on these transcripts in 3'UTR reporter assays ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S4](#SD5){ref-type="supplementary-material"}). Since NF-κB pathway mediates vFLIP inhibition of KSHV lytic replication[@R17], we determined the NF-κB status in ΔmiRs cells. The NF-κB activity was 2.2-fold weaker in ΔmiRs cells than in WT cells in a reporter assay ([Fig. 2a](#F2){ref-type="fig"}). While the NF-κB activity was increased by 3.6-fold in WT cells following T/B induction, it remained 1.8-fold lower in ΔmiRs cells than in WT cells. Consistent with these results, a weaker DNA-protein NF-κB complex band was detected by a gel shift assay in ΔmiRs cells than in WT cells with or without lytic induction ([Fig. 2b, c](#F2){ref-type="fig"}). This band was abolished by specific competitor and supershifted by antibodies to cRel, p50, and p65 but not a control antibody, confirming it as the cRel/p50/p65 NF-κB complex. Furthermore, ΔmiRs cells had substantial less p65 nuclear staining than WT cells ([Fig. 2d](#F2){ref-type="fig"}). While T/B treatment increased p65 nuclear staining in both cell types, those of ΔmiRs cells remained weaker than WT cells. Similar results were also observed with p50 and cRel ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S5](#SD6){ref-type="supplementary-material"}). Thus, the NF-κB activity was lower in ΔmiRs cells than in WT cells. We then determined whether the miR cluster could function in trans to regulate NF-κB pathway. Expression of the miR cluster increased the NF-κB activity by 2.1-fold in ΔmiRs cells ([Fig. 2e](#F2){ref-type="fig"}). Although T/B induction increased the NF-κB activity by 4.8-fold in ΔmiRs cells, the miR cluster further increased it by 1.3-fold. Similar results were observed in WT cells though these cells had higher basal NF-κB activity. Expression of the miR cluster alone in uninduced and T/B-induced KSHV-negative cells also increased the NF-κB activity by 2.0- and 1.4-fold, respectively. These results indicated that the miR cluster not only rescued the NF-κB activity in ΔmiRs cells but also itself was sufficient to enhance NF-κB pathway in KSHV-negative cells. Next, we determined whether NF-κB pathway mediates miR cluster inhibition of viral replication. While the miR cluster suppressed RTA and MCP expression, this effect was reversed by a mutant of the NF-κB complex inhibitor IκBα (NF-κB_DN) in both uninduced and T/B-induced ΔmiRs cells ([Fig. 2f, g](#F2){ref-type="fig"}). NF-κB_DN also increased the expression of RTA and MCP in uninduced and T/B-induced WT and ΔmiRs cells ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S6](#SD7){ref-type="supplementary-material"}). Thus, we concluded that inhibition of NF-κB pathway enhanced viral lytic replication, and the suppression of viral lytic replication by the miR cluster could be mediated by this pathway. To identify the target that mediated miR cluster suppression of the NF-κB pathway, we examined the IKK members. Consistent with NF-κB activity ([Fig. 2b](#F2){ref-type="fig"}), the levels of IKKα, IKKβ and IKKγ proteins were higher in WT than in mock cells ([Fig. 3a](#F3){ref-type="fig"}). However, ΔmiR cells also had IKKα, IKKβ and IKKγ protein levels at least comparable to or slightly higher than WT cells ([Fig. 3a](#F3){ref-type="fig"}) albeit their lower NF-κB activity ([Fig. 2a--d](#F2){ref-type="fig"}), indicating that the reduced NF-κB activity in ΔmiR cells was unlikely mediated by the IKK members. Upon T/B induction, the levels of all three proteins were increased but were similar among the three cell types ([Fig. 3a](#F3){ref-type="fig"}), reflecting the stimulating effect of T/B on NF-κB pathway. We further examined the IκB family members. The level of IκBβ protein was reduced in WT and ΔmiR cells ([Fig. 3a](#F3){ref-type="fig"}), which was consistent with their NF-κB activities ([Fig. 2b](#F2){ref-type="fig"}). Upon T/B induction, the expression of IκBβ protein was further reduced but to comparable levels among the three cell types ([Fig. 3a](#F3){ref-type="fig"}). Constitutive activation of NF-κB activity could increase the levels of IκBα and IκBε through a feedback loop[@R18],[@R19]. Indeed, we detected higher IκBε level in both WT and ΔmiR cells than mock cells ([Fig. 3a](#F3){ref-type="fig"}). T/B induction increased the IκBε levels in all three cell types. In contrast to IκBε, the level of IκBα in WT cells was almost the same as the mock cells ([Fig. 3a](#F3){ref-type="fig"}), suggesting that IκBα was suppressed by another factor(s). Deletion of the miR cluster significantly increased IκBα level by 1.51-fold ([Fig. 3a](#F3){ref-type="fig"}). While IκBα levels were reduced in all three cell types following T/B induction, those of ΔmiR cells remained 2.82-fold higher than WT cells. These results indicated that IκBα might be a target of the miR cluster. Indeed, overexpression of the miR cluster in KSHV-infected BCP-1 PEL cells[@R20] reduced IκBα protein level by 63% in uninduced cells and by 73% in T/B induced cells ([Fig. 3b](#F3){ref-type="fig"}). In contrast, under the same condition, the levels of IκBε were only marginally affected by the miR cluster with a reduction of 12% and 11%, respectively ([Fig. 3b](#F3){ref-type="fig"}). Consistent with these results, overexpression of the miR cluster in BCP-1 cells increased NF-κB activity by 3.0- and 1.97-fold in uninduced and T/B-induced cells, respectively ([Fig. 3c](#F3){ref-type="fig"}). To further confirm that IκBα is targeted by the miR cluster, we performed a reporter assay with a IκBα 3'UTR reporter. As shown in ([Fig. 3d](#F3){ref-type="fig"}), the IκBα 3'UTR reporter activity was 2.5-fold higher in ΔmiRs cells than in WT cells. Furthermore, the miR cluster could function in trans in ΔmiRs cells to reduce the reporter activity by as much as 50% ([Fig. 3e](#F3){ref-type="fig"}). Thus, the miR cluster directly targeted the IκBα 3'UTR. We used the 3'UTR reporter assay to screen the 14 miRs in the cluster, and identified miR-K1 as the miR that targeted IκBα ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S7a](#SD8){ref-type="supplementary-material"}). As shown in [Fig. 3f](#F3){ref-type="fig"}, miR-K1 reduced the IκBα 3'UTR reporter activity by 30% while both miR-K3 and miR-K11 did not reduce the reporter activity. The miR expression constructs were functional as they suppressed the activities of their sensor reporters containing perfect matching sequences of their respective miRs ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S7b](#SD8){ref-type="supplementary-material"}). The suppression effect of miR-K1 on IκBα 3'UTR reporter activity increased with its increasing expression level ([Fig. 3g](#F3){ref-type="fig"}; [Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S3b](#SD4){ref-type="supplementary-material"}). While deletion of the miR cluster from KSHV genome increased the IκBα 3'UTR reporter activity ([Fig. 3d](#F3){ref-type="fig"}), expression of miR-K1 in ΔmiRs cells, which resulted in miR-K1 expression level similar to that of WT cells ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S3c](#SD4){ref-type="supplementary-material"}), reversed the trend and reduced the reporter activity by 35% ([Fig. 3h](#F3){ref-type="fig"}). The interaction of a miR with its target is primarily mediated by a 6- to 7-bp seed sequence at the 5'-end of the miR[@R21]. We identified two putative miR-K1 binding sites (S1 and S2) in the IκBα 3'UTR using a miR target prediction software miRanda[@R22] ([Fig. 3i](#F3){ref-type="fig"}). Deletion of both sites from the IκBα 3'UTR totally abolished the inhibitory effect of miR-K1 on the reporter while deletion of any one sites had no detectable effects ([Fig. 3j](#F3){ref-type="fig"}). We further generated reporters with different tandem repeats of the sites. As shown in [Fig. 3k](#F3){ref-type="fig"}, the inhibitory effect of miR-K1 on the reporters increased with the number of the repeats for both sites, reaching 70% and 67% for S1 and S2, respectively, when the reporters containing three repeats of the binding sites were examined. Furthermore, expression of miR-K1 with or without the viral genome significantly reduced the IκBα protein level ([Fig. 3l, m](#F3){ref-type="fig"}; [Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S8](#SD9){ref-type="supplementary-material"}). These results clearly indicated that miR-K1 suppressed the IκBα protein by directly targeting its 3'UTR. The identification of miR-K1 as the miR targeting IκBα indicated that it should regulate NF-κB pathway. Indeed, expression of miR-K1 enhanced NF-κB activity in a dose-dependent fashion ([Fig. 4a](#F4){ref-type="fig"}; [Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S3b](#SD4){ref-type="supplementary-material"}). While deletion of the miR cluster reduced the NF-κB activity in KSHV-infected cells ([Fig. 2a](#F2){ref-type="fig"}), expression of miR-K1 was sufficient to rescue the NF-κB activity ([Fig. 4b](#F4){ref-type="fig"}). We further inhibited the function of miR-K1 in KSHV-infected PEL cells using a locked nucleic acids/DNA-mixed oligonucleotide (miR-K1 suppressor), which resulted in the reduction of NF-κB activity by 40% ([Fig. 4c](#F4){ref-type="fig"}). In contrast, we observed minimal effects on the NF-κB activity when suppressor of miR-K3 or -K8 was used ([Fig. 4c](#F4){ref-type="fig"}). As expected, the suppressors reduced the levels of their respective miRs by 82% to 99% ([Fig. 4d](#F4){ref-type="fig"}). Furthermore, miR-K1 suppressor efficiently relieved the suppressive effect of miR-K1 on the activity of its sensor reporter by 1.74-fold ([Fig. 4e](#F4){ref-type="fig"}) and significantly increased the IκBα protein level ([Fig. 4f](#F4){ref-type="fig"}), indicating that it was fully functional. Finally, we examined the role of miR-K1 in viral lytic replication. While deletion of the miR cluster enhanced viral lytic replication as reflected by increased expression levels of RTA and MCP ([Fig. 1a--c](#F1){ref-type="fig"}), expression of miR-K1 reversed the trend, and reduced the levels of RTA and MCP transcripts by 68% and 46% in uninduced ΔmiRs cells, respectively, and by 65% and 38% in T/B-induced ΔmiRs cells, respectively ([Fig. 4g, h](#F4){ref-type="fig"}). Consistently, miR-K1 suppressor increased the expression of RTA and MCP transcripts in BCP-1 cells by 2.3- and 2.6-fold, respectively, and by 1.5- and 1.4-fold following T/B induction, respectively ([Fig. 4i, j](#F4){ref-type="fig"}). In contrast, we observed minimal effects on the expression of RTA and MCP transcripts when suppressor of miR-K3 or -K8 was used ([Fig. 4i, j](#F4){ref-type="fig"}). The miR-K1 suppressor also increased the expression of lytic protein ORF59 from 2.4% to 8.2% (3.4-fold) in uninduced PEL cells, and from 9.7% to 23% (2.4-fold) following T/B induction ([Fig. 4k, l](#F4){ref-type="fig"}). In Northern hybridization, the miR-K1 suppressor increased the expression of Pan transcript, one of the most abundant viral lytic transcripts, by 3.7- and 3.2-fold, and ORF57 transcript, another viral lytic transcript, by 2.6- and 2.1-fold in uninduced and T/B-induced PEL cells, respectively ([Fig. 4m](#F4){ref-type="fig"}). These results clearly showed that miR-K1 suppressed viral lytic replication. Several recent studies have shown that miRs of herpesviruses suppress viral replication by directly targeting viral genes[@R15],[@R23]--[@R26]. Our results show that KSHV encodes a miR to suppress vial replication by regulating cellular NF-κB pathway. Thus, herpesviruses appear to exploit the miR-mediated suppression pathway as a common mechanism to inhibit viral replication and regulate latency. Both vFLIP and LANA also regulate KSHV replication[@R17],[@R27],[@R28]. Hence, KSHV has evolved multiple mechanisms to suppress viral lytic replication to achieve latency ([Fig. 4n](#F4){ref-type="fig"}). The NF-κB pathway regulates innate and adaptive immunity, cell survival, and inflammation[@R29]. Oncogenic gammaherpesviruses use multiple strategies to regulate NF-κB activity. Both EBV LMP1 and vFLIP activate NF-κB pathway to promote cell growth and survival, and contribute to viral latency[@R30]--[@R32]. Our results support a general role of NF-κB pathway in regulating the latency of gammaherpesviruses through two interrelated mechanisms, by inhibiting viral replication and by promoting cell survival ([Fig. 4n](#F4){ref-type="fig"}). METHODS {#S1} ======= Methods and any associated references are available in the online version of the paper. METHODS {#S2} ======= Construction of ΔmiRs and ΔmiRs_rt {#S3} ---------------------------------- The procedures for generating a KSHV mutant with a cluster of 14 KSHV miRs (except miR-K10a/b and -K12) deleted (ΔmiRs) and its revertant (ΔmiRs_rt) were previously described[@R13]. The strategy is illustrated in [Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S1a](#SD2){ref-type="supplementary-material"}. We first obtained a PCR product with primers 5'TTGAATACAGTTGGGGGTAGTCCGCTGGTATTCCCAGCTGAGGTTGCCTTAGTGTAGGCTGGAGCTGCTTC3' and 5'GTGCTTCTGTTTGAAGGCGAATAAAACAGGAAGCGGGTTGGACTGGCAGGGTCATATGAATATCCTCCTTAG3' using plasmid pMS102-Zeocin as a template. This PCR fragment contained a zeocin-resistance cassette (Zeo^R^) flanked by loxP sites and 50 bp sequences from the two ends of the miR cluster (genomic position: 122,055 and 119,161). The product was electroporated into *E. coli* strain DH10B containing the wild-type virus BAC36 (WT) and the helper plasmid pGET-rec to facilitate homologous recombination. Zeocin-resistant colonies containing KSHV genomes with the miR cluster deleted and replaced by the Zeo^R^ were selected. To remove the Zeo^R^, a Cre-expression plasmid pCTP-T carrying a tetracycline-resistance gene (Tc^R^), a temperature sensitive origin of replication, and the Cre recombinase gene under the control of the tetracycline-responsive promoter, was introduced into the zeocin-resistant bacteria by transformation. The bacteria were then cultured in LB media with tetracycline at 50 µg/ml for 3 h at 30°C to allow Cre expression and site-specific recombination between the two LoxP sites resulting in the excision of the Zeo^R^. The tetracycline-resistant colonies were selected and verified for the presence of KSHV genome and removal of the Zeo^R^. The Cre-expression plasmid was eliminated by culturing the bacteria at 42°C. The revertant ΔmiRs_rt was generated in two steps using ΔmiRs as a template. First, two PCR products were generated. One product was obtained using primers 5'ATACTCGAGGTGTAGGCTGGAGCTGCTTC3' and 5'GTATCTGATTTAATAAACACTAACAAGTTTTGTAAGAATCATTAGAATGCCATATGAATATCCTCCTTAG3', and pMS102-Zeocin as a template. This fragment contained the Zeo^R^ flanked by LoxP sites together with a fragment of 50 bp KSHV sequence (genome position: 122056--122106) at one end and an XhoI restriction site at the other end. A second product was obtained using primers 5'GACCCAGCTGGTTTCCATAAATGGATATACTTCCGGAAAACGAAGGAGGG3' and 5\'ATACTCGAGGTGCTTCTGTTTGAAGGCGAATAAAACAGGAAGCGGGTTGGACTGGCAGGGT3\', and BAC36 DNA as a template. This product contained the sequence of the entire miR cluster with an XhoI site at one end. Second, these two PCR products were ligated after XhoI digestion resulting in a cassette containing the miR cluster and Zeo^R^, which was then electroporated into *E. coli* DH10 containing ΔmiRs. Selection of colonies containing KSHV genomes with the miR cluster repaired followed by excision of the Zeo^R^ led to the generation of ΔmiRs \_rt. Cell culture and virus induction {#S4} -------------------------------- Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium with 10% of fetal bovine serum (FBS). For stable selection of cultures of recombinant viruses, hygromycin at 200 µg/ml was added to the media. KSHV-infected primary effusion lymphoma (PEL) BCP-1 cells[@R20] were cultured in RPMI-1640 with 10% of FBS. To induce viral lytic replication, cells were treated with 20 ng/ml of TPA and 0.25 mM sodium butyrate (T/B) for the specified lengths of time. To determine virion production, supernatants from cells induced for 5 days were collected and filtered through 0.45 µm filters. Virions were pelleted by ultracentrifugation, treated sequentially by Turbo DNAase I (Ambion, Austin, TX), and proteinase K. Virion DNA was determined by quantitative real-time PCR (qPCR) following DNA extraction. Purified BAC36 DNA was used as a copy number control. Quantitative real-time reverse transcription PCR (RT-qPCR) {#S5} ---------------------------------------------------------- Total RNA isolated with Trizol reagent (Invitrogen, Carlsbad, CA) was treated with DNase I. RT-qPCR for KSHV genes was carried out as previously described[@R33]. Real-time quantification of miRs was carried out by stem-loop RT-qPCR[@R34]. A reverse transcriptase reaction was first carried out by incubating the RNA sample with a stem-loop RT primer for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C, and then held at 4°C. qPCR was performed on a 7700HT Sequence Detection System (Applied Biosystems, Foster City, CA). The reactions were carried out in a 96-well plate at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and then 60°C for 1 min. All the reactions were run in triplicates. The differences of cycle threshold values (C~T~) between the samples (ΔC~T~) were calculated after calibration with GAPDH and converted to fold changes using one of the samples as a standard (1-fold). The primers used in RT-qPCR are listed in [Supplementary Table 1](#SD1){ref-type="supplementary-material"}. Plasmids {#S6} -------- KSHV RTA promoter-luciferase reporter plasmid was described previously[@R35]. NF-κB reporter plasmid, containing tandem repeats of consensus NF-κB binding sites, was obtained from Dr. Bill Sudgen. NF-κB DN plasmid pIκB-αM was kindly provided by Dr. Paul J. Chiao. The construction of IκBα 3'UTR luciferase reporter plasmid IκBα3UTRluc (IκBα-WT) was carried out by inserting the full-length of IκBα 3'UTR sequence downstream of the luciferase sequence into the pGL3 vector. Deletions of the two putative miR-K1 binding sites S1 and S2 from IκBα 3'UTR were performed by site-directed mutagenesis using primers 5'GTGGTACATGTAACAGCCAGGAGTGTTAAGCGTTC3' and 5'TACATGTACCACTGGGGTCAGTCACTCGAAGC3' for site 1, and primers 5'GTTACCCATGGTGTACATAATGTATTGTTGG3', 5'TACACCATGGGTAACACAAACCTTGACAGG3' for site 2 ([Fig. 3i](#F3){ref-type="fig"}). Corresponding reporters were named IκBα3UTRlucΔS1 (ΔS1), IκBα3UTRlucΔS2 (ΔS2), IκBα3UTRlucΔS1+2 (ΔS1+2), respectively. Reporters containing different numbers of tandem repeats of the miR-K1 binding sites were generated by fusing the repeats through Hind III sites and cloning the products into the pGL3 vector. To construct 3'UTR luciferase reporter plasmids of RTA and ZTA type I, II and III transcripts[@R16], we inserted their full-length 3'UTR sequences into the pGL3 vector downstream of the luciferase sequence between KpnI and XhoI sites after PCR amplification of cDNAs with the following primers RTA3\'UTR-KpnI-F: 5'TTTTGGTACCAGTGTTCGCAAGGGCGTCTGTGCCT3' and RTA3\'UTR-XhoI-R: 5'TCTC CTCGAGGTAGGGTTTCTTACGCCGGCATCGT3'. Sequences of RTA and ZTA transcripts were based on AF091348, AF091349 and AF091350. To express the miR cluster or individual KSHV miRs, the whole cluster or individual miR fragments were cloned by PCR amplification as previously described[@R36], and inserted between BglII and HindIII sites of an expression vector pSUPER.retro.puro (OligoEngine, Seattle, WA). The sensor plasmids for KSHV miRs containing two repeats of perfect matching sequences of the respective miRs were described previously[@R36]. Reporter assays {#S7} --------------- The luciferase reporter constructs and β-galactosidase expression plasmid pSV-β-gal (Promega, Madison, WI) were cotransfected into cells cultured in 24-well plates using the F2 transfection reagent (Targeting Systems, El Cajon, CA). For induction, cells transfected for 24 h were treated with T/B for 12 h. Other reporter assays were performed with the indicated expression plasmids and lengths of transfection time. Cells were then lysed, and the luciferase and β-gal activities were measured using luciferase and β-galactosidase kits (Promega). Luciferase activity was normalized to β-galactosidase activity. Results were calculated as means ± SEM from at least three independent experiments, each in triplicates except [Fig 4a](#F4){ref-type="fig"}, which the means were calculated from two independent experiments, each in triplicates. Western-blotting {#S8} ---------------- Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes as previously described[@R37]. The membranes were first blocked with 5% non-fat milk and then incubated with a primary antibody followed with a horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma, Life Science, St. Louis, MO). Specific bands were revealed with chemiluminescence substrates (Roche, Nutley, NJ) and recorded with an IS2000MM Imaging Scanner (Eastman Kodak Company, Rochester, NY). Antibodies to the following proteins were used: IKKα, IKKβ, IKKγ, IκBα, IκBβ, and IκBε, all from GeneTex (San Antonio, TX), and β-tubulin (Sigma). Immunofluorescence assay (IFA) {#S9} ------------------------------ Cells grown on cover slid were first fixed with methanol or 1% paraformaldehyde, and blocked with 10% FBS. Cells were then incubated with primary antibody, and specific signal was detected with a secondary antibody conjugated with Alexa Fluor 568 (Invitrogen). The cells were counter-stained with 4', 6'-diamidino-2-phenylindole (DAPI). Images were observed and recorded with a Zeiss Axiovert 200 M epifluorescence microscope (Carl Zeiss, Inc., Thornwood, NY). Antibodies to p65, p50 and cRel (Calbiochem, Gibbstown, NJ), ORF59 (a gift of Dr. Bala Chandran), and LANA (ABI, New York, NY) were used. Electrophoretic mobility shift assay (EMSA) {#S10} ------------------------------------------- Nuclear extracts were prepared as previously described[@R35]. Annealed double-stranded oligonucleotides containing a NF-κB consensus site (5'AGTTGAGGGACTTTCCTT3') were labeled with \[γ-^32^p\]ATP. For gel shift assay, 4 µg of the nuclear extract was incubated for 20 min with 5 × 10^5^ cpm of labeled probe in 20 µl of binding buffer containing 10 mM Tris-HCl at pH 7.6, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 1 µg/µl bovine serum albumin, and 2 µg of poly(dI-dC). A competition reaction was carried out in the same manner except that the mixture was pre-incubated with excess cold probe or an irrelevant oligonucleotide for 10 min before the addition of labeled probe. To detect NF-κB complexes, a supershift assay was carried out by adding 1 µg of a specific antibody to p65, p50, or c-Rel to the reaction followed by an additional 15 min of incubation. Corresponding IgG from a normal animal was used as a control. The reaction mixtures were separated in 6% polyacrylamide gels. Northern-blot hybridization {#S11} --------------------------- Total RNA was fractionated on a 1% formaldehyde agarose gel, transferred to nylon membranes, and hybridized with 32P-labeled riboprobes of Pan and ORF57[@R38]. miR-K1 suppressor {#S12} ----------------- We inhibited the functions of miRs using locked nucleic acids (LNAs)/DNA-mixed oligonucleotides complementary to the miR sequences (miR suppressors). An antisense oligonucleotide containing LNAs forms highly specific and stable duplexes with the complementary RNA. When bound to a miR, the oligonucleotide prevents it from interacting with the RISC protein complex and its target[@R39]. Oligonucleotide suppressors for miR-K1 (5'GCTTACA[CCCAGTTT]{.ul}CCTGTAAT3'), -K3 (5'CGCTGCC[GTCCTCAG]{.ul}AATGTGA3') and -K8 (5'CGTGCTC[TCTCAGTC]{.ul}GCGCCTA3'), and a scrambled control (5\'CATTAAT[GTCGGACA]{.ul}ACTCAAT3\'), all containing LNAs at eight consecutive centrally located bases (underlined) were designed[@R39] and synthesized (Sigma). To suppress the function of a miR, 10 nM of the LNA oligonucleotide were transfected into BCP-1 cells using the siPORT NeoFX kit (Applied Biosystems). Supplementary Material {#SM} ====================== We thank Kenneth Izumi and Anthony Griffiths for their valuable suggestions. This work was supported by grants from American Cancer Society (\#RSG-04-195) and National Institute of Health (CA096512, CA124332, CA132637 and DE017333) to S-J Gao. C. G. Kim was supported by Konkuk University in 2008. **AUTHOR CONTRIBUTIONS** Data of genetics, viral replication, NF-κB-related analysis, and microRNA suppressor were obtained by X.F.L ([Fig. 1](#F1){ref-type="fig"}--[4](#F4){ref-type="fig"}, [Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S1](#SD2){ref-type="supplementary-material"}-[S3](#SD4){ref-type="supplementary-material"}, [S5](#SD6){ref-type="supplementary-material"}, [S6](#SD7){ref-type="supplementary-material"}, [S8](#SD9){ref-type="supplementary-material"}); bioinformatics, mutagenesis of the miR target, and part of the genetics and viral replication were conducted by Z.Q.B. ([Fig. 2](#F2){ref-type="fig"}--[3](#F3){ref-type="fig"}, [Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S1](#SD2){ref-type="supplementary-material"}, [S4](#SD5){ref-type="supplementary-material"}, [S7](#SD8){ref-type="supplementary-material"}, [S8](#SD9){ref-type="supplementary-material"}); EMSA was conducted by X.P.X. ([Fig. 2](#F2){ref-type="fig"}); F.C.Y. contributed to the viral genetics and Northern-blotting ([Fig. 4](#F4){ref-type="fig"}, [Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S1](#SD2){ref-type="supplementary-material"}). C.G.K. contributed to target identification ([Supplementary Information](#SD1){ref-type="supplementary-material"}, [Fig. S7](#SD8){ref-type="supplementary-material"}). Y.F.H. contributed to bioinformatics ([Fig. 3](#F3){ref-type="fig"}). S.J.G. direct, design and analyze all the experiments and data. Manuscript was written by X.F.L. and S.J.G. **COMPETING FINANCIAL INTERESTS** The authors declare no competing financial interests. **SUPPLEMENTARY INFORMATION** Supplementary Information is linked to the online version of the paper. {#F1} {#F2} {#F3} {#F4} [^1]: These authors contributed equally to this work.

INTRODUCTION ============ Worldwide overland transport business requires several millions of an employee, most of whom consist of male truck driver ([@b48-jer-14-2-313]). Tasks of cargo truck is to drive for distribution of various cargos, therefore the continuous observation of traffic situation and locomotion due to tight schedule of distribution etc. results in an increase of mental fatigue by overwork load ([@b48-jer-14-2-313]). In addition to fatigue of truck drivers, they were always exposed to lumbar back pain and disorder with excessive vibration against whole body ([@b5-jer-14-2-313]; [@b43-jer-14-2-313]), epidemiology of which showed more cases of disorder of back pain and handicap of vertebrae disk in exposed group to vibration than as was not ([@b4-jer-14-2-313]; [@b20-jer-14-2-313]). Also [@b2-jer-14-2-313] reported that 249 of 489 (59%) truck driver experienced various the back pain of vertebrae. That is, truck driver encountering the several dangerous factors due to long-time driving steps down ordinarily not only in a condition of increased pain of muscular skeletal part of waist, neck, shoulder ect. ([@b18-jer-14-2-313]; [@b26-jer-14-2-313]; [@b38-jer-14-2-313]; [@b49-jer-14-2-313]), but also of lowered level of sensibility and motor ability. Because of truck's higher seat height made for cargo transportation in contrast to other vehicles, most drivers during up and downward stepping must be ready to suffer the falling accident both inconvenience and falling risk simultaneously ([@b21-jer-14-2-313]). [@b39-jer-14-2-313] of preceded study related with downward stepping on bus stair reported that 27.00±1.32 cm of 1st stair, 26.50±2.17 cm of 2nd stair, and 37.66±0.28 cm of 3rd stair height respectively, the 3rd stair height of which was too high and very inconvenient as of 3.70 N/BW of peak vertical force (PVF) in descending. Particularly it was reported that wrong downward stepping may increase an injury risk at knee joint due to situation of interrupted-visual information during the nighttime by irregular driving schedule ([@b8-jer-14-2-313]; [@b30-jer-14-2-313]; [@b40-jer-14-2-313]). Visual information at landing provides preliminary confirming of spatial information against landing point and controls preparatory role of joint and muscle activity to absorb an impulse force at landing ([@b45-jer-14-2-313]), but interrupted visual information of the height against landing point results in reduced stability according to increase of ground reaction force (GRF) in *x--y* axis, loading rate and PVF ([@b25-jer-14-2-313]). Therefore injury risk on muscular-skeletal system may be increased when did not absorb an impulse force occurring at landing of foot ([@b35-jer-14-2-313]). In spite of subsidiary hand rail attached to exit door to resolve an inconvenience, most driver prefers to jump directly from seat ([@b15-jer-14-2-313]). It is necessary quantitative materials for predicts and prevents the risk possibility of injury during landing from cargo truck. That is, the aim of the study was to compare and analyze the kinetic variables during downward foot-on according to the foothold heights under interrupted-visual information on 25-t cargo truck. MATERIALS AND METHODS ===================== Subject ------- Skilled adult male drivers (n=10; mean year, 29.3±6.18 years; mean heights, 177.78±2.30 cm; mean weights, 76.17±2.34 kg) engaged in cargo truck driving over 1 year participated in the experiment. Experimental design for this was reviewed and approved from Bioethics Committee of Jeju National University (JJNU-IRB-2016-024-001) and all of whom understood and consented on the purpose and details of the study. Experimental procedure ---------------------- After 25-t cargo truck owner's consents, the truck made of 3 footholds for downward stepping was set up within indoor truck house with three-dimensional cinematography and GRF (AMTI-OR-7, Advanced Mechanical Technology Inc., Watertown, MA, USA) equipments under lateral side of driver seat. Height difference of between GRF plate and ground surface was adjusted on wheel and leveled with wooden box similar to height of GRF plate ([Fig. 1](#f1-jer-14-2-313){ref-type="fig"}). Then GRF (1 ea) data was collected at sample rate at 1,000 Hz, similar wooden box was installed widely to reduce data errors occurring by stepping anxiety due to narrowed landing area. The 4 ea Camcorder camera (HDR/HDV 1980i, Sony Corp, Tokyo, Japan) and its light facility were installed within controlled spatial range which can capture an entire downward stepping motion, and set up at 60 frame/sec of camera speed and 1/500 sec of exposure time respectively. Body segment parameter ([@b37-jer-14-2-313]) to calculate center of mass and velocity of whole body and total 19 digitizing points on body were applied to lower extremities model of the study ([Fig. 2](#f2-jer-14-2-313){ref-type="fig"}). The experiment was performed in order of the total 4 types of utilization of handrail on the 1st (1), 2nd (2), 3rd (3) stair according to whether secured or interrupted of visual information and jumping from driver's seat directly (4) (1 subject: 2×4 trials) ([Fig. 3](#f3-jer-14-2-313){ref-type="fig"}). Experimental procedure was measured the same trial repeatedly during the daytime in case of secured-visual information and the nighttime in case of interrupted-visual information respectively. Then at the nighttime, all subjects wore an eye-patch additionally for complete interrupted visual information from camera's light facility. On the basis of that impulse force increased according to height's increase between foot and ground surface during downward stepping of bus ([@b22-jer-14-2-313]), The distance between truck and GRF plate was fixed on one's preferred distance through preliminary practice. Definition of analysis phase ---------------------------- Dimensionless model of leg stiffness was known as proper criterion on the motion evaluation in a situation of an increasing movement of leg's length in human activities, which stiffness variables was made up the vertical GRF force (normalized body weight) and change rate (%) of leg length and was evaluated in a way of ignoring the unit of two variables ([@b46-jer-14-2-313]). K l e g F m a x ( l o \- l m i n o ) l o is defined as normalized value divided PVF with body weight, also *F~max~* is normalized value of change rate (%) of leg length, *l~o~* is shortest length of leg at ground landing as an estimate of leg length, then leg length delivered from COP to center of gravity (COG) of pelvic was cited from muscular-skeletal system model ([@b9-jer-14-2-313]). Analysis and process of data ---------------------------- The average and the standard deviation of the calculated variables were obtained using IBM SPSS Statistics ver. 21.0 (IBM Co., Armonk, NY, USA), performed repeated measures two-way analysis of variance according to interrupted visual information and foothold heights and performed the post hoc test (Duncan) at (*P*\<0.05) in case of significant level respectively. RESULTS ======= The results from kinetic variables and stiffness of lower leg according to interrupted-visual information (1st, 2nd, and 3rd stair) and foothold heights from cargo truck was as ([Table 1](#t1-jer-14-2-313){ref-type="table"}). Kinetic variables and stiffness of lower leg according to interrupted-visual information (1st, 2nd, and 3rd stair) and foothold heights showed significant difference (*P*\<0.001), followed in order of driver seat\>3rd\>2nd\>1st stair. Also PVF and loading rate showed the same results, followed in order of driver seat\>3rd\>1st, 2nd stair at PVF, and driver seat\>3rd\>1st stair (*P*\<0.05) respectively. The results from COP according to interrupted-visual information (1st, 2nd, and 3rd stair) and foothold heights from cargo truck was as ([Table 2](#t2-jer-14-2-313){ref-type="table"}). Medial lateral (ML)-COP, anterior posterior (AP)-COP, and COP area according to interrupted-visual information (1st, 2nd, and 3rd stair) did not showed significant difference (*P*\>0.05), but and foothold heights showed significant level (*P*\<0.05), and followed in order of driver seat\>3rd\>1st stair of ML-COP, driver seat\>1st, 2nd, 3rd stair of AP-COP, and driver seat\>3rd, 2nd\>1st of COP area respectively. The results from stiffness of leg and kinetic variables according to interrupted-visual information (1st, 2nd, and 3rd stair) and foothold heights from cargo truck was as ([Table 3](#t3-jer-14-2-313){ref-type="table"}). Medial lateral stability index (MLSI), APSI, vertical (V)SI, and dynamic postural stability index (DPSI) showed significant difference (*P*\>0.05), and followed in order of driver seat\>3rd\>1st stair of MLSI, APSI, VSI, and DPSI, but driver, 3rd\>1st, 2nd (*P*\<0.05) respectively. DISCUSSION ========== There is nowadays many types of information and model on exercise prescription and rehabilitation diagnosis for vibration exposure and back pain of body resulted from long time driving. Clear diagnosis and prescription are very important to maximize the effectiveness of exercise rehabilitation depending on the type of injury in truck driver ([@b21-jer-14-2-313]). But causes and effects and quantitative materials between impact types and injury occurrence is still not enough in spite of higher injury possibility and fatal wound during downward stepping from cargo truck. Spring-mass model defined lower leg of body as linear spring without no mass, and it had close relationship with change rate of leg length during supporting phase and PVF ([@b13-jer-14-2-313]). It was reported that stiffness of lower leg showed rather difference in line with ground conditions on which foot contacts, and it was in inverse proportion to stiffness of surface touched during exercise ([@b1-jer-14-2-313]; [@b16-jer-14-2-313]). Therefore some level of stiffness should be maintained for the fittest performance of human movement ([@b3-jer-14-2-313]; [@b12-jer-14-2-313]; [@b24-jer-14-2-313]; [@b27-jer-14-2-313]; [@b32-jer-14-2-313]; [@b44-jer-14-2-313]; [@b47-jer-14-2-313]), but it may be failed to control of leg stiffness by rapid change of PVF and length of lower leg during jumping or downward stepping from vertical height of driver's seat to solid surface. That is, downward stepping from 1st stair of truck with noninterrupted visual information showed similar value with 35 (dimensionless) of stiffness value during running, proportional increasing trend of 2nd and 3rd, and showed about 2 times of 44 (dimensionless) of added body weight (30%) during running in case of downward stepping from driver seat ([@b46-jer-14-2-313]). Also PVF and loading rate showed significant increase according to foothold height, and showed similar pattern with leg stiffness in the study. Particularly leg stiffness, PVF, and loading rate showed larger increased pattern in interrupted-visual information than was not case. These result partly coincided with report of [@b42-jer-14-2-313] which could not be controlled and shown the more variability of impulse force in case of interrupted-visual information and be controlled the consistent performance of movement during landing. Loading rate is value of PVF divided by applied time, which means elapsed time to PVF point by body weight ([@b10-jer-14-2-313]), and high loading rate means not only limited absorption of impulse at landing but also pressure of stress against lower leg for a short time ([@b17-jer-14-2-313]). Also because leg stiffness is closely related with PVF and change rate of lower leg (%) ([@b11-jer-14-2-313]; [@b14-jer-14-2-313]; [@b32-jer-14-2-313]), increased lower leg's stiffness means a degree of stiffness and rigidity of lower leg. Therefore the more flexed type of knee joint during locomotion may reduce impulse force, vertical stiffness, impact absorption, impulse force transferred to skull ([@b19-jer-14-2-313]; [@b33-jer-14-2-313]; [@b34-jer-14-2-313]), but in case of downward stepping motion from high foothold height of cargo truck, an impact absorption mechanism may be neglected unconsciously. Particularly visual information may make preparation for activities of joint and muscle to absorb an impact occurring at landing by preliminary recognition on spatial distance against landing point ([@b42-jer-14-2-313]; [@b45-jer-14-2-313]), but interrupted-visual information may take a mechanism to limit the movement of ankle in the course of securing of stability ([@b28-jer-14-2-313]; [@b29-jer-14-2-313]). In that the former mechanism can adopt manageable strategy prior to ground landing in various impact situations ([@b42-jer-14-2-313]), interrupted-visual information on the foothold heights during downward foot-on from cargo truck enables to increase the leg stiffness, PVF, and loading rate for continuous supplementation of deficit feedback on visual information. COP variables as stability index in the study was defined as the center of action of GRF vector occurring alongside with rectangular coordinates (two-dimensional \[ML, AP\]) against horizontal plane and was evaluated in unit of time-serial function ([@b23-jer-14-2-313]). That is, it did not showed difference in case of interrupted visual information, but showed increasing trend according to truck's foothold heights. COP area integrated by ML-COP and AP-COP showed increasing trend due to increase of the moved trace of COP on the direction of ML and AP for posture control after landing according to truck's foothold heights. The stability index by DPSI method (Maximum force on 3 direction GRF \[ML, AP, and V\] divided by elapsed time) which can be evaluated in dynamic situation meant improved higher stability index when coming to close to 0 value in each direction, but the higher index value meant lowering stability index ([@b50-jer-14-2-313]). Decrease of stability meant the type occurring stronger impulse force during shorter time than that of landing of foot. MLSI, APSI, VSI, and DPSI of the study showed the higher foothold height and interrupted visual information, the more decrease in stability. When comparing of 2 variables, due to increase of impact types (PVF, loading rate) during landing on solid ground condition, increase of COP area also assumes to be correlated with equilibrium and movement of COG. whereas COP variables compared with interrupted visual information as main effector did not show difference, show more reduced COP area in case of 1st, 2nd, 3rd stair and showed rapid increasing trend in case of driver seat than noninterrupted visual information. Predictable ability of an estimating time getting into contact with ground surface of foot through visual information on the landing height during downward stepping is critical factor to reduction of impulse force ([@b42-jer-14-2-313]; [@b41-jer-14-2-313]). In fact, it was reported that occurrence time of impulse force after landing was shorter than reaction time of muscle to sensory signal during absorbing an impulse ([@b36-jer-14-2-313]). Therefore it was assumed that reduction of DPSI according to increase of foothold height of truck was related with the failure of sufficient time securement for posture control during landing on ground ([@b6-jer-14-2-313]; [@b51-jer-14-2-313]). Furthermore interrupted visual condition of the study influenced on the increasing trend of stability index (MLSI, APSI, VSI, and DPSI) and rapid increasing trend of COP area (ML, AP, and area), but increase of COP area should done because of difficulty of stability securement during short time of landing, also it might result in failure of posture control against increase of vertical load. Particularly because prediction of falling injury with COP variables ([@b31-jer-14-2-313]), downward stepping from 2nd, 3rd stair and jumping directly from driver seat in situation of interrupted-visual information may be cause of secondary falling injury. Because threshold level of leg stiffness may differentiate from an individual traits and ground conditions, its absolute criterion may not fix, but injury may occur whenever too low or high of it normally ([@b7-jer-14-2-313]). Driver's habit confirming the spatial information against landing position during downward stepping from truck time enables to control properly the leg stiffness and PVF with preparatory prediction of air phase time and control of impulse force. Consequently the result of the study will be helpful to not only therapist of exercise rehabilitation but availability on diagnosis, evaluation, prediction, and types of impulse-falling injury-skeletal fracture. This work was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea (NRF-2016S1A5B5A07915699). **CONFLICT OF INTEREST** No potential conflict of interest relevant to this article was reported. {#f1-jer-14-2-313} {#f2-jer-14-2-313} {#f3-jer-14-2-313} ###### Change of leg stiffness and kinetic variables during downward step in cargo truck Section Visual conditions (V) Descending type (D) Total average Source *F* *P*-value ------------------------------- ----------------------- --------------------- --------------- --------------- --------------- -------------- -------- -------- ------- Leg stiffness (dimensionless) Open 36.20±10.39 58.56±11.00 71.02±7.86 83.33±9.74 62.28±20.00 D 47.943 0.001 Closed 57.51±8.26 69.01±6.54 79.47±12.63 92.32±18.68 74.58±17.68 V 24.259 0.001 Total average 46.86±14.25 63.79±10.31 75.24±11.12 87.83±15.22 68.43±19.75 D×V 1.475 0.109 Peak vertical force (N/BW) Open 1.87±0.63 3.28±1.12 4.95±1.56 7.57±2.48 4.42±2.64 D 24.798 0.001 Closed 3.04±0.99 4.35±2.07 6.27±2.11 9.59±2.78 5.81±3.20 V 30.668 0.001 Total average 2.45±1.00 3.81±1.71 5.61±1.92 8.57±2.76 5.11±3.00 D×V 0.723 0.545 Loading rate (N/BW/sec) Open 30.84±15.84 58.03±29.64 127.56±55.06 239.06±64.11 113.87±92.43 D 16.767 0.001 Closed 69.28±29.80 155.20±169.68 200.44±107.83 270.77±124.53 173.92±136.36 V 10.080 0.003 Total average 50.05±30.46 106.61±128.60 164.00±91.32 254.91±97.76 143.89±119.62 D×V 0.655 0.585 Values are presented as mean±standard deviation. D, main effect of the descending types; V, main effect of the visual conditions; D×V, interaction; BW, body weight. ###### Change of center of pressure (COP) variables during downward step in cargo truck Section Visual conditions (V) Descending type (D) Total average Source *F* *P*-value ----------------------------- ----------------------- --------------------- --------------- ------------ ------------- ------------ ------- -------- ------- Medial-lateral COP (cm) Open 1.47±0.52 1.94±0.93 2.50±1.80 2.37±0.83 2.07±1.14 D 4.439 0.009 Closed 1.14±0.89 1.81±0.55 1.83±0.57 2.43±0.67 1.80±0.80 V 1.993 0.167 Total average 1.30±0.73 1.88±0.69 2.16±1.34 2.40±0.74 1.94±0.99 D×V 0.686 0.567 Anterior-posterior COP (cm) Open 10.80±7.97 8.75±2.38 9.48±3.28 10.88±3.71 9.98±4.74 D 2.916 0.047 Closed 7.45±2.67 7.71±2.68 8.38±2.96 13.16±4.40 9.20±3.91 V 0.815 0.373 Total average 9.17±6.02 8.23±2.52 8.93±3.09 12.02±4.13 9.59±4.33 D×V 1.741 0.176 COP area (cm^2^) Open 12.38±3.66 15.71±5.38 19.27±7.99 24.23±9.61 17.90±8.10 D 27.800 0.001 Closed 8.82±8.75 13.34±4.71 14.42±4.95 30.14±4.86 16.68±10.02 V 0.590 0.448 Total average 10.60±6.78 14.53±5.07 16.85±6.93 27.18±8.01 17.29±9.07 D×V 2.343 0.089 Values are presented as mean±standard deviation. D, main effect of the descending types; V, main effect of the visual conditions; D×V, interaction. ###### Change of dynamic posture stability index during downward step in cargo truck Section Visual conditions (V) Descending type (D) Total average Source *F* *P*-value ------------------------------------ ----------------------- --------------------- --------------- --------------- -------------- ------------- -------- -------- ------- Medial lateral stability index Open 1.33±1.04 1.74±1.17 3.13±1.41 12.94±8.87 4.78±6.51 D 20.446 0.001 Closed 2.05±1.58 5.22±9.61 8.02±4.99 16.08±5.99 7.84±7.98 V 6.649 0.014 Total average 1.63±1.35 3.48±6.89 5.57±4.36 14.51±7.54 6.31±7.39 D×V 36.000 0.664 Anterior posterior stability index Open 2.76±2.79 3.94±1.63 8.11±5.18 28.56±11.83 10.84±12.34 D 39.575 0.001 Closed 6.86±2.78 8.24±8.24 16.13±9.45 35.09±15.94 16.58±15.05 V 9.334 0.004 Total average 4.81±3.43 6.09±6.19 12.12±8.48 31.83±14.07 13.71±13.98 D×V 0.249 0.861 Vertical stability index Open 24.28±14.26 37.33±17.80 71.31±25.28 138.05±45.61 67.74±52.34 D 19.174 0.001 Closed 38.20±21.05 81.88±77.37 105.65±67.75 167.36±79.82 98.27±79.10 V 7.931 0.008 Total average 31.24±18.90 59.61±59.45 88.48±52.79 152.71±65.03 83.01±68.39 D×V 0.346 0.792 Dynamic posture stability index Open 28.38±17.16 43.02±20.20 82.56±29.80 179.56±61.74 83.38±69.37 D 24.615 0.001 Closed 47.12±24.33 95.36±90.15 129.80±75.41 218.53±100.48 122.70±98.38 V 8.801 0.005 Total average 37.75±22.63 69.19±69.02 106.18±60.84 199.05±83.59 103.04±86.86 D×V 0.311 0.817 Values are presented as mean±standard deviation. D, main effect of the descending types; V, main effect of the visual conditions; D×V, interaction.

Direct submission or co-submission {#sec0001} ================================== Co-submissions are papers that have been submitted alongside an original research paper accepted for publication by another Elsevier journal Specifications Table {#sec0002} ==================== Subject AreaComputer ScienceMore specific subject areaIdentifying a FunctionMethod nameMethod: Split Difference Pattern Triangles: 1st Difference and Difference Zero TrianglesName and reference of original methodSystem of Equations [http://www.math.cmu.edu/\~bkell/21110--2010s/formula.html](http://www.math.cmu.edu/~bkell/21110-2010s/formula.html){#interref0001} Method of Differences.  Brilliant.org. Retrieved 21:28, May 9, 2020, from <https://brilliant.org/wiki/method-of-differences/> Newton\'s Interpolation Formula <https://www.encyclopediaofmath.org/index.php/Newton_interpolation_formula>Resource availability Method details {#sec0003} ==============   Introduction {#sec0004} ============ Often in algebra, we see sequences and need to find the polynomial that fits that sequence. The current method known involves setting up an equation similar to:$$y = ax^{3} + bx^{2} + \text{cx} + d$$and solving a system of equations. This method uses a pattern to determine the coefficients of the polynomial rather than the extensive method that we use today. Experimental and computational details {#sec0005} ====================================== Split difference {#sec0006} ---------------- ### Finding the differences {#sec0007} #### Splitting the sequence into its differences {#sec0008} Given are the first few terms of the sequence:$$9,{}49,{}169,{}441,{}961,{}1849$$ There is a function that can resemble this sequence. The first step would be to split the sequence into its differences. To do so, the differences of the terms would be found. Then, the differences of those differences are found, and this process can be repeated till all the differences are the same. Example:Row 42424Row 37296120Row 280152248368Row 140120272520888Row 09491694419611849 #### Determine the degree of the polynomial {#sec0009} If the function is a polynomial of some whole number degree, the successive differences between the differences should at one point be the same. The number of rows of differences obtain would be the degree of the polynomial. Example: Row 4 shows constant differences. Therefore, the degree of the polynomial equation that represents the given sequence is 4. #### Finding the 0th differences {#sec0010} For this method it is necessary to start at the 0th term and its successive differences. When given a sequence that starts at the first term, we can use its difference table to find the 0th differences. Once the triangle of differences is found, the 0th term can be found by working backwards and finding the 0th differences. Example:Row 4242424Row 3487296120Row 23280152248368Row 1840120272520888Row 019491694419611849 Now, you should have the following differences for each row:$$1,{}8,{}32,{}48,{}24$$ ### Creating the term zero difference triangle {#sec0011} Begin with the triangle:$$\begin{matrix} {\mspace{9720mu} 1} & \\ {\mspace{3240mu} 0} & {\mspace{900mu} 1} \\ \end{matrix}$$ The next step is to add another row. This is done by adding the numbers above it, similar to the rule used in Pascal\'s Triangle. However, now we will multiply each sum by their own term number. The leftmost number of each row will have the term number of 0. This process must be repeated till there are one plus the degree found in part one number of rows. Like Pascal\'s Triangle, this pattern triangle is the same every time you use this method. Repeating this pattern will give you the triangle:Row 01Row 101Row 2022Row 30166Row 401143624 ### Apply and solve {#sec0012} #### Finding the coefficients {#sec0013} The final step is to piece together parts 1 and 2 to determine the function. To do so, the 0th differences found in part 1 is used. First, the final difference is divided by the last term of the last row of the triangle in part 2. That value will be the leading coefficient. The rest of the numbers of that row is multiplied by that coefficient and subtracted from the other differences respectively. While going up the triangle, this process will be repeated till all of the numbers have been used up. Example: Begin with the differences found in Part 1.$$1,{}8,{}32,{}48,{}24$$ Divide the last term of the differences by the corresponding term in the pattern triangle and subtract. Repeat until all the numbers in the pattern triangle are used.$$\begin{array}{ccl} & & {1\mspace{6mu}\mspace{6mu} 8\mspace{6mu}\mspace{6mu} 32\mspace{6mu}\mspace{6mu} 48} \\ & & {\mspace{6mu}\frac{24}{24}} \\ & & {{= 1}} \\ & & \frac{0\mspace{6mu}\mspace{6mu} 1\mspace{6mu}\mspace{6mu} 14\mspace{6mu}\mspace{6mu} 36}{1\mspace{6mu}\mspace{6mu} 7\mspace{6mu}\mspace{6mu} 18\mspace{6mu}\mspace{6mu}\frac{12}{6}} \\ & & {{= 2}} \\ & & \frac{0\mspace{6mu}\mspace{6mu} 2\mspace{6mu}\mspace{6mu} 12}{1\mspace{6mu}\mspace{6mu} 5\mspace{6mu}\mspace{6mu}\frac{6}{2}} \\ & & {{= 3}} \\ & & \frac{0\mspace{6mu}\mspace{6mu} 3}{1\mspace{6mu}\mspace{6mu}\frac{2}{1}} \\ & & {\mspace{6mu}{= 2}} \\ & & \frac{0}{\frac{1}{1}} \\ & & {\quad{= 1}} \\ \end{array}$$ #### Determine the equation {#sec0014} Here, we use the empty polynomial equation of the degree you found in Part 1, as shown below:$$y = ax^{4} + bx^{3} + cx^{2} + \text{dx} + {}e$$ Substitute the variables in the equation above with the quotients found earlier in Part 3 (a is the first quotient). Example:$$y{} = {}x^{4} + {}2x^{3} + {}3x^{2} + {}2x{} + {}1$$ Alternate form of split difference {#sec0015} ================================== There is a similar method to split difference which can be found to be easier in some circumstances. Finding the differences {#sec0016} ----------------------- Repeat the Part 1 process from the standard Split Difference method. However, there is no need to find the 0th term or the 0th differences. Example:Row 42424Row 37296120Row 280152248368Row 140120272520888Row 09491694419611849 Creating the first term difference triangle {#sec0017} ------------------------------------------- Begin with the triangle:$$\begin{matrix} 1 \\ {1\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 1} \\ \end{matrix}$$ Create another row by adding the numbers above times the term number each number holds, starting with left as 1. For example, the second number in the next row would be (1 \* 1) + (1 \* 2). Once again, create as many rows as the degree plus 1. Like Pascal\'s Triangle, this pattern triangle is the same every time you use this method.Row 01Row 111Row 2132Row 317126Row 411550624 Apply and solve - Here, the method works same as it did in part 1 {#sec0018} ----------------------------------------------------------------- ### Finding the coefficients {#sec0019} The final step is to piece together parts 1 and 2 to determine the function. To do so, the 0th differences found in part 1 is used. First, the final difference is divided by the last term of the last row of the triangle in part 2. That value will be the leading coefficient. The rest of the numbers of that row is multiplied by that coefficient and subtracted from the other differences respectively. While going up the triangle, this process will be repeated till all of the numbers have been used up. Example: Begin with the differences found in Part 1.$$9,{}40,{}80,{}72,{}24$$ Divide the last term of the differences by the corresponding term in the pattern triangle and subtract. Repeat until all the numbers in the pattern triangle are used. Example:$$\begin{array}{ccl} & & {9\mspace{6mu}\mspace{6mu} 40\mspace{6mu}\mspace{6mu} 80\mspace{6mu}\mspace{6mu} 72} \\ & & {\mspace{6mu}\mspace{6mu}\frac{24}{24}} \\ & & {\mspace{6mu}{= 1}} \\ & & \frac{1\mspace{6mu}\mspace{6mu} 15\mspace{6mu}\mspace{6mu} 50\mspace{6mu}\mspace{6mu} 60}{8\mspace{6mu}\mspace{6mu} 25\mspace{6mu}\mspace{6mu} 30\mspace{6mu}\mspace{6mu}\frac{12}{6}} \\ & & {\,\mspace{6mu}{= 2}} \\ & & \frac{2\mspace{6mu}\mspace{6mu} 14\mspace{6mu}\mspace{6mu} 24}{6\mspace{6mu}\mspace{6mu} 11\mspace{6mu}\mspace{6mu}\frac{6}{2}} \\ & & {\mspace{6mu}{= 3}} \\ & & \frac{3\mspace{6mu}\mspace{6mu} 9}{3\mspace{6mu}\mspace{6mu}\frac{2}{1}} \\ & & {\mspace{6mu}{= 2}} \\ & & \frac{2}{\frac{1}{1}} \\ & & {\quad{= 1}} \\ \end{array}$$ ### Determine the equation {#sec0020} Here, we use the empty polynomial equation of the degree you found in Part 1, as shown below:$$y = ax^{4} + bx^{3} + cx^{2} + \text{dx} + e$$ Substitute the variables in the equation above with the quotients found earlier in Part 3 Example:$$y = {}x^{4} + {}2x^{3} + {}3x^{2} + {}2x + {}1$$ Current methods {#sec0021} =============== System of equations [@bib0001] {#sec0022} ------------------------------ The current method works by using a system of equations to solve the polynomial. One must set up multiple equations following the structure:$$y = ax^{4} + bx^{3} + cx^{2} + \text{dx} + e$$ They will then substitute x for 1 and y for the first term and so on till they have as many equations as coefficients. Using the elimination method, this method requires you to find the coefficients one by one. However, this method can become very tedious, especially when dealing with higher degree polynomials. It is also very difficult when dealing with non-integer coefficients. Difference table [@bib0002] {#sec0023} --------------------------- The Split Difference Method is certainly based off the ideas behind this method. This method requires creating the difference table for the sequence to find the leading coefficient and highest degree. Then, the method calls for subtracting this value from each term in the sequence and drawing the difference table once more to find the next highest degree. This process is repeated until all the terms are found. However, this method is extremely time consuming as it requires one to create multiple difference tables. Newton\'s interpolation method [@bib0003] {#sec0024} ----------------------------------------- This method involves creating a special difference table where the 'difference' between each successive term is found using a special equation. While this method may be fast, it doesn\'t provide one with a simplified polynomial function, but rather a quite large one that will take too much time to simplify at higher degrees. Split difference method benefits {#sec0025} -------------------------------- This method compared to any previous method runs much faster, especially at higher degree equations. The first step of finding the differences and degree is the same as you would in any other method because it is necessary to determine the degree of the function. The triangle is one that doesn\'t change based on the scenario and therefore can be memorized for quick use. The only extra calculation occurs in the final step which is comprised of simple arithmetic and very little of it. This minimizes the issue of making an accidental error which tends to be common in the system of equations method. By reusing the difference table that would need to have been made anyways and limiting the method to only one difference table, we get a much faster process than that of the System of Equations and the Difference Table method. Additionally, we get a simplified result unlike that of Newton\'s Interpolation Method. Proof of pattern triangle {#sec0026} ========================= Proof of the first term difference triangle {#sec0027} ------------------------------------------- $$f\left( x \right) = A_{d}x^{d} + A_{d - 1}x^{d - 1} + A_{d - 2}x^{d - 2}{+ \ldots +}A_{0}x^{0}$$xf(x)/Δ~0~Δ~1~Δ~2~Δ~3~Δ~4~Δ~n~1f(1)f(2) -- f(1)2f(2)f(3) -- 2f(2) + *f*(1)f(3) -- f(2)f(4) -- 3f(3) + 3f(2) -- f(1)3f(3)f(4) -- 2f(3) + *f*(2)f(5) -- 4f(4) + 6f(3) -- 4f(2) + *f*(1)f(4) -- f(3)f(5) -- 3f(4) + 3f(3) -- f(2)$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}f\left( {n - k + 1} \right)$4f(4)f(5) -- 2f(4) + *f*(3)f(6) -- 4f(5) + 6f(4) -- 4f(3) + *f*(2)f(5) -- f(4)f(6) -- 3f(5) + 3f(4) -- f(3)5f(5)f(6) -- 2f(5) + *f*(4)f(6) -- f(5)6f(6) Given the degree, d, is n when Δ*~n~* is constant, the leading coefficient, A~d~, is:$$\frac{\sum_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}f\left( {n - k + 1} \right)}{n!}$$ Let g(x) = *f*(x) -- A~d~x^d^xg(x) or Δ~0~Δ~1~Δ~2~Δ~3~Δ~n~1g(1)g(2) -- g(1)2g(2)g(3) -- 2g(2) + *g*(1)g(3) -- g(2)g(4) -- 3g(3) + 3g(2) -- g(1)3g(3)g(4) -- 2g(3) + *g*(2)$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}g\left( {n - k + 1} \right)$g(4) -- g(3)g(5) -- 3 g(4) + 3 g(3) -- g(2)4g(4)g(5) -- 2g(4) + *g*(3)$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}g\left( {n - k + 2} \right)$g(5) -- g(4)g(6) -- 3g(5) + 3g(4) -- g(3)5g(5)g(6) -- 2g(5) + *g*(4)g(6) -- g(5)6g(6) Substitute f(x) -- A~d~x^d^ for g(x).xg(x) or Δ~0~Δ~1~Δ~2~Δ~3~Δ~n~1f(1) -- A~d~1^d^(f(2) -- A~d~2^d^) -- (f(1) -- A~d~1^d^)2f(2) -- A~d~2^d^(f(3) -- A~d~3^d^) -- 2(f(2) -- A~d~2^d^) +\ (f(1) -- A~d~1^d^)(f(3) -- A~d~3^d^) -- (f(2) -- A~d~2^d^)(f(4) -- A~d~4^d^) -- 3(f(3) -- A~d~3^d^) +\ 3(f(2) -- A~d~2^d^) -- (f(1) -- A~d~1^d^)3f(3) -- A~d~3^d^(f(4) -- A~d~4^d^) -- 2(f(3) -- A~d~3^d^) +(f(2) -- A~d~2^d^)$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 1} \right) -} \right.$${A_{d}\left( {n - k + 1} \right)^{d}}\rbrack$(f(4) -- A~d~4^d^) -- (f(3) -- A~d~3^d^)(f(5) -- A~d~5^d^) -- 3(f(4) -- A~d~4^d^) +3(f(3) -- A~d~3^d^) -- (f(2) -- A~d~2^d^)4f(4) -- A~d~4^d^(f(5) -- A~d~5^d^) -- 2(f(4) -- A~d~4^d^) +(f(3) -- A~d~3^d^)$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 2} \right) -} \right.$${A_{d}\left( {n - k + 2} \right)^{d}}\rbrack$(f(5) -- A~d~5^d^) -- (f(4) -- A~d~4^d^)(f(6) -- A~d~6^d^) -- 3(f(5) -- A~d~5^d^) +3(f(4) -- A~d~4^d^) -- (f(3) -- A~d~3^d^)5f(5) -- A~d~5^d^(f(6) -- A~d~6^d^) -- 2(f(5) -- A~d~5^d^) +(f(4) -- A~d~4^d^)(f(6) -- A~d~6^d^) -- (f(5) -- A~d~5^d^)6f(6) -- A~d~6^d^ Given the degree of g(x), [d]{.smallcaps}-1, is n when Δ*~n~* is constant, the leading coefficient, A~d-1~, is:$$\frac{\sum_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 1} \right) - A_{d}\left( {n - k + 1} \right)^{d}} \right\rbrack}{n!}$$ Using the previous two coefficients, we can let h(x) = *f*(x) -- A~d~x^d^ -- A~d-1~x^d-1^ . The first term of Δ~n~ for h(x) would be:$$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 1} \right) - A_{d}\left( {n - k + 1} \right)^{d} - \mspace{6mu} A_{d - 1}\left( {n - k + 1} \right)^{d - 1}} \right\rbrack$$ If this process is repeated until every coefficient is found, the function will only have the leading coefficient. We can call this function z(x).$$z\left( x \right) = f\left( x \right) - A_{d}x^{d} - A_{d - 1}x^{d - 1} - \cdots - A_{1}x^{1} = A_{0}x^{0}$$ The first term of Δ~n~ for z(x) would be:$$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 1} \right) - A_{d}\left( n - k + 1 \right)^{d} - \mspace{6mu} A_{d - 1}\left( {n - k + 1} \right)^{d - 1} - \ldots - A_{1}\left( {n - k + 1} \right)^{1}} \right\rbrack$$ Since z(x) is a constant coefficient function, there is only one Δ~n~ to pay attention to - Δ~0~. All other columns will be 0. Thus, we can simplify the summation above to:$$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 1} \right) - \sum\limits_{i = 0}^{d}A_{d - i}\left( {n - k + 1} \right)^{d - i}} \right\rbrack$$ Now we can define a function, P~m~(x) which represents a part of the original polynomial, f(x) with degree d, where m represents the degree of the function. The general formula for first difference of each column Δ~n~ for P~m~(x) would be:$$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 1} \right) - \sum\limits_{i = 0}^{d - m - 1}A_{d - i}\left( {n - k + 1} \right)^{d - i}} \right\rbrack$$ Now if we write the first term of each column Δ~n~ for any degree, d we get:   P~M~012N0$f\left( 1 \right) - \left\lbrack {\sum\limits_{i = 0}^{d - 1}A_{d - i}\left( 1 \right)^{d - i}} \right\rbrack$1$f\left( 1 \right) - \left\lbrack {\sum\limits_{i = 0}^{d - 2}A_{d - i}\left( 1 \right)^{d - i}} \right\rbrack\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\left\lbrack f\left( {2 - k} \right) - \sum\limits_{i = 0}^{d - 2}A_{d - i}\left( {2 - k} \right)^{d - i} \right\rbrack$2$f\left( 1 \right) - \left\lbrack {\sum\limits_{i = 0}^{d - 3}A_{d - i}\left( 1 \right)^{d - i}} \right\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\left\lbrack {f\left( {2 - k} \right) -} \right.$${\sum\limits_{i = 0}^{d - 3}A_{d - i}\left( {2 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {3 - k} \right) -} \right.$${\sum\limits_{i = 0}^{d - 3}A_{d - i}\left( {3 - k} \right)^{d - i}}\rbrack$...D--3$f\left( 1 \right) - \left\lbrack {\sum\limits_{i = 0}^{2}A_{d - i}\left( 1 \right)^{d - i}} \right\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\left\lbrack {f\left( {2 - k} \right) -} \right.$${\sum\limits_{i = 0}^{2}A_{d - i}\left( {2 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {3 - k} \right) -} \right.$${\sum\limits_{i = 0}^{2}A_{d - i}\left( {3 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 1} \right) -} \right.$${\sum\limits_{i = 0}^{2}A_{d - i}\left( {n - k + 1} \right)^{d - i}}\rbrack$D--2$f\left( 1 \right) - \left\lbrack {\sum\limits_{i = 0}^{1}A_{d - i}\left( 1 \right)^{d - i}} \right\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\left\lbrack {f\left( {2 - k} \right) -} \right.$${\sum\limits_{i = 0}^{1}A_{d - i}\left( {2 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {3 - k} \right) -} \right.$${\sum\limits_{i = 0}^{1}A_{d - i}\left( {3 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 1} \right) -} \right.$${\sum\limits_{i = 0}^{1}A_{d - i}\left( {n - k + 1} \right)^{d - i}}\rbrack$D--1f(1) -- A~d~1^d^f(2) -- f(1) -- A~d~2^d^ + A~d~1^d^$\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {3 - k} \right) -} \right.$${\sum\limits_{i = 0}^{0}A_{d - i}\left( {3 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 1} \right) -} \right.$${\sum\limits_{i = 0}^{0}A_{d - i}\left( {n - k + 1} \right)^{d - i}}\rbrack$Df(1)f(2) -- f(1)f(3) -- 2f(2) + *f*(1)$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}f\left( {n - k + 1} \right)$ Taking the difference between the rows, without the last value in each row, we get:P~M~ -- P~M-1~012NP~0~ - 0P~1~ -- P~0~A~1~1^1^P~2~-- P~1~A~2~1^2^A~2~(2^2^ -- 1^2^)P~3~ -- P~2~A~3~1^3^A~3~(2^3^ -- 1^3^)A~3~(3^3^ -- 2(2)^3^ + 1^3^)...P~D-3~ -- P~D-4~$A_{d - 3}\sum\limits_{k = 0}^{0}\left( {- 1} \right)^{k}\begin{pmatrix} 0 \\ k \\ \end{pmatrix}\left( {1 - k} \right)^{d - 3}$$A_{d - 3}\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} 1 \\ k \\ \end{pmatrix}\left( {2 - k} \right)^{d - 3}$$A_{d - 3}\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} 2 \\ k \\ \end{pmatrix}\left( {3 - k} \right)^{d - 3}$$A_{d - 3}\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left( {n - k + 1} \right)^{d - 3}$P~D-2~ -- P~D-3~$A_{d - 2}\sum\limits_{k = 0}^{0}\left( {- 1} \right)^{k}\begin{pmatrix} 0 \\ k \\ \end{pmatrix}\left( {1 - k} \right)^{d - 2}$$A_{d - 2}\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} 1 \\ k \\ \end{pmatrix}\left( {2 - k} \right)^{d - 2}$$A_{d - 2}\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} 2 \\ k \\ \end{pmatrix}\left( {3 - k} \right)^{d - 2}$$A_{d - 2}\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left( {n - k + 1} \right)^{d - 2}$P~D-1~ -- P~D-2~$A_{d - 1}\sum\limits_{k = 0}^{0}\left( {- 1} \right)^{k}\begin{pmatrix} 0 \\ k \\ \end{pmatrix}\left( {1 - k} \right)^{d - 1}$$A_{d - 1}\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} 1 \\ k \\ \end{pmatrix}\left( {2 - k} \right)^{d - 1}$$A_{d - 1}\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} 2 \\ k \\ \end{pmatrix}\left( {3 - k} \right)^{d - 1}$$A_{d - 1}\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left( {n - k + 1} \right)^{d - 1}$P~D~ -- P~D-1~$A_{d}\sum\limits_{k = 0}^{0}\left( {- 1} \right)^{k}\begin{pmatrix} 0 \\ k \\ \end{pmatrix}\left( {1 - k} \right)^{d}$$A_{d}\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} 1 \\ k \\ \end{pmatrix}\left( {2 - k} \right)^{d}$$A_{d}\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} 2 \\ k \\ \end{pmatrix}\left( {3 - k} \right)^{d}$$A_{d}\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left( {n - k + 1} \right)^{d}$ With the last value in each row being:$$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 1} \right) - \sum\limits_{i = 0}^{d - m - 1}A_{d - i}\left( {n - k + 1} \right)^{d - i}} \right\rbrack\mspace{6mu} = m!\mspace{6mu}$$ This pattern is similar to the pattern triangle which shows the values as:$$\begin{matrix} 1 \\ {1\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 1} \\ {1\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 3\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 2} \\ {1\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 7\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 12\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 6} \\ {1\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 15\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 50\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 60\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 24} \\ \end{matrix}$$ The difference between this pattern triangle and the table we have above is that the constants behind each number in the pattern triangle are set as 1. Simplify the following equation$$\left( {n + 1} \right)\left\lbrack {\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left( {n - k + 1} \right)^{m}} \right\rbrack + \left( {n + 2} \right)\left\lbrack {\sum\limits_{k = 0}^{n + 1}\left( {- 1} \right)^{k}\begin{pmatrix} {n + 1} \\ k \\ \end{pmatrix}\left( {n - k + 2} \right)^{m}} \right\rbrack$$ Expand the summation:$$\begin{array}{ccl} & = & {\left( n + 1 \right)\left\lbrack \left( \begin{array}{l} n \\ 0 \\ \end{array} \right)\left( n + 1 \right)^{m} - \left( \begin{array}{l} n \\ 1 \\ \end{array} \right)\left( n \right)^{m} + \left( \begin{array}{l} n \\ 2 \\ \end{array} \right)\left( n - 1 \right)^{m} - \cdots + \left( - 1 \right)^{n}\left( \begin{array}{l} n \\ n \\ \end{array} \right)\left( 1 \right)^{m} \right\rbrack} \\ & & {+ \left( n + 2 \right)\left\lbrack \left( \begin{array}{l} {n + 1} \\ 0 \\ \end{array} \right)\left( n + 2 \right)^{m} - \left( \begin{array}{l} {n + 1} \\ 1 \\ \end{array} \right)\left( n + 1 \right)^{m} + \left( \begin{array}{l} {n + 1} \\ 2 \\ \end{array} \right)\left( n \right)^{m} - \cdots + \left( - 1 \right)^{n + 1}\left( \begin{array}{l} {n + 1} \\ {n + 1} \\ \end{array} \right)\left( 1 \right)^{m} \right\rbrack} \\ \end{array}$$ Distribute:$$\begin{array}{ccl} & = & {s\left( n + 1 \right)\left( \begin{array}{l} n \\ 0 \\ \end{array} \right)\left( n + 1 \right)^{m} - \left( n + 1 \right)\left( \begin{array}{l} n \\ 1 \\ \end{array} \right)\left( n \right)^{m} + \left( n + 1 \right)\left( \begin{array}{l} n \\ 2 \\ \end{array} \right)\left( n - 1 \right)^{m} - \cdots + \left( n + 1 \right)\left( - 1 \right)^{n}\left( \begin{array}{l} n \\ n \\ \end{array} \right)\left( 1 \right)^{m}} \\ & & {+ \left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ 0 \\ \end{array} \right)\left( n + 2 \right)^{m} - \left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ 1 \\ \end{array} \right)\left( n + 1 \right)^{m} + \left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ 2 \\ \end{array} \right)\left( n \right)^{m} - \cdots} \\ & & {+ \left( n + 2 \right)\left( - 1 \right)^{n + 1}\left( \begin{array}{l} {n + 1} \\ {n + 1} \\ \end{array} \right)\left( 1 \right)^{m}} \\ \end{array}$$ Group like terms:$$\begin{array}{ccl} & = & {\left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ 0 \\ \end{array} \right)\left( n + 2 \right)^{m} + \left( n + 1 \right)^{m}\left\lbrack \left( n + 1 \right)\left( \begin{array}{l} n \\ 0 \\ \end{array} \right) - \left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ 1 \\ \end{array} \right) \right\rbrack} \\ & & {- \left( n \right)^{m}\left\lbrack \left( n + 1 \right)\left( \begin{array}{l} n \\ 1 \\ \end{array} \right) - \left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ 2 \\ \end{array} \right) \right\rbrack + \left( n - 1 \right)^{m}\left\lbrack \left( n + 1 \right)\left( \begin{array}{l} n \\ 2 \\ \end{array} \right) - \left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ 3 \\ \end{array} \right) \right\rbrack - \cdots} \\ & & {+ \left( - 1 \right)^{n}1^{m}\left\lbrack \left( n + 1 \right)\left( \begin{array}{l} n \\ n \\ \end{array} \right) - \left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ {n + 1} \\ \end{array} \right) \right\rbrack} \\ \end{array}$$ Expand the combinations:$$\begin{array}{ccl} & = & {\left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ 0 \\ \end{array} \right)\left( n + 2 \right)^{m} + \left( n + 1 \right)^{m}\left\lbrack \left( n + 1 \right)\frac{n!}{0!n!} - \left( n + 2 \right)\frac{\left( n + 1 \right)!}{1!n!} \right\rbrack} \\ & & {- \left( n \right)^{m}\left\lbrack \left( n + 1 \right)\frac{n!}{1!\left( n - 1 \right)!} - \left( n + 2 \right)\frac{\left( n + 1 \right)!}{2!\left( n - 1 \right)!} \right\rbrack} \\ & & {+ \left( n - 1 \right)^{m}\left\lbrack \left( n + 1 \right)\frac{n!}{2!\left( n - 2 \right)!} - \left( n + 2 \right)\frac{\left( n + 1 \right)!}{3!\left( n - 2 \right)!} \right\rbrack - \cdots} \\ & & {+ \left( - 1 \right)^{n}1^{m}\left\lbrack \left( n + 1 \right)\frac{n!}{n!0!} - \left( n + 2 \right)\frac{\left( n + 1 \right)!}{\left( n + 1 \right)!0!} \right\rbrack} \\ \end{array}$$ Factor out like terms from each group:$$\begin{array}{ccl} & = & {\left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ 0 \\ \end{array} \right)\left( n + 2 \right)^{m} + \left( n + 1 \right)^{m + 1}\left\lbrack \frac{\left( n + 1 \right)!}{0!\left( n + 1 \right)!} - \frac{\left( n + 2 \right)!}{1!\left( n + 1 \right)!} \right\rbrack} \\ & & {- \left( n \right)^{m + 1}\left\lbrack \frac{\left( n + 1 \right)!}{1!\left( n \right)!} - \frac{\left( n + 2 \right)!}{2!\left( n \right)!} \right\rbrack + \left( n - 1 \right)^{m + 1}\left\lbrack \frac{\left( n + 1 \right)!}{2!\left( n - 1 \right)!} - \frac{\left( n + 2 \right)!}{3!\left( n - 1 \right)!} \right\rbrack - \cdots} \\ & & {+ \left( - 1 \right)^{n}1^{m}\left\lbrack \frac{\left( n + 1 \right)!}{\left( n + 1 \right)!0!} - \frac{\left( n + 2 \right)!}{\left( n + 1 \right)!0!} \right\rbrack} \\ \end{array}$$ Simplify into a summation:$$\left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ 0 \\ \end{array} \right)\left( n + 2 \right)^{m} + \sum\limits_{k = 0}^{n}\left( - 1 \right)^{k}\left( n - k + 1 \right)^{m + 1}\left\lbrack \left( \begin{array}{l} {n + 1} \\ k \\ \end{array} \right) - \left( \begin{array}{l} {n + 2} \\ {k + 1} \\ \end{array} \right) \right\rbrack$$ Simply the inner combinations:$$\begin{array}{ccl} {\left( \begin{array}{l} {n + 1} \\ k \\ \end{array} \right)\text{-}\left( \begin{array}{l} {n + 2} \\ {k + 1} \\ \end{array} \right)} & & {= \frac{\left( n + 1 \right)!}{k!\left( n - k + 1 \right)!} - \frac{\left( n + 2 \right)!}{\left( k + 1 \right)!\left( n - k + 1 \right)!}} \\ & & {= \frac{\left( k + 1 \right)\left( n + 1 \right)!}{\left( k + 1 \right)!\left( n - k + 1 \right)!} - \frac{\left( n + 2 \right)!}{\left( k + 1 \right)!\left( n - k + 1 \right)!}} \\ & & {= \left( n + 1 \right)!\left\lbrack \frac{k + 1\text{-}n\text{-}2}{\left( k + 1 \right)!\left( n - k + 1 \right)!} \right\rbrack = - \left\lbrack \frac{\left( n + 1 \right)!}{\left( k + 1 \right)!\left( n - k \right)!} \right\rbrack = - \left( \begin{array}{l} {n + 1} \\ {k + 1} \\ \end{array} \right)} \\ \end{array}$$ The expression above can be simplified to:$$\begin{array}{ccl} & & {= \left\lbrack \sum\limits_{k = 0}^{n + 1}\left( - 1 \right)^{k}\left( \begin{array}{l} {n + 1} \\ k \\ \end{array} \right)\left( n - k + 2 \right)^{m + 1} \right\rbrack = \left( n + 2 \right)\left( \begin{array}{l} {n + 1} \\ 0 \\ \end{array} \right)\left( n + 2 \right)^{m} + \sum\limits_{k = 0}^{n}\left( - 1 \right)^{k + 1}\left( \begin{array}{l} {n + 1} \\ {k + 1} \\ \end{array} \right)\left( n - k + 1 \right)^{m + 1}} \\ & & {= \sum\limits_{k = \text{-}1}^{n}\left( - 1 \right)^{k + 1}\left( \begin{array}{l} {n + 1} \\ {k + 1} \\ \end{array} \right)\left( n - k + 1 \right)^{m + 1} = \sum\limits_{k = 0}^{n + 1}\left( - 1 \right)^{k}\left( \begin{array}{l} {n + 1} \\ k \\ \end{array} \right)\left( n - k + 2 \right)^{m + 1}} \\ & & {\therefore\left( n + 1 \right)\left\lbrack \sum\limits_{k = 0}^{n}\left( - 1 \right)^{k}\left( \begin{array}{l} n \\ k \\ \end{array} \right)\left( n - k + 1 \right)^{m} \right\rbrack + \left( n + 2 \right)\left\lbrack \sum\limits_{k = 0}^{n + 1}\left( - 1 \right)^{k}\left( \begin{array}{l} {n + 1} \\ k \\ \end{array} \right)\left( n - k + 2 \right)^{m} \right\rbrack} \\ \end{array}$$ Therefore, this follows the pattern of the First Term Difference Triangle Term zero difference triangle {#sec0028} ----------------------------- The first pattern triangle can be derived in a similar way except we start with the *x* = 0 term instead of *x* = 1 since the pattern triangle uses the 0th differences rather than the 1st. The difference table would for the sequence would be:xf(x) or Δ~0~Δ~1~Δ~2~Δ~3~Δ~4~Δ~n~0f(0)f(1) -- f(0)1f(1)f(2) -- 2f(1) + *f*(0)f(2) -- f(1)f(3) -- 3f(2) + 3f(1) -- f(0)2f(2)f(3) -- 2f(2) + *f*(1)f(4) -- 4f(3) + 6f(2) -- 4f(1) + *f*(0)f(3) -- f(2)f(4) -- 3f(3) + 3f(2) -- f(1)$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}f\left( {n - k} \right)$3f(3)f(4) -- 2f(3) + *f*(2)f(5) -- 4f(4) + 6f(3) -- 4f(2) + *f*(1)f(4) -- f(3)f(5) -- 3f(4) + 3f(3) -- f(2)$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}f\left( {n - k + 1} \right)$4f(4)f(5) -- 2f(4) + *f*(3)f(6) -- 4f(5) + 6f(4) -- 4f(3) + *f*(2)f(5) -- f(4)f(6) -- 3f(5) + 3f(4) -- f(3)5f(5)f(6) -- 2f(5) + *f*(4)f(6) -- f(5)6f(6) Now if we write the first term of each column Δ~n~ for any degree, d we get:   P~M~012N0$f\left( 0 \right)\text{--}\left\lbrack {\sum\limits_{i = 0}^{d - 1}A_{d - i}\left( 0 \right)^{d - i}} \right\rbrack$1$f\left( 0 \right)\text{--}\left\lbrack {\sum\limits_{i = 0}^{d - 2}A_{d - i}\left( 0 \right)^{d - i}} \right\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\left\lbrack {f\left( {1 - k} \right) -} \right.$${\sum\limits_{i = 0}^{d - 2}A_{d - i}\left( {1 - k} \right)^{d - i}}\rbrack$2$f\left( 0 \right)\text{--}\left\lbrack {\sum\limits_{i = 0}^{d - 3}A_{d - i}\left( 0 \right)^{d - i}} \right\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\left\lbrack {f\left( {1 - k} \right) -} \right.$${\sum\limits_{i = 0}^{d - 3}A_{d - i}\left( {1 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {2 - k} \right) -} \right.$${\sum\limits_{i = 0}^{d - 3}A_{d - i}\left( {2 - k} \right)^{d - i}}\rbrack$...d--3$f\left( 0 \right)\text{--}\left\lbrack {\sum\limits_{i = 0}^{2}A_{d - i}\left( 0 \right)^{d - i}} \right\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\left\lbrack {f\left( {1 - k} \right) -} \right.$${\sum\limits_{i = 0}^{2}A_{d - i}\left( {1 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {2 - k} \right) -} \right.$${\sum\limits_{i = 0}^{2}A_{d - i}\left( {2 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k} \right) -} \right.$${\sum\limits_{i = 0}^{2}A_{d - i}\left( {n - k} \right)^{d - i}}\rbrack$d--2$f\left( 0 \right)\text{--}\left\lbrack {\sum\limits_{i = 0}^{1}A_{d - i}\left( 0 \right)^{d - i}} \right\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\left\lbrack {f\left( {1 - k} \right) -} \right.$${\sum\limits_{i = 0}^{1}A_{d - i}\left( {1 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {2 - k} \right) -} \right.$${\sum\limits_{i = 0}^{1}A_{d - i}\left( {2 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k} \right) -} \right.$${\sum\limits_{i = 0}^{1}A_{d - i}\left( {n - k} \right)^{d - i}}\rbrack$d--1f(0) -- A~d~0^d^f(1) -- f(0) -- A~d~1^d^ + A~d~0^d^$\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {2 - k} \right) -} \right.$${\sum\limits_{i = 0}^{0}A_{d - i}\left( {2 - k} \right)^{d - i}}\rbrack$$\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k} \right) -} \right.$${\sum\limits_{i = 0}^{0}A_{d - i}\left( {n - k} \right)^{d - i}}\rbrack$df(0)f(1) -- f(0)f(2) -- 2f(1) + *f*(0)$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}f\left( {n - k} \right)$ The last difference is each row is still equal to that row factorial, which gives the degree of that partial polynomial. Taking the difference between the rows, without the last value in each row, we get:P~M~ -- P~M-1~012NP~0~ - 0P~1~-- P~0~A~1~0^1^P~2~ -- P~1~A~2~0^2^A~2~(1^2^ -- 0^2^)P~3~ -- P~2~A~3~0^3^A~3~(1^3^ -- 0^3^)A~3~(2^3^ -- 2(1)^3^+ 0^3^)...P~D-3~ -- P~D-4~$A_{d - 3}\sum\limits_{k = 0}^{0}\left( {- 1} \right)^{k}\begin{pmatrix} 0 \\ k \\ \end{pmatrix}\left( {- k} \right)^{d - 3}$$A_{d - 3}\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} 1 \\ k \\ \end{pmatrix}\left( {1 - k} \right)^{d - 3}$$A_{d - 3}\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} 2 \\ k \\ \end{pmatrix}\left( {2 - k} \right)^{d - 3}$$A_{d - 3}\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left( {n - k} \right)^{d - 3}$P~D-2~ -- P~D-3~$A_{d - 2}\sum\limits_{k = 0}^{0}\left( {- 1} \right)^{k}\begin{pmatrix} 0 \\ k \\ \end{pmatrix}\left( {- k} \right)^{d - 2}$$A_{d - 2}\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} 1 \\ k \\ \end{pmatrix}\left( {1 - k} \right)^{d - 2}$$A_{d - 2}\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} 2 \\ k \\ \end{pmatrix}\left( {2 - k} \right)^{d - 2}$$A_{d - 2}\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left( {n - k} \right)^{d - 2}$P~D-1~ -- P~D-2~$A_{d - 1}\sum\limits_{k = 0}^{0}\left( {- 1} \right)^{k}\begin{pmatrix} 0 \\ k \\ \end{pmatrix}\left( {- k} \right)^{d - 1}$$A_{d - 1}\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} 1 \\ k \\ \end{pmatrix}\left( {1 - k} \right)^{d - 1}$$A_{d - 1}\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} 2 \\ k \\ \end{pmatrix}\left( {2 - k} \right)^{d - 1}$$A_{d - 1}\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left( {n - k} \right)^{d - 1}$P~D~ -- P~D-1~$A_{d}\sum\limits_{k = 0}^{0}\left( {- 1} \right)^{k}\begin{pmatrix} 0 \\ k \\ \end{pmatrix}\left( {- k} \right)^{d}$$A_{d}\sum\limits_{k = 0}^{1}\left( {- 1} \right)^{k}\begin{pmatrix} 1 \\ k \\ \end{pmatrix}\left( {1 - k} \right)^{d}$$A_{d}\sum\limits_{k = 0}^{2}\left( {- 1} \right)^{k}\begin{pmatrix} 2 \\ k \\ \end{pmatrix}\left( {2 - k} \right)^{d}$$A_{d}\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left( {n - k} \right)^{d}$ With the last value in each row being:$$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k} \right) - \sum\limits_{i = 0}^{d - m - 1}A_{d - i}\left( {n - k} \right)^{d - i}} \right\rbrack\mspace{6mu} = {\mspace{6mu} m}!{\mspace{6mu}\mspace{6mu}\mspace{6mu}}$$ This pattern is similar to the pattern triangle which shows the values as:$$\begin{matrix} 1 \\ {0\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 1} \\ {0\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 1\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 2} \\ {0\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 1\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 6\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 6} \\ {\mspace{6mu} 0\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 1\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 14\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 36\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 24} \\ {\mspace{6mu}\mspace{6mu}\mspace{6mu} 0\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 1\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 30\mspace{6mu}\mspace{6mu}\mspace{6mu}\mspace{6mu} 150\mspace{6mu}\mspace{6mu}\mspace{6mu} 240\mspace{6mu}\mspace{6mu}\mspace{6mu} 120.} \\ \end{matrix}$$ The difference between this pattern triangle and the table we have above is that the constants behind each number in the pattern triangle are set as 1. Simplify the following equation:$$\left\lbrack {n + 1} \right\rbrack\left\lbrack {\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left( {n - k} \right)^{m} + \sum\limits_{k = 0}^{n + 1}\left( {- 1} \right)^{k}\begin{pmatrix} {n + 1} \\ k \\ \end{pmatrix}\left( {n - k + 1} \right)^{m}} \right\rbrack$$ Expand the summation:$$\begin{array}{ccl} & = & {\left( {n + 1} \right)\left\lbrack \begin{pmatrix} n \\ 0 \\ \end{pmatrix}\left( n \right)^{m} - \mspace{6mu}\begin{pmatrix} n \\ 1 \\ \end{pmatrix}\left( {n - 1} \right)^{m} + \mspace{6mu}\begin{pmatrix} n \\ 2 \\ \end{pmatrix}\left( {n - 2} \right)^{m} - \ldots + \mspace{6mu}\left( {- 1} \right)^{n}\begin{pmatrix} n \\ n \\ \end{pmatrix}\left( 0 \right)^{m} + \begin{pmatrix} {n + 1} \\ 0 \\ \end{pmatrix}\left( {n + 1} \right)^{m} \right.} \\ & & \left. {- \mspace{6mu}\begin{pmatrix} {n + 1} \\ 1 \\ \end{pmatrix}\left( n \right)^{m} + \begin{pmatrix} {n + 1} \\ 2 \\ \end{pmatrix}\left( {n - 1} \right)^{m} - \ldots + \mspace{6mu}\left( {- 1} \right)^{n + 1}\begin{pmatrix} {n + 1} \\ {n + 1} \\ \end{pmatrix}\left( 0 \right)^{m}} \right\rbrack \\ \end{array}$$ Distribute:$$\begin{array}{ccl} & = & {\left( n + 1 \right)\left( \begin{array}{l} n \\ 0 \\ \end{array} \right)\left( n \right)^{m} - \left( n + 1 \right)\left( \begin{array}{l} n \\ 1 \\ \end{array} \right)\left( n - 1 \right)^{m} + \left( n + 1 \right)\left( \begin{array}{l} n \\ 2 \\ \end{array} \right)\left( n \right)^{m} - \cdots + \left( n + 1 \right)\left( - 1 \right)^{n}\left( \begin{array}{l} n \\ n \\ \end{array} \right)\left( 0 \right)^{m}} \\ & & {+ \left( n + 1 \right)\left( \begin{array}{l} {n + 1} \\ 0 \\ \end{array} \right)\left( n + 1 \right)^{m} - \left( n + 1 \right)\left( \begin{array}{l} {n + 1} \\ 1 \\ \end{array} \right)\left( n \right)^{m} + \left( n + 1 \right)\left( \begin{array}{l} {n + 1} \\ 2 \\ \end{array} \right)\left( n - 1 \right)^{m} - \cdots} \\ & & {+ \left( n + 1 \right)\left( - 1 \right)^{n + 1}\left( \begin{array}{l} {n + 1} \\ {n + 1} \\ \end{array} \right)\left( 0 \right)^{m}} \\ \end{array}$$ Group like terms:$$\begin{array}{ccl} & = & {\left( \begin{array}{l} {n + 1} \\ 0 \\ \end{array} \right)\left( n + 1 \right)^{m + 1} + \left( n + 1 \right)\left\lbrack \left( n \right)^{m}\left\lbrack \left( \begin{array}{l} n \\ 0 \\ \end{array} \right) - \left( \begin{array}{l} {n + 1} \\ 1 \\ \end{array} \right) \right\rbrack - \left( n - 1 \right)^{m}\left\lbrack \left( \begin{array}{l} n \\ 1 \\ \end{array} \right) - \left( \begin{array}{l} {n + 1} \\ 2 \\ \end{array} \right) \right\rbrack \right.} \\ & & {+ \left. \left( n - 2 \right)^{m}\left\lbrack \left( \begin{array}{l} n \\ 2 \\ \end{array} \right) - \left( \begin{array}{l} {n + 1} \\ 3 \\ \end{array} \right) \right\rbrack - \cdots + \left( - 1 \right)^{n}0^{m}\left\lbrack \left( \begin{array}{l} n \\ n \\ \end{array} \right) - \left( \begin{array}{l} {n + 1} \\ {n + 1} \\ \end{array} \right) \right\rbrack \right\rbrack} \\ \end{array}$$ Expand the combinations:$$\begin{array}{ccl} & & {\! = \!\left( {n\! + \! 2} \right)\begin{pmatrix} {n\! + \! 1} \\ 0 \\ \end{pmatrix}\left( {n\! + \! 2} \right)^{m}\! + \!\left( {n\! + \! 1} \right)^{m}\left\lbrack {\left( {n\! + \! 1} \right)\frac{n!}{0!\mspace{6mu} n!} - \left( {n + 2} \right)\frac{\left( {n + 1} \right)!}{1!\mspace{6mu} n!}} \right\rbrack} \\ & & {\quad - \left( n \right)^{m}\left\lbrack {\left( {n + 1} \right)\frac{n!}{1!\left( {n - 1} \right)!} - \left( {n + 2} \right)\frac{\left( {n + 1} \right)!}{2!\left( {n - 1} \right)!}} \right\rbrack} \\ & & {\quad + \left( {n - 1} \right)^{m}\left\lbrack {\left( {n + 1} \right)\frac{n!}{2!\left( {n - 2} \right)!} - \left( {n + 2} \right)\frac{\left( {n + 1} \right)!}{3!\left( {n - 2} \right)!}} \right\rbrack - \ldots} \\ & & {\quad + \left( {- 1} \right)^{n}1^{m}\left\lbrack {\left( {n + 1} \right)\frac{n!}{n!0!} - \left( {n + 2} \right)\frac{\left( {n + 1} \right)!}{\left( {n + 1} \right)!0!}} \right\rbrack} \\ \end{array}$$ Combine the difference of factorials.$$\begin{array}{ccl} & = & {\begin{pmatrix} {n + 1} \\ 0 \\ \end{pmatrix}\left( {n + 1} \right)^{m} + \left( {n + 1} \right)\left\lbrack \left( n \right)^{m}\left\lbrack \frac{\left( {1 - \left( {n + 1} \right)} \right)\left( n \right)!}{1!\left( n \right)!} \right\rbrack - \left( {n - 1} \right)^{m}\left\lbrack \frac{\left( {2 - \left( {n + 1} \right)} \right)\left( n \right)!}{2!\left( {n - 1} \right)!} \right\rbrack \right.} \\ & & \left. + \left( {n - 2} \right)^{m}\left\lbrack \frac{\left( {3 - \left( {n + 1} \right)} \right)\left( n \right)!}{3!\left( {n - 2} \right)!} \right\rbrack - \;\ldots + \left( {- 1} \right)^{n - 1}1^{m}\left\lbrack \frac{\left( {n - \left( {n + 1} \right)} \right)\left( n \right)!}{\left( n \right)!1!} \right\rbrack + 0 \right\rbrack \\ & = & {\begin{pmatrix} {n + 1} \\ 0 \\ \end{pmatrix}\left( {n + 1} \right)^{m} + \left( {n + 1} \right)\left\lbrack \left( n \right)^{m}\left\lbrack \frac{- \left( n \right)\left( n \right)!}{1!\left( n \right)!} \right\rbrack - \left( {n - 1} \right)^{m}\left\lbrack \frac{- \left( {n - 1} \right)\left( n \right)!}{2!\left( {n - 1} \right)!} \right\rbrack \right.} \\ & & \left. + \left( {n - 2} \right)^{m}\left\lbrack \frac{- \left( {n - 2} \right)\left( n \right)!}{3!\left( {n - 2} \right)!} \right\rbrack - \ldots + \left( {- 1} \right)^{n - 1}1^{m}\left\lbrack \frac{- 1\left( n \right)!}{\left( n \right)!1!} \right\rbrack + 0 \right\rbrack \\ \end{array}$$ Factor out like terms.$$\begin{array}{ccl} & = & {\begin{pmatrix} {n + 1} \\ 0 \\ \end{pmatrix}\left( {n + 1} \right)^{m} + \left( {n + 1} \right)\left\lbrack \left( n \right)^{m + 1}\left\lbrack \frac{- \left( n \right)!}{1!\left( n \right)!} \right\rbrack - \left( {n - 1} \right)^{m + 1}\left\lbrack \frac{- \left( n \right)!}{2!\left( {n - 2} \right)!} \right\rbrack \right.} \\ & & \left. + \;\left( {n - 2} \right)^{m + 1}\left\lbrack \frac{- \left( n \right)!}{3!\left( {n - 3} \right)!} \right\rbrack - \ldots + \left( {- 1} \right)^{n - 1}1^{m + 1}\left\lbrack \frac{- 1\left( n \right)!}{\left( n \right)!1!} \right\rbrack + 0 \right\rbrack \\ \end{array}$$ Distribute.$$\begin{array}{ccl} & = & {\begin{pmatrix} {n + 1} \\ 0 \\ \end{pmatrix}\left( {n + 1} \right)^{m + 1} + \left\lbrack \left( n \right)^{m + 1}\left\lbrack \frac{- \left( {n + 1} \right)!}{1!\left( n \right)!} \right\rbrack - \left( {n - 1} \right)^{m + 1}\left\lbrack \frac{- \left( {n + 1} \right)!}{2!\left( {n - 2} \right)!} \right\rbrack \right.} \\ & & \left. + \left( {n - 2} \right)^{m + 1}\left\lbrack \frac{- \left( {n + 1} \right)!}{3!\left( {n - 3} \right)!} \right\rbrack - \ldots + \left( {- 1} \right)^{n - 1}1^{m + 1}\left\lbrack \frac{- 1\left( {n + 1} \right)!}{\left( n \right)!1!} \right\rbrack + 0 \right\rbrack \\ \end{array}$$ Simplify into a summation.$$\begin{array}{l} {= \begin{pmatrix} {n + 1} \\ 0 \\ \end{pmatrix}\left( {n + 1} \right)^{m} + \sum\limits_{k = 1}^{n + 1}\left( {- 1} \right)^{k + 1}\begin{pmatrix} {n + 1} \\ k \\ \end{pmatrix}\left( {n - k + 1} \right)^{m + 1} = \mspace{6mu}\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k + 1}\begin{pmatrix} {n + 1} \\ k \\ \end{pmatrix}\left( {n - k + 1} \right)^{m + 1}} \\ {\therefore\left\lbrack {n + 1} \right\rbrack\left\lbrack {\sum\limits_{k = 0}^{n}\left( {- 1} \right)^{k}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left( {n - k} \right)^{m} + \sum\limits_{k = 0}^{n + 1}\left( {- 1} \right)^{k}\begin{pmatrix} {n + 1} \\ k \\ \end{pmatrix}\left( {n - k + 1} \right)^{m}} \right\rbrack = \sum\limits_{k = 0}^{n + 1}\left( {- 1} \right)^{k}\begin{pmatrix} {n + 1} \\ k \\ \end{pmatrix}\left( {n - k + 1} \right)^{m + 1}} \\ \end{array}$$ This follows the pattern of the Term Zero Difference Triangle Proof of application of pattern triangle {#sec0029} ---------------------------------------- From the previous proof, we know that the first term of each column Δ~n~ for any degree is:$$\sum\limits_{k = 0}^{n}\left( - 1 \right)^{n}\begin{pmatrix} n \\ k \\ \end{pmatrix}\left\lbrack {f\left( {n - k + 1} \right) - \sum\limits_{i = 0}^{d - m - 1}A_{d - i}\left( {n - k + 1} \right)^{d - i}} \right\rbrack$$ We know that$$\Delta_{d,n} - \Delta_{d - 1,n} = A_{d}\sum\limits_{k = 0}^{n}\left( - 1 \right)^{k}\left( \begin{array}{l} n \\ k \\ \end{array} \right)\left( n - k + 1 \right)^{d}$$ where the summation gives each term in the pattern triangle. Thus, the following equation:$$\Delta_{d - 1,n} = \Delta_{d,n} - A_{d}\sum\limits_{k = 0}^{n}\left( - 1 \right)^{k}\left( \begin{array}{l} n \\ k \\ \end{array} \right)\left( n - k + 1 \right)^{d}$$ will give you the differences of the polynomial function one degree lower (removing the highest degree variable like shown in the proof of the pattern triangle for g(x)). Since A~d~ is the last term of each row divided by n!, the equation above can be simplified to:$$\Delta_{d - 1,n} = \Delta_{d,n} - \left\lbrack \frac{\sum_{k = 0}^{n}\left( - 1 \right)^{n}\left( \begin{array}{l} n \\ k \\ \end{array} \right)f\left( n - k + 1 \right)}{n!} \right\rbrack\sum\limits_{k = 0}^{n}\left( - 1 \right)^{k}\left( \begin{array}{l} n \\ k \\ \end{array} \right)\left( n - k + 1 \right)^{d}$$ This equation above means that the first term of each column in the difference table of the function P~d-1~(x) can be found by taking the first term of each column in the difference table of the function P~d~(x) and subtracting the quantity of each respective value in the pattern triangle times the value of the leading coefficient of P~d~(x), which can also be found by knowing the differences of P~d~(x). It is important to understand that there can be an infinite amount number of polynomial equations that can represent the function. For example, if you were given the first two terms of a sequence -- 0,1 -- any equation of form f(x) = (x-1)^d^ will match the function, where d is all real numbers. Therefore, if given the first n terms of a sequence, one can determine the lowest degree polynomial function of maximum degree, n -- 1, that represents the sequence. Conclusion {#sec0030} ========== In summary, the "Split Difference" method can be used to determine the equation to fit a given sequence. This method uses a pattern triangle and applies it to the difference table used to find the degree of a polynomial in order to speed up this process of determining a representative polynomial function. The experimental results have shown this method to be fast and very accurate.A HEADINGS IN APPENDICESA.1 INTRODUCTIONA.2 EXPERIMENTAL AND COMPUTATIONAL DETAILSA.2.1 SPLIT DIFFERENCE2.1.1 Finding the Differences*2.1.1.1 Splitting the Sequence into its Differences2.1.1.2 Determine the Degree of the Polynomial2.1.1.3 Finding the 0th Differences*2.1.2 Creating the Difference Zero Triangle2.1.3 Apply and Solve*2.1.3.1 Finding the Coefficients2.1.3.2 Determine the Equation*A.2.2 ALTERNATIVE METHOD2.2.1 Finding the Differences2.2.2 Creating the First Difference Triangle2.2.3 Apply and Solve*2.2.3.1 Finding the Coefficients2.2.3.2 Determine the Equation*A.3 CURRENT METHODSA.3.1 System of EquationsA.3.2 Difference TableA.3.3 Newton\'s InterpolationA.3.4 Split Difference Method BenefitsA.4 PROOFA.4.1 Proof of First Difference Pattern TriangleA.4.2 Proof of Application of Pattern TriangleA.4.3 Proof of Split Difference MethodsA.5 CONCLUSIONS Declaration of Competing Interests ================================== The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Zingg R, Fischer M. The consolidation of nanomedicine. WIREs Nanomed Nanobiotechnol. 2019;11:e1569 10.1002/wnan.1569 1. INTRODUCTION {#wnan1569-sec-0001} =============== Ever since the U.S. Food and Drug Administration (FDA) approved a nanotherapeutic product for the first time back in 1995---Doxil, an anticancer drug using PEGylated nano‐liposomes---the dawn of nanomedicine has been proclaimed. In fact, however, despite a number of clinical successes that have impacted cardiology and oncology in particular, nanotechnology has not yet revolutionized the diagnosis and treatment of diseases on a large scale. For pharmaceuticals, in vivo imaging and in vitro diagnostics, nanotechnology is poised to contribute significantly, but the technology has yet to bear its fruits. Many argue that this results at least partly from the uncertainties regarding the health risks nanomaterials may pose (Agarwal, Bajpai, & Sharma, [2018](#wnan1569-bib-0001){ref-type="ref"}; Jongandand & Borm, [2008](#wnan1569-bib-0015){ref-type="ref"}). While this is a problem for nanotechnology as a whole, the legal regulation of nanomedicine introduces a special set of constraints worth an independent study (Bregoli et al., [2016](#wnan1569-bib-0006){ref-type="ref"}; Burgess et al., [2010](#wnan1569-bib-0008){ref-type="ref"}). Safety concerns, relating to nanomaterial‐induced toxicity effects in particular, have nanopharmaceuticals go through more elaborate and more costly testings (Venkatraman, [2014](#wnan1569-bib-0029){ref-type="ref"}). The lack of validated methods for toxicity testing assays and the limited understanding of the interaction of nanomaterials with biological systems complicate the establishment of accepted health risk assessments (Halappanavar, Vogel, Wallin, & Yauk, [2018](#wnan1569-bib-0013){ref-type="ref"}). From a regulatory standpoint, the FDA has failed to set forth practical assays, testing, or data requirements (Bawa, Barenholz, & Owen, [2016](#wnan1569-bib-0004){ref-type="ref"}). The resulting uncertainty may explain nanomedicine\'s underperformance at clinical trial stage, a primary reason for its slow translation into approved therapeutic therapies (Weissig & Guzman‐Villanueva, [2015](#wnan1569-bib-0031){ref-type="ref"}; Yu & Bae, [2018](#wnan1569-bib-0032){ref-type="ref"}). In December 2017, the agency finally released a non‐binding draft guidance for industry in order to address the safety and efficacy challenges of approving complex drugs containing nanomaterials.[1](#wnan1569-note-0001){ref-type="fn"} Still, this leaves room for a number of uncertainties concerning possible approval pathways (Emily, Ioanna, Scott, & Beat, [2018](#wnan1569-bib-0010){ref-type="ref"}). This article advances that, in light of these uncertainties, nanomedicine consolidated rather than expanded. In short, we contend that innovators mostly sought to deal with the uncertainties inherent to nanomedicine by trying to build upon approved technologies. Typically, firms would favor scientifically crowded fields where other nanomedicine inventors already paved the way. Accordingly, the industry would seek to tailor nano‐enabled products to known markets rather than to explore the technology\'s potential to its full extent (Bosetti & Vereeck, [2012](#wnan1569-bib-0005){ref-type="ref"}; Neuman & Chandhok, [2016](#wnan1569-bib-0022){ref-type="ref"}; Pelaz et al., [2017](#wnan1569-bib-0024){ref-type="ref"}). While we realize that there undoubtedly are advantages to leveraging closely related research to leap forward, that is, to stand on the shoulders of giants, we contend that too close an adherence to past research might have prevented nanomedicine from diving into unexplored fields. We believe this phenomenon may, for example, be observed with respect to advances---or lack thereof---in nanopharmaceuticals. Of the 50 nanopharmaceuticals that had received FDA approval in 2016, almost all were nanoformulations of existing drugs rather than providing a novel pharmacological effect themselves. Their clinical benefits have been mainly limited to reductions in toxicity rather than improvements in efficacy, not fully realizing the high expectations of the scientific and medical community (Joseph, Artish, Tian, & Andrew, [2017](#wnan1569-bib-0016){ref-type="ref"}; Ventola, [2012](#wnan1569-bib-0030){ref-type="ref"}). Despite being commercial successes, Doxil did not revolutionize chemotherapy, nor did other nanocarrier therapeutics such as Abraxane and Ambiosome or nanorcystalline drugs such as Rapamune (Venkatraman, [2014](#wnan1569-bib-0029){ref-type="ref"}). To fully turn the potential of nanopharmaceuticals into clinical formulations, additional advances in, and deeper understanding of, drug‐loading capacities, drug‐release control, clearance or degradation at target site, cellular uptake, and interaction with biological systems are needed (Nassiri & Abdollahi, [2016](#wnan1569-bib-0021){ref-type="ref"}; Park, [2013](#wnan1569-bib-0023){ref-type="ref"}; Qiao et al., [2019](#wnan1569-bib-0026){ref-type="ref"}). 2. THE EMERGENCE OF NANOMEDICINE {#wnan1569-sec-0002} ================================ Nanomedicine patents granted in the United States can be used to investigate this claim. Patent data can be extracted using PatentsView, a database sourced from United States Patent and Trademark Office provided data. Nanomedicine patents are classified together with nanobiotechnology in class B82Y 5/00 of the International Patent Classification (Jürgens & Herrero‐Solana, [2017](#wnan1569-bib-0017){ref-type="ref"}).[2](#wnan1569-note-0002){ref-type="fn"} While patents granted for nanomedicine inventions have been on the rise, the numbers are still quite low. The annual rate of filing stabilized at around 150 applications a year since 1996 (see Figure [1](#wnan1569-fig-0001){ref-type="fig"}). Note that the most recent statistics are incomplete since patent applications are published 18 months after their earliest filing date only. {#wnan1569-fig-0001} Overall, nanomedicine patents thus only represent 3,497 out of the total 48,212 patents granted in nanotechnology (1974--2016). When filing for a patent, all applicants, their attorneys and agents have a duty to disclose all information known to be material to patentability, other patents in particular (Title 37 of the Code of Federal Regulations, §1.56\[a\]). Failure to comply bears drastic consequences: a patent may be declared unenforceable for its term (Erstling, [2011](#wnan1569-bib-0011){ref-type="ref"}). Therefore, citations serve as a good indicator for the prior art that was relied on when inventing (Barirani, Agard, & Beaudry, [2013](#wnan1569-bib-0002){ref-type="ref"}). Corresponding information was extracted from PatentsView, as well. Through this, we gain an overview of the emergence of nanomedicine, that is, what prior art it built upon over time. Note that patents cited by nanomedicine patents may not necessarily pertain to nanomedicine themselves. 3. THE CONSOLIDATION OF NANOMEDICINE {#wnan1569-sec-0003} ==================================== Figure [2](#wnan1569-fig-0002){ref-type="fig"} depicts the evolution of nanomedicine in terms of patent citation networks. Therein, nodes represent patents, and links between nodes are established when one of the patents cites the other. When examining these networks of patent citations, one can observe a trend toward consolidation. In the early stages from 1974 to 1990, three small "islands" of prior art had formed in isolation. Then they bridged in 1995. From then on, nanomedicine grew increasingly closer. Nowadays, despite spanning a variety of areas, almost all patents are linked to each other either directly or indirectly through common prior art. Furthermore, although the vast majority of cited patents are non‐nanomedicinal (88%), a majority of patents refers to at least one nanomedicine patent (up to 80% in 2014). Rather than branching out in specialized subfields, nanomedicine has been increasingly consolidating. ) Nanomedicine patents; () non‐nanomedicine patents](WNAN-11-na-g002){#wnan1569-fig-0002} In fact, the central nodes in the networks are mostly nanomedicinal: Out of the 10 most frequently cited patents, seven are classified as nanomedicine. Four of these patents relate to mixing essentially water‐insoluble drugs with cyclodextrin---molecules with a size of about 1 nm---in order to obtain amorphous complexes that are themselves in turn highly water‐soluble (Patents 1, 4, 7, and 8 according to Table [1](#wnan1569-tbl-0001){ref-type="table"}). In other words, they pertain to new formulations of known drugs that significantly enhance their absorption by the human body. Patent 3 according to Table [1](#wnan1569-tbl-0001){ref-type="table"} relates to using surface modifiers in order to keep an average particle diameter of a drug below 400 nm, vastly improving the drug\'s bioavailability. Patent 5 addresses selective targeting of radionuclides to solid tumor areas within the body. Similarly, Patent 6 pertains to providing block copolymers that can be used for controlled delivery of biologically active materials. As apparent from this analysis, the building blocks of nanomedicine indeed seem to be inventions covering new formulations of known active substances rather than independent, new products. ###### Top 10 most cited patents in nanomedicine Patent number Year Technology Title Citations ---- --------------- ------ -------------- ------------------------------------------------------------------------------------------------------------ ----------- 1 US4727064 1993 Nanomedicine Pharmaceutical preparations containing cyclodextrin derivatives 140 2 US5270163 1997 Other Methods for identifying nucleic acid ligands 91 3 US5145684 2007 Nanomedicine Surface modified drug nanoparticles 63 4 US4596795 1985 Nanomedicine Administration of sex hormones in the form of hydrophilic cyclodextrin derivatives 60 5 US4863713 1993 Nanomedicine Method and system for administering therapeutic and diagnostic agents 60 6 US5543158 2004 Nanomedicine Biodegradable injectable nanoparticles 57 7 US5024998 2001 Nanomedicine Pharmaceutical formulations for parenteral use 55 8 US4983586 1990 Nanomedicine Pharmaceutical formulations for parenteral use 53 9 US5256395 2010 Other Affinity enhancement immunological reagents for in vivo detection and killing of specific target cells 50 10 US5143854 2011 Other Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof 48 4. PUSHING THE BOUNDARIES IN NANOMEDICINE {#wnan1569-sec-0004} ========================================= Despite being such a broad and interdisciplinary field at the frontier of the life sciences, research in nanomedicine so far seems to have rather consolidated than expanded. It may have been uncertainties in safety, regulatory and ethical requirements that have led innovators to engage in nanomedicine that relies on past nanomedical innovation, eventually at the expense of fostering advances in novel fields. To reduce said uncertainty, a number of steps could be taken. Firstly, federal and international regulatory agencies should start by establishing regulatory definitions or common working descriptions of key terms like "nanotechnology," "nanomaterial," and "nanomedicine" to ensure harmonized governance (Bartlett et al., [2015](#wnan1569-bib-0003){ref-type="ref"}; Bawa et al., [2016](#wnan1569-bib-0004){ref-type="ref"}; Pita, Ehmann, & Papaluca, [2016](#wnan1569-bib-0025){ref-type="ref"}; Tinkle et al., [2014](#wnan1569-bib-0028){ref-type="ref"}). Secondly, regulatory bodies should seek to move from a non‐binding draft recommendation regime to a definite and enforceable one. Current regulations in nanomedicine comprise only a body of reflection papers in Europe and Japan, and industry guidelines in Canada and the United States (Bremer‐Hoffmann, Halamoda‐Kenzaoui, & Borgos, [2018](#wnan1569-bib-0007){ref-type="ref"}). Specific regulations and protocols for preclinical development and characterization would provide stakeholders with the certainty they seek (Bawa et al., [2016](#wnan1569-bib-0004){ref-type="ref"}; Marchant & Abbott, [2013](#wnan1569-bib-0020){ref-type="ref"}; Sainz et al., [2015](#wnan1569-bib-0027){ref-type="ref"}). Thirdly, to counter the lack of product safety data, the agencies should develop a comprehensive database of information, and provide firms with incentives for pre‐market voluntary submissions (Diamond, [2008](#wnan1569-bib-0009){ref-type="ref"}; Ventola, [2012](#wnan1569-bib-0030){ref-type="ref"}). With better data, the regulator is in a better position to assess the submitted nanoproducts, thereby increasing the efficiency of its procedures. In essence, regulatory bodies must shift from their approach considering nanoparticles as small versions of larger molecules to one where they recognize their fundamental different properties (Bawa et al., [2016](#wnan1569-bib-0004){ref-type="ref"}; Fischer, [2018](#wnan1569-bib-0012){ref-type="ref"}; Ventola, [2012](#wnan1569-bib-0030){ref-type="ref"}). Alternatively, the public funder must take action. Early initiatives in nanomedicine and nanobiotechnology had already identified the reticence of pharmaceutical firms in embracing novel and risky‐seeming nanomedicinal research (Jackman, Lee, & Cho, [2016](#wnan1569-bib-0014){ref-type="ref"}; Keelan, Leong, Ho, & Iyer, [2015](#wnan1569-bib-0018){ref-type="ref"}; Lenoir & Herron, [2015](#wnan1569-bib-0019){ref-type="ref"}). The National Cancer Institute illustratively launched the Alliance for Nanotechnology in Cancer in 2004 to ignite nano‐scale products for cancer diagnosis, prevention, and treatment. The most promising research avenues developed by the Alliance are then handed off to private sector partners for effective clinical translation and commercialization.[3](#wnan1569-note-0003){ref-type="fn"} The Alliance has funneled taxpayer funds into early‐stage research, spawning a number of advances in nanoparticle vaccines (PRINT particle design by Liquidia Technologies), in‐vitro blood diagnostics (T2Hemostasis devices which utilize the proprietary T2 Magnetic Resonance by T2 Biosystems), as well as drug targeting and delivery more broadly (AxioCore nanofiber drug delivery by Arsenal Vascular; Lenoir & Herron, [2015](#wnan1569-bib-0019){ref-type="ref"}). Such targeted initiatives have the potential to lead to patents in novel but uncertain fields, pushing boundaries for‐profit firms would not necessarily be willing to cross. CONFLICT OF INTEREST {#wnan1569-sec-0005} ==================== The authors have declared no conflicts of interest for this article. RELATED WIREs ARTICLES {#wnan1569-sec-0006} ====================== <https://doi.org/10.1002/wnan.1416> <https://doi.org/10.1002/wnan.1527> <https://doi.org/10.1002/wnan.1465> US Food and Drug Administration, Drug Products, Including Biological Products, that Contain Nanomaterials, Guidance for Industry, December 2017. European Patent Office. Nanotechnology and Patents. 2013. National Cancer Institute, Cancer Nanotechnology Plan 2015.