The primary molecular endpoint for many Duchenne muscular dystrophy (DMD) clinical trials is the induction, or increase in production, of dystrophin protein in striated muscle. For accurate endpoint analysis, it is essential to have reliable, robust and objective quantification methodologies capable of detecting subtle changes in dystrophin expression. In this work, we present further development and optimisation of an automated, digital, high-throughput script for quantitative analysis of multiplexed immunofluorescent (IF) whole slide images (WSI) of dystrophin, dystrophin associated proteins (DAPs) and regenerating myofibres  in transverse sections of DMD, Becker muscular dystrophy (BMD) and control skeletal muscle biopsies. The script enables extensive automated assessment of myofibre morphometrics, protein quantification by fluorescence intensity and sarcolemmal circumference coverage, colocalisation data for dystrophin and DAPs and regeneration at the single myofibre and whole section level. Analysis revealed significant variation in dystrophin intensity, percentage coverage and amounts of DAPs between differing DMD and BMD samples. Accurate identification of dystrophin via a novel background subtraction method allowed differential assessment of DAP fluorescence intensity within dystrophin positive compared to dystrophin negative sarcolemma regions. This enabled surrogate quantification of molecular functionality of dystrophin in the assembly of the DAP complex. Overall, the digital script is capable of multiparametric and unbiased analysis of markers of myofibre regeneration and dystrophin in relation to key DAPs and enabled better characterisation of the heterogeneity in dystrophin expression patterns seen in BMD and DMD alongside the surrogate assessment of molecular functionality of dystrophin. Both these aspects will be of significant relevance to ongoing and future DMD and other muscular dystrophies clinical trials to help benchmark therapeutic efficacy. There is a growing need for reliable, replicable, automated, high-throughput, digital image analysis techniques that can be utilised for the pathological assessment of diagnostic and clinical trial samples. Improvement in accuracy and reliability of digital analysis platforms presents an alternative to manual, labour intensive and potentially subjective histological assessments, or semi-quantitative expert assessment. When coupled with high-throughput slide scanning to generate whole slide images (WSI), in both brightfield and fluorescent paradigms, digital image analysis facilitates the assessment of entire tissue landscapes, alleviating potential bias from manual selection of discreet regions of interest for analysis, and enabling up-scaled assessment of fibres in a biopsy from a few hundred to many thousands. Subsequent image analysis using modern tissue phenomics software can unlock unparalleled multiparametric data analytics for biological tissues , 23, 37.DMD gene that preclude the production of the protein dystrophin [. Lack of dystrophin results in severe contraction induced muscle damage, causing continuous cycles of muscle degeneration and regeneration, ultimately leading to depletion of muscle mass, fibrofatty replacement and loss of muscle function in affected patients [During the last decade, there has been a rapid expansion in the number of Duchenne muscular dystrophy (DMD) clinical trials . DMD is strophin , 28. Dysstrophin . Lack ofpatients . Regenerpatients .DMD transcript using antisense oligonucleotides to restore the reading frame of the gene, leading to the production of shortened dystrophin protein [. Other therapeutic approaches currently being assessed include stop codon read-through agents [The primary molecular endpoint for many DMD clinical trials and the proof of concept for therapeutic approaches is the induction or increase in the production of dystrophin . For exa protein . Other th agents , myoblash agents , 18. Irrh agents , 6, 35. h agents , 29, 30.h agents , requirih agents , 38. Whih agents , 35, 38.Through a previous study, the foundation for a high-throughput, operator-independent digital method was laid; this focussed on the sarcolemmal intensity of dystrophin through analysis of multiplexed laminin or spectrin, providing the membrane mask, with dystrophin . The tecTo address some of these needs, a new series of image analysis methods implemented in Definiens Developer XD (Munich) have been developed, applicable to digital scans of entire sections of skeletal muscle and tailored specifically to the assessment and study of DMD and BMD biopsies. Data generated from this new method allows not only quantification of dystrophin at the sarcolemma but provides vital information of colocalisation and quantification of key DAPs in relation to sarcolemmal dystrophin. Furthermore, the acquisition procedure has been refined and a novel method for background subtraction has been implemented in an attempt to accurately identify all true dystrophin signal present at the sarcolemma. In addition, automated assessment of the levels of myofibre regeneration as assessed by the number of fibres expressing fetal and/or developmental myosin is presented , 40–42, Serial unfixed frozen sections (7 μm-thick) were cut from 3 control, BMD and DMD frozen muscle biopsies with a Leica CM 1850 UV cryostat . Slides were air dried for 45 min before staining and a Super pap pen  was used to create hydrophobic barriers around the tissue.Samples were incubated with 200-300 μl of primary 1 h-RT) and 200-300 μl of secondary (30 min-RT) cocktail of antibodies diluted in PBS . Volumes varied based on the size of the tissue sections to ensure they were completely covered. Details of the antibody combinations and specifications used are listed in Table  h-RT andSlides were scanned within 24 h of immunostaining on a ZEISS Axio Scan.Z1 Slide Scanner . Laminin α2 staining in the 568 channel was used as a reference protein for coarse and fine focus map generation using the 10x and 20x objectives respectively. After creation of the focus map, 3 channel  WSIs were captured using the 20x objective with offline stitching. The scanner used for this study is calibrated annually with its LEDs measured to an internal reference standard to ensure stable and constant output power over the entire lifetime of each LED. Additionally, as long as only one LED is turned on, the photodiode acts as a closed-loop power stabilization in the μs range. It is capable of controlling the power of the active LED even during the exposure time of an image. This guarantees a stable light output even if the LED is used in short millisecond ranging exposures.One-way ANOVA test with Tukey's multiple comparison correction was used to compare means of each sample with the means of every sample for each experimental condition. GraphPad Prism 7.0 software was used for statistical analysis and graph design. Data are presented as mean ± standard deviation.The method described was developed for the analysis of WSI of immunofluorescent (IF) stained transversely cut muscle sections, composed of a mask stain (laminin α2) identifying the myofibre boundary , dystrophin stain, and either one of two additional relevant sarcolemmal markers (α-sarcoglycan or β-dystroglycan), or a sarcoplasmic markers (f/d myosin). The image analysis processing is comprised of three distinct stages: identification of muscle tissue and exclusion of artefacts; identification of muscle fibres within the tissue; and characterisation of morphological features and staining profiles of individual muscle fibres. The analysis has been implemented using Definiens Developer XD (Munich), version 2.7.0. A workflow of the methodology can be found in supplementary file 1. A summary of the salient features of the analysis is outlined below.For each identified myofibre, a series of morphological descriptors and staining characteristics were calculated and recorded. The morphological descriptors include fibre area, sarcoplasm area, sarcolemma area, fibre width, sarcoplasm width, mean and maximum sarcolemma thickness. The thresholding for sarcolemma thickness was used to optimise the recognition of true myofibre-rich regions by the mask, and not used as a variable endpoint in measuring dystrophin intensity. The staining characteristics included both direct measurements of staining intensity within the sarcolemma and sarcoplasm for all image channels, and empirically enhanced characteristics. The reporting of stain properties is adapted to the two stain combinations under investigation: mask with primary and secondary sarcolemma stain (dystrophin + α-sarcoglycan/β-dystroglycan); and mask with a primary sarcolemma and secondary sarcoplasmic stain (dystrophin + f/d myosin).mask-mod layer, Supp. file 1).For all sarcolemma stains a background subtraction method was developed to identify only significant levels of staining as positive. A number of background removal methods were investigated, such as global thresholds based on the intensity of staining within non-mask regions or within sarcoplasmic regions, but this was insufficient due to the heterogeneity of staining often observed  . Fluorescence intensity was reported as raw values for the sarcolemma (without subtracting the cytoplasmic background). In thresholding for sarcolemmal positivity, it was assumed that the cytoplasmic and sarcolemma region background signal values are equal. Direct comparisons between cytoplasmic background and sarcolemmal background would not be possible due to masking of the sarcolemmal background by true dystrophin signal. By utilising this method, we do not need to set arbitrary intensity thresholds for positivity and are able to adapt dynamically to variations across the section in background fluorescence and non-specific signal.The Circumference Positivity (the sum of (border length-2)/2 for each positive object in a fibre, as a proportion of the circumference of sarcolemma) was used to provide coverage measurements as it has been shown to provide both quantitatively and qualitatively robust measurement of positive sarcolemmal protein coverage, providing a high degree of consistency between similarly scored fibres and with pathologist classification.Each fibre was then classified into one of 4 classes according to the Circumference Positivity: 0–25%, 25–50, 50–75% and 75–100% coverage. Fibres with 0–25% positivity were classified as protein negative whilst fibres with > 25% coverage as protein positive.. Similar assessment was performed for different thresholds of identification of positive sarcolemmal stain. The final formula provided the greatest level of concordance with a pathologist’s manual assessment of the raw fluorescent image against the generated classification map for protein positive and protein negative fibres , dystrophin and a tertiary protein marker of interest . Four regions were randomly selected from CTRL, BMD and DMD samples and ‘auto exposure’ values were generated by the Zeiss AxioScan fluorescent slide scanner for dystrophin and each tertiary protein marker. The auto-exposure values obtained for each protein were then averaged in controls, BMD and DMD samples, along with a global average from all samples.Separate images were then captured using the 4 different exposure times  and subsequently assessed visually by the pathologist to gauge the final ‘optimal’ exposure setting that would allow detection of a range of dystrophin signal from trace to bright ‘revertant’ fibres  with mi). When assessed for % positivity of dystrophin, all 10 replicates gave an average of 99% positivity with minimum and maximum values of 98.5 and 100% positive  were immunostained and analysed on 10 separate days across a 5-week period. 5 sections were stained for dystrophin, laminin α2 and α-sarcoglycan while 5 sections were stained for dystrophin, laminin α2 and β-dystroglycan. Across the 10 replicates the average mean dystrophin intensity was 49,119 AU (arbitrary units) with a maximum and minimum average of 51,662 and 46,669 respectively. This represents a good degree of reproducibility with only 10% variability in fluorescence intensity across the entire period . Minimal variation in mean fluorescent intensity was seen across the 5 replicates for each protein with each sample consistently returning 98–100% protein positivity for analysed fibres, in accordance with manual observation. Maximum and minimum intensity values were 45,603 AU and 52,730 for α-sarcoglycan analysis and 44,074 and 51,533 for β-dystroglycan. Variation in α-sarcoglycan intensity was 13.5% across the 5 experiments while variation in β-dystroglycan was 18%.Similar results were observed for replicate analysis of α-sarcoglycan and β-dystroglycan Fig. c-f. Mini. This demonstrated the script was not aberrantly detecting an abundance of false positive regenerating myofibres that are not present in healthy samples and that a reliable threshold for positivity had been set.For f/d myosin analysis, 5 replicates from the same control sample were immunostained for laminin α2 and a cocktail of f/d myosin on 5 separate days. Each experiment returned a minimum of 0% positive and a maximum of 2% positive f/d myosin positive fibres. Fig. g. This dHaving determined the optimal technical methods for immunostaining, acquisition and quantification, we then performed a small proof of concept study using images generated from 3 CTRL, 3 BMD and 3 DMD samples. The levels of dystrophin, DAPC stain and f/d-myosins were quantified and demographics for all samples are listed in Table . The percentage variation within each group was 9, 59 and 50% respectively for CTRL, BMD and DMD samples, with no statistically significant variation between CTRL samples. However, significant variation was observed between all BMD samples, and also observed between DMD_1/DMD_3 and DMD_2. This variation can also be observed when intensity is plotted as a cumulative frequency against percentage of total fibres for each sample .All CTRL, BMD and DMD samples were immunostained for laminin α2 and dystrophin with each sample containing 2 serial sections to act as technical replicates. Mean dystrophin fluorescence intensity, % of dystrophin positive fibres and the cumulative frequency of sarcolemmal dystrophin circumference coverage was then assessed. The average fluorescence intensity of the 3 CTRL samples was 54,759 AU, BMDs was 29,829 AU and DMDs was 9686 AU . The percentage variation within each sample group  was 0.5, 23.2 and 83.8% respectively. Significant differences in percentage positivity within each disease group were observed between BMD_1/BMD_2 and BMD_3 and DMD_1/DMD_3 and DMD_2. No significant differences were observed between CTRL samples.For percentage positivity (> 25% dystrophin immunostaining at the sarcolemma), CTRLs showed an average of 99% dystrophin positive myofibres, BMDs 91% and DMDs 25%. However, more substantial variability between individual patients was apparent, with BMD_1 showing 99% positivity compared to 76% for BMD_3. This variability was also apparent for the DMD samples, where DMD_1 and DMD_3 showed similar 11 and 10% positivity, respectively, while DMD_2 showed a substantially higher 56% dystrophin positive fibres . Here, variation in levels of percentage positivity among the BMD and DMD samples can more clearly be observed. DMD_1 and DMD_3 follow very similar profiles while DMD_2 shows a similar pattern of positivity to BMD_3. All 3 CTRLs show uniformity in their percentage coverage in comparison to the BMD and DMD samples. Variation can also be observed between BMD_1 and BMD_2 which was not as clearly apparent when simply analysing % positive fibres (> 25% coverage of dystrophin). In addition, for each sample, classification maps of dystrophin positivity are also generated to highlight fibre variability in percentage coverage across the entire section and provide a visual map of the analysed data. Sarcolemmal circumference coverage was used to place fibres in 4 categories. Protein negative (< 25% coverage) or varying degrees of protein positive . Fig. This variation is observed better when the sarcolemmal circumference coverage for dystrophin positivity is plotted as a cumulative frequency graph Fig. d. Here, Alongside primary analysis of dystrophin, we analysed key proteins within the DAPC that interact with dystrophin. Samples were immunostained for a combination of laminin α2, dystrophin, and either α-sarcoglycan or β-dystroglycan. alongside the % positivity of myofibres for either protein in each sample . Additionally, fluorescence intensity of these tertiary markers (α-sarcoglycan and β-dystroglycan) was assessed in regions of myofibre sarcolemma that has been classified as either dystrophin positive or dystrophin negative . This paired, ordered processing allows comparative quantification of the level of DAPs in the presence or absence of dystrophin at the sarcolemma.Mean and cumulative sarcolemmal fluorescence intensity of α-sarcoglycan/β-dystroglycan was assessed ple Fig. c+f. Addiive Fig. g-h. This with 28.6 and 2% variation. However, variation in fluorescence intensity within the CTRL, BMD and DMD groups was not statistically significant. All samples showed 98–100% myofibre positivity for both proteins with no significant differences observed .Average CTRL mean fluorescence intensity for α-sarcoglycan and β-dystroglycan was 40,268 AU and 34,773 AU respectively with 13.5 and 16.4% variation between the 3 CTRL samples. Average BMD intensity was 23,615 AU (α-sarcoglycan) and 20,458 AU (β-dystroglycan) with 15.5 and 36.1% variation between samples while the average DMD values were 21,096 AU (α-sarcoglycan) and 13,153 AU (β-dystroglycan) ). DMD_2 showed a greater increase in fluorescence intensity of α-sarcoglycan in dystrophin positive regions than DMD_1 and DMD_3. However, this difference was not observed when assessing levels of β-dystroglycan intensity in dystrophin positive regions.When assessing colocalisation with dystrophin, all samples showed an increase in fluorescence intensity for both DAPs in regions of the sarcolemma identified as dystrophin positive compared to dystrophin negative regions Fig. g-h. DMD_A f/d myosin cocktail was used to identify myofibres undergoing regeneration. The number of f/d myosin positive fibres was then determined to give percentage positivity values for the entire section, giving an overall assessment of the regeneration in each sample.. Fig. As expected, all 3 controls showed little to no f/d myosin positive fibres with an average value of 1% positivity. BMD_1 and BMD_2 had 4 and 6% positivity respectively whilst BMD_3 presented with 29% of fibres positive for f/d myosin. The 3 DMD samples had a combined average of 32% myofibre positivity for f/d myosin with DMD_2 having the highest value of 42% Fig. a. Fig. 5DMD. However, this heterogeneity of protein expression has so far been poorly characterised using formal quantitative techniques. It is essential to understand the varying levels of dystrophin expression in different BMD patients as this will be vital to help benchmark the success of therapeutic interventions. We have shown that in just 3 patients for each disease group, there is considerable heterogeneity with differences observed in dystrophin intensity, percentage coverage and amounts of DAPs. Analysis of a larger cohort of BMD biopsies employing the current method is ongoing, aimed at characterising the heterogeneity of dystrophin expression, mapping it to the individual genotypes, and correlating to clinical outcome. This will be vital for future clinical trials in DMD, allowing for more accurate benchmarking of results against well characterised BMD phenotypes.With the rapid advancement in the number of DMD clinical trials, and several drugs recently approved  the high levels of trace dystrophin in DMD_2 are not present in the IF images and as such would not be detected during digital analysis  compared to dystrophin positive sarcolemma in the CTRLs.Induction of functional dystrophin should improve sarcolemmal integrity and reduce susceptibility to myofibre necrosis and consequent regeneration in a moderately affected muscle. The ‘myosin module’ of our script enables correlative analysis between the amount of functional dystrophin and the extent of regeneration. In a majority of instances, mutations disrupting the DMD reading frame result in a severe clinical phenotype with little or no dystrophin expression in the biopsy assessed by dystrophin antibodies against a C-terminus epitope and high levels of myofibre regeneration. There are rare but well-recognised exceptions to the DMD reading frame hypothesis . For insDespite improvements on a previous version, there are certain limitations to this script. At present, the fibre recognition module is designed to recognise transverse fibres in cross-sectional orientation. Longitudinal myofibres will not be detected and as such are excluded from the analysis. To ensure the greatest percentage of fibre detection, muscle tissue biopsy blocks must be mounted in cross-sectional orientation allowing for the greatest number of transverse fibres. It is also paramount for tissue sections to be of the highest quality with as little artefactual damage as possible. While we have implemented various elements to account for certain artefactual features, heavily damaged tissue or the introduction of new artefacts will interfere with the analysis, resulting in unreliable final results. Additionally, many small newly regenerating/atrophic fibres in certain pathological samples will not be identified due to the morphometric nature of these myofibres.A novel, high throughput system for quantitative analysis of dystrophin and its associated protein complex members, alongside automated assessment of myofibre regeneration via the presence of aberrant myosin isoforms, is presented. WSI capture and automated analysis allows deep and comprehensive assessment of many thousands of myofibres within each individual muscle biopsy section using multiple parameters, such as mean fluorescence intensity, percentage positive myofibres for sarcolemmal and sarcoplasmic proteins and changes in DAPC expression in dystrophin positive and dystrophin negative regions of the sarcolemma. Combining this with our novel approach to individual myofibre background subtraction for identification of true fluorescent signal allows for accurate dystrophin quantification, even that of trace levels, and investigation of dystrophin functionality with its ability to correctly localise binding partners. Analysis of a very small cohort revealed remarkable heterogeneity in dystrophin expression patterns between different BMD and DMD patients. The ability to detect trace dystrophin, as well as quantitation of colocalised dystrophin and DAPs, will enhance our understanding of the heterogeneity of dystrophin expression in biopsies in the context of the individual genotypes in BMD and DMD, as well as a surrogate marker for molecular functionality of induced dystrophin following therapy. These aspects will be relevant to ongoing and future clinical trials in DMD.Additional file 1.


NR3C1, FKPB5, CRHR1, and AVPR1B). We found a significant association (p < 0.001) between CRHR1 gene variants rs878886 and rs16940665, or haplotype rs878886*C–rs16940665*T, and tianeptine antidepressant response and remission according to the hospital anxiety and depression scale. Analyses, including a structural equation model with simple mediation, suggest a moderate effect of sociodemographic characteristics and depressive disorder features on treatment response in individuals carrying the antidepressant responder allele rs8788861 . These findings suggest direct pharmacological consequences of CRHR1 polymorphisms in the antidepressant tianeptine response and remission, in MDE patients. This study replicates the association of the CRHR1 gene, involved in the HPA axis, with (1) a specificity attributed to treatment response, (2) a lower risk of chance finding, and in (3) an ecological situation.Polymorphisms of genes involved in the hypothalamic–pituitary–adrenocortical (HPA) axis have been associated with response to several antidepressant treatments in patients suffering of depression. These pharmacogenetics findings have been reported from independent cohorts of patients mostly treated with selective serotonin reuptake inhibitors, tricyclic antidepressant, and mirtazapine. Tianeptine, an atypical antidepressant, recently identified as a mu opioid receptor agonist, which prevents and reverses the stress induced by glucocorticoids, has been investigated in this present pharmacogenetics study. More than 3200 Caucasian outpatients with a major depressive episode (MDE) from real-life settings were herein analyzed for clinical response to tianeptine, a treatment initiated from 79.5% of the subjects, during 6–8 weeks follow-up, assessing polymorphisms targeting four genes involved in the HPA axis ( Furthermore, the negative feedback is controlled by the glucocorticoid receptors (GR) and interacting proteins, like FKBP5 protein5. Interestingly, ketamine (an NMDA antagonist) has been shown to significantly improve depression symptoms within patients with treatment-resistant depression, may be partly due to its action on inflammation7. Those data support the hypothesis of HPA axis dysfunction within MDD patients and consequently the hypothesis of improving depression by acting on this system. Convergent evidence showed that antidepressant treatments may normalize or modulate the abnormal functionality of the HPA system in MDD patients11. It seems legitimate to suppose that response to antidepressant treatment could be different according to antidepressant type and genetic polymorphisms of the HPA axis.Major depressive disorder (MDD) is usually considered as being associated with a deregulation of the hypothalamic–pituitary–adrenocortical (HPA) axis. The hyperactivity of the HPA system might be testified by an elevated neurotransmission of the corticotropin-releasing hormone (CRH/CRF) and arginine vasopressin (AVP) peptides, which stimulate the production of adrenocorticotropic hormone and cortisol, leading to a low-grade inflammation in patients with MDDFKBP5 gene (which encodes a chaperon protein to GR) associated with clinical response in European Caucasian MDD patients to various antidepressant treatments, including selective serotonin reuptake inhibitors (SSRI), tricyclic antidepressant (TCA), and mirtazapine (drug targeting serotonergic and noradrenergic receptors)12. The amino acid change N363S in the glucocorticoid receptor, encoded by the single polymorphism nucleotide (SNP) rs56149945/rs6195 in the NR3C1 gene, was investigated among 367 depressed patients having received either SSRI, TCA, or mirtazapine13. This NR3C1 variant was associated with early response after 2 weeks of treatment13. Another pharmacogenetics association was observed between three SNPs encompassing NR3C1 gene and response to escitalopram (SSRI) or nortriptyline (a norepinephrine reuptake inhibitor) among 760 patients of the Genome-based Therapeutic Drugs for Depression study (GENDEP study)14. Haplotypes of three variants of the CRH receptor type 1 CRHR1 gene were associated with an SSRI (fluoxetine) or a TCA (desipramine), in highly anxious Mexican-American patients15. The pharmacogenetics association of CRHR1 gene with response and remission was confirmed in the Sequenced Treatment Alternatives to Relieve Depression, STAR*D cohort, a sample of 1768 MDD patients treated initially by an SSRI 16. In this study, polymorphisms in CRH-binding protein gene (CRHBP), CRH receptor 2 gene (CRHR2), CRH gene, and arginine vasopressin receptor 1A gene (AVPR1A) were also associated with treatment response or remission16.Accordingly, several polymorphisms of genes, involved in HPA axis, have been associated with response to several antidepressant treatments. These results of pharmacogenetics were observed for several polymorphisms of FKBP5 gene was indeed identified as the second gene having the largest effect size reaching a significant threshold17, testifying in favor of a potential use in clinical practice. Nevertheless, antidepressants have variable mechanisms of action, and other antidepressants  are worth being analyzed for their pharmacogenetics factors. We also need to further distinguish the (direct) pharmacogenetics effects from (intermediate) clinical variables, as many are known to be involved in treatment response . Indeed, a gene involved in the HPA axis could be associated with higher vulnerability to severe, comorbid, or chronic depressive disorder. All these traits are strongly related to poorer chances of treatment response18. Finally, if pharmacogenetic associations are replicated and not attributed to a clinical intermediate factor, it is important to assess if such information is predictive in “real life”, meaning in a large cohort of usual outpatients (i.e. with few exclusion criteria) evaluated in an ecological situation.We therefore have converging evidence that genes can be used to efficiently participate in the prediction of antidepressants response, and many of these genes are involved in the HPA regulation. Nevertheless, replication study was not systematic, although independent replication is considered as one of the most solid way to eliminate or, at least, reduce chance finding, especially for samples with modest size. Assessing various biomarkers predicting antidepressant response in a large meta-analysis, 20. Tianeptine is able to inhibit the dendritic remodeling caused by stress or glucocorticoids20. Finally, tianeptine has been shown to be a mu opioid receptor (MOR) agonist21 and its antidepressant effects may be mediated by this receptor22. This is the most interesting role of tianeptine since opioid system is known to partly regulate HPA axis. Indeed, an MOR antagonist would increase HPA axis activation23 and an MOR agonist would mediate this activation24. Clinical efficacy and tolerability of tianeptine have been demonstrated in depressed patients25. Our goal was thus to investigate the pharmacogenetics of tianeptine response by screening genes coding the proteins that play a major role in the HPA axis, in an ecological sample to test the predictive capacity of this set of HPA genes.Tianeptine is an antidepressant with structural similarities with TCAs, but with different pharmacological properties. Tianeptine is an atypical antidepressant because its mechanisms of action clearly challenge the monoamine hypothesis, triggering a cascade of cellular adaptations that leads to antidepressant response. Interestingly, tianeptine prevents and reverses the stress induced by glucocorticoids , and it increases dendritic length and branching of neurons in the amygdalaWe therefore collected a sample of outpatients treated with tianeptine for a major depressive episode (MDE). More than 3200 Caucasian outpatients were analyzed for clinical response to tianeptine and assessed by the genotyping of different SNPs targeting several genes involved in the HPA axis.27 to constitute a novel cohort of antidepressant treated patients, named GENESE. This time, we included in the research tools a device allowing to collect saliva in order to perform pharmacogenetic analyses. A total of 3771 outpatients were enrolled. For this pharmacogenetic study, we restricted the analyses to the 3212 Caucasians (85.18%) with available DNA  to reduce the risk of stratification biases at the genetic level, due to the variability of the allelic percentages of genetic variants in different ethnic groups. The sample size of 3212 Caucasian outpatients was sufficient to detect a pharmacogenetics difference of 5% between the responder and non-responder groups.We used a French network of psychiatrists and general physicians who already collected large samples of depressed outpatientsN = 2419 outpatients) and was prescribed as a switch from another antidepressant treatment for 18.8% of subjects (N = 573 outpatients) while this information was missing for 1.7% (N = 50). Dosage of tianeptine ranged between 12.5 and 37.5 mg/day, according to prescription recommendations. All other concomitant treatments for current somatic problems or associated symptoms of depression  were permitted, based on clinical judgment (see section “Treatments”). At the first visit, practitioners validated the diagnosis of MDE according to DSM-IV criteria. The practitioners also collected demographic data, major depressive disorder history, lifetime suicide attempt and alcohol or substance dependence at the first visit during a face-to-face interview. Non-inclusion criteria were age under 18 years old, non-Caucasian ethnicity, alcohol and substance dependence, or any other psychiatric pathology from axis I other than current MDE. The study was performed according to French regulatory guidelines and current codes of Good Clinical Practice. This study was approved by a local independent ethical committee . Each patient was informed about the aims and procedures of the study and provided written, signed consent.GENESE is a large prospective, naturalistic cohort of French outpatients diagnosed with MDE and treated with tianeptine (from whom they were naïve for the present episode). Tianeptine was initiated for 79.5% of subjects  and Depression (HADS-D) subscales.Depression was assessed with a French version of the hospital anxiety and depression scale (HADS) 16 items at baseline V1. The second visit (V2) was followed in a window between 6 and 8 weeks and a novel evaluation of the HADS was done. Furthermore, outpatients made a self-evaluation of the HADS at V1 and V2 and at 15 and 30 days after the beginning of the treatment. This scale has a good performance assessing depression severity in both psychiatric and primary care patients, a good change-sensitivityA positive treatment response (Resp+) was defined as the reduction of at least 50% of the HADS score at V2 compared to V1. Remission of depression was defined as a HADS score lower than a cutoff of 8 at V2. Finally, an early partial response was defined with a difference of reduction of the HADS higher than 25% after 2 weeks according to the report of the outpatient.p > 0.186).Associated treatments that may have an impact on treatment response  were also recorded, but they had the same frequency in both response groups (N = 62) of outpatients also received a mood stabilizer, 2.7% (N = 83) a mood stabilizer and benzodiazepine, 50.1% (N = 1525) only benzodiazepine, and 45.1% (N = 1372) no mood stabilizer nor benzodiazepine. Patients with mood stabilizers, benzodiazepines, or both, had lower chances of treatment response, respectively, 39.3%, 41.6%, and 41.2% were responders versus 44.6% without .On the other hand, 2.1%  and because the majority of patients were treated in accordance with guidelines (i.e. 37.5 mg a day for more than 82% in both groups), later analyses systematically checked the initial results restricting the analyses to patients treated with the recommended dosage of 37.5 mg/day.NR3C1, FKPB5, CRHR1, and AVPR1B) were screened using TaqMan SNP genotyping assays on an Applied Biosystem 7900HT Fast Real-Time PCR System (Life Technologies).Genomic DNA from outpatients was extracted according to the protocol of the manufacturer from salivary sample collected with Oragene DNA kit (DNA Genotek Inc). A total of 16 SNPs encompassing four pharmacogenetics candidate genes  for continuous variables and chi-square test for categorical variables. Furthermore, logistic regression was carried out for sociodemographic and clinical features as intermediate variables with CRHR1 SNP rs878886 in predictor for treatment response. General linear models were computed to generate risk factors associated with the positive treatment response in interaction with or without the CRHR1 SNP rs878886 response allele. Those analyses were performed with SPSS statistical software .Categorical variables were presented as percentages, and quantitative variables as means with standard deviation. Comparisons of sociodemographic, clinical features, and genotype between patients’ groups were performed using Student’s We used a structural equation model with simple mediation, which assumes that the effect of sociodemographic characteristics and depressive disorder features on treatment response is the same in people with and without rs8788861. This was done in the goal to examine whether differences in the prevalence of sociodemographic characteristics and depressive disorder features, associated with rs8788861, explain the greater response to tianeptine among individuals with this SNP see . All anaA total of 3212 Caucasian outpatients were treated by tianeptine for an MDE (Table N = 2419). A total of 42.8% outpatients were responders to tianeptine treatment. For the group of responders, the baseline of HADS score was 29.6 and decreased to 9.3 at V2, while in the group of non-responders, the V1 HADS score was 26.5 and decreased only to 20. MDE outpatients with mood stabilizers, benzodiazepines, or both were responders  but they have significantly lower chances of treatment response versus 44.6% responders without mood stabilizer or benzodiazepines (p = 0.007).Tianeptine was a treatment initiated in 79.5% of the MDE outpatients (p < 0.001). The duration of previous MDE was significantly lower in the group of responders (30.43 ± 22.50 months) compared to the group of non-responders (34.47 ± 29.34). Finally, the percentage of subjects who attempted suicide was significantly lower in the group of responders . Responders were more frequently patients carrying the C allele of rs878886 . For the T allele of rs16940665, the OR associated with antidepressant response was of 1.34 . These associations remained significant after correction for multiple tests  and a random split of the sample size .Pharmacogenetics analyze were also performed according to the self-report after 2 and 4 weeks of treatment. Only SNP rs878886 of CRHR1 gene showed a significant association  . The frequent haplotype rs878886*C–rs16940665*T was detected in 75.7% of the subjects. This haplotype was over-represented in 78.1% of responders compared to 73.9% of non-responders . The OR for haplotype rs878886*C–rs16940665*T was of 1.26 . This haplotype confers an attributable risk of pharmacogenetics response of 16.23%.The variants rs878886 and rs16940665 were located in the same haplotype block (linkage disequilibrium of 0.99 and p = 0.003), conferring an OR = 1.32  and an attributable risk of 19.48%.The haplotype rs878886*C–rs16940665*T was also significantly over-represented in the remitters (79.8%) compared to the non-remitters (75.0%)  were associated with CRHR1 rs878886 were observed for young age and first MDE, respectively, p = 0.004 and p = 0.046 . This non-significant indirect effect implies that differences in prevalence of sociodemographic characteristics and depressive disorder features associated with rs8788861 do not explain the greater response to tianeptine among individuals with rs8788861.The total indirect effect (obtained by summing the product of the effects of rs8788861 on each sociodemographic characteristic and depressive disorder feature with their effects on response to antidepressant) was not significant , suggesting that the effect of at least certain sociodemographic characteristics and depressive disorder features on response to tianeptine could differ between individuals with and without rs8788861. The sensitivity analysis excluding participants with missing data yielded similar results with a total indirect effect not significant  and a significant positive direct effect of rs8788861   of rs8788861 on response to tianeptine  and the residual direct effect of rs8788861 was not significant , indicating that the total effect of rs8788861 on response to tianeptine  was well explained by this moderated mediation model. The sensitivity analysis excluding participants with missing data yielded similar results, with total indirect effects  and a non-significant residual direct effect of rs8788861  . Secondly, we focused not only on treatment response but also we assessed remission, two concepts that are overlapping but nevertheless not identical . And lastly, thanks to a large sample size of 3212 MDE outpatients, we randomly split the cohort in two subsamples and confirmed that our results were independently detected  or a TCA (desipramine)16. These observations were found in small cohorts of few hundreds to up to two thousands (i.e. STAR*D) depressed patients, from different ethnic origins, and under different antidepressant treatments. In contrast, some other studies did not reported pharmacogenetics association between different CRHR1 polymorphisms and antidepressant treatments30. Thus, no associations with CRHR1 variants were observed in 159 depressive outpatients treated by citalopram (SSRI) during 4 weeks30. Furthermore, the sequencing of CRHR1 in 272 unrelated Mexican Americans with MDE allowed to identify a total of 56 polymorphisms among the gene, including 23 new variants and one that changes the amino acid sequence of the protein. However, none of them were associated with an antidepressant treatment response31. Finally, in a 6-week study in duloxetine treated (a serotonin norepinephrine reuptake inhibitor) of 241 MDE patients, only rs4792888 of CRHR1 showed a trend of pharmacogenetics association (p = 0.054)32. These discrepancies could be due to the involvement of different polymorphisms located in other genes in the biological pathway of the HPA axis or they could depend on mechanism of action of antidepressant used.Several independent studies have reported pharmacogenetics associations between different polymorphisms of FKBP5 and NR3C1 were found to be associated with antidepressant response in independent studies17. In the present study, we did not observed a pharmacogenetics association for FKBP5 and NR3C1 genes, may be due to the type of antidepressant used or to the outpatients sample. Other variants of genes involved in the HPA axis, including CRHR2 and CRHBP, have been reported associated with antidepressant, mostly SSRI or TCA, response or remission17. In a recent work, the analysis of 636 patients from the International Study to Predict Optimized Treatment in Depression (iSPOT-D), treated 8-week with antidepressants escitalopram (SSRI), sertraline (SSRI), or extended-release venlafaxine (SNRI), have showed a pharmacogenetics association with CRHBP variant rs2836514333. Again, discrepancies have been observed between pharmacogenetics association and these genes involved in the biological pathway of the HPA axis.Thus, polymorphisms in the genes 29. Several studies have reported that variants in CRHR1 have been associated with specific cortisol responses and stress-system processes35. While other works have supported that polymorphisms of other genes of CRH axis, like CRHBP gene, affect the cortisol responses37. Variants in CRHR1 gene have been found associated with several clinical features, like stress, panic, emotion, work memory, and disorders including MDE but also general anxiety disorder42. Furthermore, these association have been found also in a strong interaction with early life stress and childhood maltreatment43. In our study, early adverse events have not been collected. Because early adverse events were not collected, we could not assess their differences between responders and non-responders, which represents a limit of the present work. Interestingly, alleles of rs878886 of CRHR1 gene have been associated with fear acquisition in humans45. These studies showed that G-allele carriers have a reduced acquisition of cue-specific fear-conditioned responses compared to C/C homozygotes. The cued-fear acquisition deficits is also under the genetic interaction of variations of the SL6A4/5HTT gene, which codes for the serotonin transporter45. This fear acquisition under the control of CRHR1 and SLC6A4 variants may be also a limitation of this study due to the impact of fear control between the responders and non-responders. These implications, direct or indirect, of CRHR1 variants in the clinical features and disorders, in interaction with life events, can modulate the clinical phenotypes of the MDE subjects. And these CRHR1 variants can also impact, directly or indirectly, antidepressant treatment response.These divergent observations could be due to different clinical characteristics or different activity of the HPA axis in patients. Thus, it has been reported that fluoxetine (SSRI) responders and remitters exhibited significantly lower circadian cortisol levels than those who did not respond. Furthermore, MDD treated patients who abandoned before the third week presented a low cortisol levels20. Interestingly, it has been showed that serotonin from the dorsal raphe nucleus enhances fear and anxiety and activates a subpopulation of corticotropin-releasing factor neurons in the bed nucleus of the stria terminalis46. This circuit could be modulated by tianeptine to improve a better adaptation, in contrast to the clinical observations of early adverse events to SSRI treatment in some patients. Another propriety of tianeptine is its MOR agonist that acts specifically on the opioidergic system22. Indeed, a recent study has shown, in mice model, that the antidepressant- and opioid-like behavioral effects of tianeptine were mediated by its MOR agonist22. Interestingly, cortisol response is modulated by the endogenous opioid, beta-endorphin, via the MOR and variant in the MOR gene OPRM1 affects the cortisol stress response23 and, as mentioned previously, opioid system partly regulates HPA axis activation.Here, we investigate the pharmacogenetics treatment response of the atypical antidepressant tianeptine. Tianeptine enhances a cascade of cellular adaptations that prevents and reverses the dendritic remodeling induced by the stress or glucocorticoids and increases dendritic length and branching of neurons in the amygdalaCRHR1 gene. This pharmacogenetics work shows CRHR1 association with (1) more specifically attributed to treatment response, (2) with a lower risk of chance finding, and (3) in a more ecological situation. Our results should encourage studies on the link between opioid system and HPA axis. Furthermore, recent findings on ketamine support an interaction between these systems. Indeed, it has been shown that ketamine increases the effectiveness of the signaling induced by opiates47 and in a very recent randomized controlled trial48, they found that reduction of depression was lower when naltrexone (an MOR antagonist), versus placebo, was administered before ketamine administration. Regarding those data and results of our study, it seems that there is a real interaction between HPA and opioid systems and this interaction may modulate response to antidepressant treatment and evolution of depression.To conclude, our study was able to strongly replicate a pharmacogenetics association of an atypical antidepressant tianeptine treatment for MDE, with one of the gene involved in the HPA axis, Supplementary Materials And MethodsPharmacogenetic association in 2 random samples of the splited GENESE population between SNPs of genes regulating the CRF system and response or remission to antidepressant treatmentPharmacogenetic association between SNPs of genes regulating the CRF system and remission to antidepressant treatmentImpact of pharmacogenetics variant rs878886*C on sociodemographic and clinical features and interactions to the treatment responseMediation model of the relationship between rs8788861, differential associations with sociodemographic characteristics and depressive disorder features, and response to tianeptine in individuals without missing dataModerated mediation model of the effects of rs8788861 and sociodemographic characteristics and depressive disorder features on response to tianeptine in individuals without missing data


A pH-sensitive poly(acrylic acid) composite hydrogel was successfully synthesized via gamma irradiation and reinforced with cellulosic materials of different sizes. Cellulose was extracted from rice husks via alkali and bleaching treatment, and an acid hydrolysis treatment was performed to extract cellulose nanocrystals (CNCs). Morphological observation of cellulose and CNCs using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed diameters of 22–123 μm and 5–16 nm, respectively. The swelling properties of the fabricated poly(acrylic acid)/cellulosic hydrogels were found to respond to changes in pH, and CNC-reinforced hydrogels performed better than cellulose-reinforced hydrogels. The highly crystalline CNC provided a greater storage modulus, hence acting as a better reinforcing material for poly(acrylic acid)-based hydrogels. SEM showed that hydrogels reinforced with the CNC nanofillers contained a homogeneous pore distribution and produced better interfacial interactions than those reinforced with the cellulose microfillers, thus performing better as hydrogels. These findings demonstrate that gamma-irradiated poly(acrylic acid) hydrogels reinforced with CNCs exhibit a better stimuli response toward pH than poly(acrylic acid) hydrogels reinforced with cellulose. Recently, hydrogels that can inherently display dramatic changes in response to environmental stimuli such as temperature, pH, and certain chemicals have generated considerable research interest ,2. FurthOwing to the presence of a carbon–carbon double bond (C=C) in acrylic acid (AA), its reactivity is very high , and it Conventional chemical crosslinking improves mechanical and thermal stability, although the use of chemical crosslinking to induce crosslinking leads to concerns about toxicity, as well as a limited swelling capability and slow stimuli response ,16. PhysCellulose nanocrystals (CNCs) are promising biopolymers used as reinforcing materials for their superior mechanical properties, high biocompatibility, hydrophilicity, and low density . The extThis study aimed to prepare a hydrogel with favorable properties by gamma irradiation of the PAA composites reinforced with either cellulose or CNCs. The samples were characterized using Fourier-transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The effect of the filler size  on the gel fraction, swelling behavior, and elastic properties of the hydrogels was also investigated.Rice husk for use as raw fibers was supplied byPadiberas Nasional Berhad (BERNAS), Shah Alam, Selangor, Malaysia. Glacial acetic acid, acrylic acid, and sulfuric acid were purchased from Sigma-Aldrich (M) . Sodium hydroxide and sodium chlorite were purchased from SYSTERM-chemAR .2) aqueous solution at pH 4.5 under reflux at 100 °C for 4 h, as described by Ooi et al. [Rice husk fibers were ground to a powder, which was subsequently washed with distilled water before undergoing chemical treatment. The powder was treated with 4% sodium hydroxide (NaOH) aqueous solution under reflux at 90 °C for 3 h; this process was performed in triplicate. This alkali-treated powder was then bleached with a 1.7% sodium chlorite  solution. A previously published procedure was followed to prepare the CNCs [Subsequently, acid hydrolysis treatment were performed in order to prepare CNCs. Under controlled condition, 15 g of cellulose powder were continuously stirred in 60% sulphuric acid (Hthe CNCs . The CNCAA was mixed with distilled water and either cellulose or CNCs to produce hydrogels via gamma radiation. The PAA hydrogel fabrication reinforced with 4% or 8% cellulose is briefly outlined as follows. Cellulose (0.20 g) was mixed with distilled water, ultrasonicated  for 50 min at 25 °C and homogenized  for 30 min at 8000 rpm to ensure a uniform suspension. The cellulose suspension was then heated to 50 °C before adding an AA (4.8 g) solution and stirring with a magnetic stirrer for 30 min. The solution was then transferred to a petri dish and finally exposed to gamma irradiation  at a dose of 30 kGy  to obtain the hydrogel; this dose was selected based on previous studies . PAA hydTEM was used to determine the dimensions and morphology of the CNCs. The CNC suspension was ultrasonicated for 50 min to disperse the agglomerated particles. One drop of diluted suspension (1 wt %) was deposited on the surface of the copper grid and coated with a thin carbon film. It was then negatively stained with 2% uranyl acetate solution for 5 min and allowed to dry at room temperature. The sample was observed at 35,000× magnification using a Philips CM30 microscope  operated at 100 kV accelerating voltage. Cellulose was placed on an Al stub and incubated in an oven at 60 °C, and then the surface morphology of the thus-obtained samples was observed at 250× magnification and 15 kV accelerating voltage with SEM .SEM (Philips XL 30) was also used to observe the morphology of the hydrogel cross-sections. The samples were first immersed in distilled water for 2 d to allow the hydrogels to swell. The samples  were then freeze-dried at −50 °C for 36 h and coated with gold (0.01–0.1 μm thick). The morphology was then observed at 200× magnification and 15 kV accelerating voltage.−1 to 400 cm−1.FTIR spectroscopy ) was used to identify the functional groups present in the samples. All samples were dried before subjected to FTIR. Four samples were tested, which were the original rice husk and the rice husk after each treatments. In addition, monomer AA, PAA hydrogels, PAA/8% cellulose hydrogels, and PAA/8% CNC hydrogels were also examined. FTIR spectra were recorded from 4000 cm1). The dried samples were then soaked in distilled water at room temperature for three days in order to remove any extra soluble material. The samples were then removed from the distilled water and dried again in an oven at 37 °C until there was no change in weight (W2). The gel fraction (GF) could then be calculated from Equation (1):Gel fraction analysis was performed to determine the gelation percentage for each hydrogel composition. The samples were dried in an oven at 37 °C until a constant weight was obtained (Wi). Next, they were separately immersed in 50 mL of distilled water (pH 6.7) at 25 °C and 37 °C for 72 h to evaluate the effect of temperature or in 50 mL of buffer solution at pH 3, 7, and 11 to investigate the response to pH. The samples were then removed from the solutions at certain time intervals and weighed again as swollen samples (Wf). The swelling degree (SD) could be calculated using Equation (2):A swelling test was performed in order to investigate the swelling properties of hydrogels toward different temperature and pH response. First, the hydrogel samples were dried in an oven to obtain a constant weight  to determine the influence of the AA/filler ratio on the elastic properties of the hydrogels. All samples were cut into discs , and the storage modulus was recorded with 0.05% shear strain at 25 °C, a 1.0 mm parallel plate gap, and an angular frequency from 0.1 to 100 Hz.The needle-like structure of the CNCs is clearly observed in −1 to 3324 cm−1 corresponds to the O–H stretching of intermolecular hydrogen bonding, while the peak at 1638 cm-1 is attributed to O–H bending. The peaks around 2895 cm−1, 1429 cm−1, and 1052 cm−1 are attributed to C–H stretching, symmetry CH2 bending, and C–O stretching of glucopyranic rings, respectively. These peaks therefore confirm that the cellulose component of the rice husk is not removed during the chemical treatment processes [rocesses .−1 in the FTIR spectrum of the alkali-treated rice husk indicates the removal of the C=O bonds from the carboxylic acid and ester groups of hemicellulose, indicating that the alkaline treatment successfully removes hemicellulose [−1, associated with the C=C stretching of the aromatic ring of lignin, decreases after the first treatment and completely disappears after the second treatment, indicating the removal of lignin during bleaching [However, the disappearance of the peak at 1738 cmellulose ,28. The leaching . The FTIleaching ,28.−1 and 1235 cm−1 in −1 to 2750 cm−1 is attributed to the OH group. The FTIR spectrum of the AA monomer exhibits a distinct peak at 1615 cm−1, which corresponds to C=C stretching. In the PAA FTIR spectrum, this peak is absent, and a new peak attributed to C–C bonds emerges at 1450 cm−1, revealing that the C=C group has been transformed to a C–C covalent bond and that successful crosslinking has occurred during the process of hydrogel formation [The peaks at 1701 cmormation .During crosslinking, gamma rays produce free radicals and convert the C=C bonds into C–C bonds . A hydroThe FTIR spectra for both PAA/cellulose and PAA/CNC hydrogels do not significant differ from that of the PAA hydrogel, which suggests that crosslinking only occurs between the AA monomers without affecting any other functional groups of the cellulose or CNC backbones. It therefore appears that the cellulosic material is entrapped within the PAA 3D matrix, thus forming semi-interpenetrating networks (semi-IPNs) of PAA/cellulose or PAA/CNC hydrogels. There is no characteristic peak of cellulosic material visible in the FTIR spectra for both PAA/cellulose and PAA/CNC hydrogels due to low concentration of fillers. Previously, Lim et al.  reportedOne important parameter for evaluating the properties of hydrogels is their capability to swell in water without the solvation of polymeric content. The hydrophilic properties of cellulose, which are due to the presence of OH groups, attract water molecules, thus increasing the extent of swelling . Both 4%However, the size of the reinforcement filler plays a key role in determining the amount of water uptake and absorption speed. Reinforced PAA/CNC hydrogels, which contain nanosized CNCs, exhibited a greater degree of swelling than hydrogels reinforced with microsized cellulose. The nanosize of CNCs allows a better dispersion, thus facilitating the formation of more rigid and stable pores while also providing greater interstitial volume and surface area . Based oWith regard to the different temperatures , the sweThe influence of swelling time related to cellulose and CNC composition, as expressed as swelling percentage for all hydrogels, was investigated a. The tr−). When the pH exceeds 4.25, the swelling percentage increases as the COOH groups begin to ionize and form COO− anions, as shown in the Equation (3) [The variations in the swelling percentage for hydrogels at pH 3, 7, and 11 are shown in tion (3) . As the tion (3) .(3)RCOOA uniform, homogeneous distribution of hydrogel pore structures is expected to allow water molecules to absorb in all directions, thus increasing the swelling percentage. The PAA/CNC hydrogels exhibit a smoother surface than the PAA/cellulose hydrogels, which indicates that if the fillers are smaller, the interfacial interactions will be better owing to the more homogeneous dispersion of fillers within the polymer matrix. However, the excess amount of fillers in the PAA hydrogels with both 8% cellulose and CNC leads to the congestion of pores, as presented in The storage modulus (G′) is typically defined as a measurement of the energy stored in a material and represents the elastic portion of its flow behavior while the loss modulus (G″) is a measurement of viscosity of a solid material. Elastic materials can immediately change their shape when a shear force is imposed upon them and return to their original shape when the shear force is released. For both the cellulose and CNC-filler-reinforced hydrogels, G′ and G″ increases significantly, as shown in The modification of cellulose fibers loaded during hydrogel production affects the hydrogel performance. As the size of cellulose fibers is reduced (from cellulose to CNCs), the swelling and rigidity of the resulting PAA hydrogels increases owing to the formation of more pores and interfacial interactions. The above results demonstrate that hydrogels reinforced with nanosized CNCs from the acid hydrolysis of cellulose result in improved hydrogel performance and greater potential as biocomposite materials. Significantly, PAA/CNC hydrogels also show a remarkable response toward pH, thus suggesting their potential as a drug-delivery system.


The marine sedimentary subsurface is a vast and inhospitable ecosystem, often described as a place where microbes “race to their death,” as microbial cells are buried and available energy is severely diminished with increasing depth/age. By combining a variety of biogeochemical and molecular methods to describe the energetics and genetics of the bacteria specialized in anaerobic ammonium oxidation, we show that despite prolonged exposure to highly unfavorable conditions for tens of thousands of years, these bacteria exhibit remarkable net population growth when reaching their niche: the nitrate–ammonium transition zone. This common, yet understudied, geochemical transition zone represents an oasis in the sedimentary energetic desert, and the growth it supports is of major importance for the global nitrogen cycle. Scalindua species which we name Candidatus Scalindua sediminis, so far exclusively found in marine sediments. It has the capacity to utilize urea and cyanate and a mixotrophic lifestyle. Our results demonstrate that specific microbial groups are not only able to survive unfavorable conditions over geological timescales, but can proliferate in situ when encountering ideal conditions with significant consequences for biogeochemical nitrogen cycling.No other environment hosts as many microbial cells as the marine sedimentary biosphere. While the majority of these cells are expected to be alive, they are speculated to be persisting in a state of maintenance without net growth due to extreme starvation. Here, we report evidence for in situ growth of anaerobic ammonium-oxidizing (anammox) bacteria in ∼80,000-y-old subsurface sediments from the Arctic Mid-Ocean Ridge. The growth is confined to the nitrate–ammonium transition zone (NATZ), a widespread geochemical transition zone where most of the upward ammonium flux from deep anoxic sediments is being consumed. In this zone the anammox bacteria abundances, assessed by quantification of marker genes, consistently displayed a four order of magnitude increase relative to adjacent layers in four cores. This subsurface cell increase coincides with a markedly higher power supply driven mainly by intensified anammox reaction rates, thereby providing a quantitative link between microbial proliferation and energy availability. The reconstructed draft genome of the dominant anammox bacterium showed an index of replication (iRep) of 1.32, suggesting that 32% of this population was actively replicating. The genome belongs to a  The deep sedimentary biosphere is populated by microbes that once resided in the surface layers but over time became buried deeper and deeper into the sediments as new material settled on the seafloor . Below tEnergy availability is one of the most fundamental factors limiting life; however, it has not been explicitly demonstrated to control microbial abundances in the deep biosphere . The gloHere we present the geochemistry, microbial ecology, and energetics of anammox bacteria in the understudied nitrate–ammonium transition zone (NATZ), which marks the transition of the dominant porewater N species from nitrate to ammonium. We provide strong indications for in situ growth of anammox bacteria and link the growth to increased power supply in ∼80,000-y-old subsurface sediments. Through genomic characterization of the dominant anammox bacterium, we also reveal the potential features that enable them to survive and grow in the subsurface. By consuming most of the upward flux of ammonium from deep anoxic sediments, the growth and activities of sedimentary anammox bacteria and their associated organisms have important implications for the global fluxes of nitrogen between marine sediments and the overlying ocean.SI Appendix, Table S1) and performed geochemical measurements and microbiological analyses with high vertical resolution. Although the cores are separated by more than 300 nautical miles, they exhibited similar geochemical profiles (Dataset S1) summarized as follows: 1) O2 monotonically decreased and was depleted at a depth of 0.4 to 1.2 m below seafloor (mbsf), while dissolved Mn2+ built up right below the oxygen depletion depths; 2) NO3− was abundant in the oxic zone and depleted in layers below the oxygen depletion depth; and 3) NH4+ was abundant in the deep anoxic sediment but undetectable in the oxic zone. These geochemical profiles all displayed a well-defined NATZ, where nitrate diffusing downward from the oxic zone and ammonium diffusing upward from deeper anoxic sediments are coconsumed, presumably by the anammox process . In order to investigate the global occurrence of this particular GTZ, we searched through geochemical profiles from earlier studies (see SI Appendix for details) and identified the NATZ at 63 additional sites . These sites were mainly located on continental slopes and midocean ridges of the Atlantic, Pacific, and Indian oceans (We retrieved four sediment cores (2.0 to 3.6 m long) from the seabed of the Arctic Mid-Ocean Ridge (AMOR) at water depths of 1,653 to 3,007 m  in the four AMOR cores. The applied boundary conditions  and model parameters , allowed a simulation matching the measured profiles of total organic carbon (TOC), dissolved inorganic carbon (DIC), O2, NO3−, NH4+, and Mn2+ . In particular, as indicated by root mean square errors , the modeled concentrations of NO3− and NH4+ showed only minor deviations from the measured ones (1.1 to 3.1 μM for NO3− and 0.5 to 4.6 μM for NH4+), suggesting that the modeled profiles and reaction rates provided a realistic estimation of the in situ nitrogen cycling processes . Our model predicts that anammox is restricted to the NATZs across all of the four cores . This confined window of anammox in the NATZ is due to the combination of the coexisting reactants (NH4+ and NO2−/NO3−) and the absence of oxygen inhibition.We applied a one-dimensional reaction-transport model  to simulur cores D, althoun = 67) with an NATZ, 56 sites  have no upward ammonium efflux from the NATZs , suggesting that most of the ammonium from deep anoxic sediments is being consumed there. At these sites, the downward fluxes of NO3− are higher than the corresponding upward diffusive flux of NH4+  from the four AMOR cores . We found that all sequences affiliated with known anammox bacteria belonged to the genus Scalindua and are represented by only three OTUs , suggesting the presence of an anammox population with low diversity  in all cores, but was not detected in the upper oxic zone and only occasionally detected (<0.1%) below the NATZs  and the modeled rate of anammox . Similar−3 to 10−1 fmol NH4+ cell−1 d−1 , which is one to four orders of magnitude lower than those measured in laboratory bioreactors for other anammox bacteria . These levels of proton pumping rates are lower than those required (104 to 105 protons per second) for the rotation of a single bacterial flagellum in Escherichiacoli  from the GC08 NATZ . This genome was ∼3.0 Mbp, with 2,879 coding sequences across 71 scaffolds, thus over 1 Mbp smaller than other known Scalindua genomes . The assembled 16S rRNA gene sequence  of this genome has a 100% match with that of the dominant Scalindua (OTU_5) found in our 16S rRNA amplicon analysis  with previously characterized Scalindua species from other marine habitats, including Candidatus Scalindua rubra  showed that this genome, together with sequences from other marine sediments, represents a lineage within the genus of Scalindua. We propose a provisional taxon name for this uncultivated anammox bacterium, “Candidatus Scalindua sediminis,” based on its origin and prevalence (see below) in deep marine sediments.In order to study the ecophysiology and potential adaptation mechanisms of anammox bacteria in the subsurface, we performed metagenome sequencing and analysis for four selected depths  reduction zone)] of GC08, because the most prominent anammox relative abundance increase was observed in this core F. Througanalysis A, suggesua rubra  and Candua AMX11  from seajaponica  and Candprofunda  enrichedediments , and othediments A. Phylogediments B and theCa. S. sediminis-like bacteria (97% 16S rRNA nucleotide identity) were present in 156 samples (as of January 2020), all of which were from marine sediments. The spatial distribution pattern of this Scalindua bacterium resembles that of NATZ, i.e., both were mainly found in continental slope sediments or midocean ridges in the Atlantic and Pacific oceans . This resemblance might be explained by the observation that Ca. Scalindua sediminis mainly lives in the NATZ  short reads archive , an algorithm that uses the slope of genome coverage between the origin of replication and the terminus to estimate the ratio of active replicating cells in a population , suggesting that 32% of this population was actively replicating at the sampling time, assuming each cell replicates two copies of its genome during growth  and thus unfortunately prevented iRep calculations outside the NATZ (≥5× coverage is required) . Recently, a seawater-derived Scalindua bacterium grown in a laboratory bioreactor also exhibited similar iRep values (iRep of ∼1.2 to 1.4)  . In prinCa. S. sediminis genome has the core genetic machinery for anammox metabolism, including hydrazine synthesis from NO and NH4+ catalyzed by hydrazine synthase (HZS), and hydrazine degradation to dinitrogen gas (N2) by hydrazine dehydratases (five variants of HZO) (cd1 cytochrome nitrite reductase (NirS) was missing in the genome, we detected a Scalindua nirS gene in an unbinned 18,718-bp contig in the bulk metagenome assembly from the GC08 NATZ with a similar coverage as the rest of the contigs of Ca. S. sediminis , suggesting this contig may be part of the missing proportion of the Ca. S. sediminis genome. Therefore, Ca. S. sediminis can potentially use this protein to reduce nitrite to nitric oxide (NO). It can use the Wood–Ljungdahl pathway reversely to fix CO2 (The  of HZO) C. Althou fix CO2 C. For su fix CO2 C.Ca. S. sediminis has the potential to utilize urea and cyanate, suggesting a versatile metabolic lifestyle. For the urea metabolism, it encodes a urease operon (UreABC) and a urea-specific ABC transporter, as well as several urease accessory proteins (UreDEFG) that could facilitate the transport and intracellular degradation of urea to NH4+  and other uncultured Planctomycetes , catalyzing the degradation of cyanate to NH4+ and CO2 , but also provide it with an internal source of ammonium allowing it to persist under the severe competition disadvantage relative to ammonia-oxidizing Thaumarchaeota . Due to the gene-dose effect   , they maCa. S. sediminis has to persist for up to 80,000 y in sediments above the NATZ where the anammox metabolism is not favorable and therefore are likely to employ other metabolic strategies to stay alive. The Ca. S. sediminis genome has a cytochrome cbb3 menaquinol oxidase, a feature that also exists in other anammox genomes . Because Ca. S. sediminis has the complete pathway for glycolysis, the near-complete the tricarboxylic acid (TCA) cycle (only fumarate hydratase missing) and the electron-transport chain for respiration , and V-type ATPase) (Dataset S2), it is possible that this bacterium can degrade organic acid using nitrate as the electron acceptor, as observed in other characterized anammox bacteria , they have no known genetic machinery to use alternative electron acceptors other than O2 . Using qPCR, we also detected 104 to 106 copies g−1 of narG gene in the NATZs of all four AMOR cores , comparable to anammox abundances. These narG-bearing nitrate-reducing bacteria could reduce nitrate to nitrite by using organic matter as electron donor and fulfill most of the nitrite demand of Scalindua . The presence of nirS and nirK genes encoding nitrite reductases  suggests that a proportion of nitrite could be reduced further to gaseous nitrogen  and integrating the microbial traits into existing reaction-transport models  and adapt to energy-limiting conditions. Experimental characterization is needed to confirm the importance of these alternative substrates and their interactions with the heterotrophic denitrifiers in the same zone. Considering the widespread occurrence of NATZs  and GC05  were collected from the middle section of the Knipovich Ridge, while GC08  and GC09  were collected from the central and northeastern ends of the Mohns Ridge, respectively . Oxygen concentrations were measured immediately using a needle-type fiber-optic oxygen microsensor  inserted manually into sediments. Pore water extractions were conducted with Rhizons samplers at 5-cm intervals over the first half meter and at 25- or 30-cm intervals below that depth. Subsamples for microbiological analysis were taken from the same depths as porewater extraction by using sterile 10-mL cutoff syringes. Details about onboard core handling, porewater analysis, and onshore solid phase analyses are provided in SI Appendix, Materials and Methods.Sediment cores used in this study were retrieved using a gravity corer from the seabed of the AMOR with water depths of 1,653 to 3,007 m, during the CGB Summer Cruise 2014 (GC08 and GC09) and 2016 (GC04 and GC05) onboard the Norwegian R/V 2) in the solid phase. The model considers two sets of reactions: 1) primary reactions during organic matter degradation: aerobic degradation (R1), heterotrophic denitrification (R2), and MnO2 reduction (R3); as well as 2) secondary reactions, including nitrification (R4), Mn(II) oxidation with oxygen (R5), and anammox (R6). Model simulations assume chemical species, including all implicit reactive intermediates, are at steady state. The applied upper and lower boundary conditions are listed in SI Appendix, Table S3. Model parameters  were calibrated by visually comparing the model simulation outputs against measured depth profiles of O2, NH4+, NO3−, DIC, Mn(II), and TOC, the combination of which was suggested to be sufficient to constrain this type of model with reasonable precision . Porewater profiles were manually checked and discarded if they 1) do not reach the nitrate-depletion depth  or 2) contain too few datapoints (<6).Geochemical profiles of porewater nitrate and ammonium indicating a NATZ in marine sediments were either retrieved from published literature or obtained from the PANGAEA database (∆Gr0) was calculated using thermodynamic data of standard Gibbs free energy of formation of each reactant/product, corrected to near in situ pressure and temperature using the R package CHNOSZ (∆Gr) was then calculated following the description in LaRowe and Amend . The abundance of denitrifying bacteria was quantified by targeting the narG (encoding the periplasmic NarG), nirS and nirK genes , using the protocol described in Zhao et al.  with minor modifications. Amplicon libraries of the 16S rRNA gene were prepared using a two-round PCR amplification strategy with the primers of 515F/806r, as described in Zhao et al. . Genes in the genome of Ca. S. sediminis were predicted using Prodigal  and sequenced (2 × 150 bp) by an Illumina HiSeq 2500 sequencer. The quality of the reads and presence of adaptor sequences were first checked using FastQC v.0.11.5  and then gbtools  based onProdigal . Genome Prodigal , eggNOG Prodigal , and BlaProdigal  using thProdigal . DetaileAll available high-quality anammox bacterial genomes were downloaded from the NCBI database and were included in the phylogenomic analysis, which was based on marker genes consisting of 14 syntenic ribosomal proteins , demonstrated to undergo limited lateral gene transfer . These sScalindua OTUs identified via BLASTn  were included in the comparative genomic analysis using Anvi’o v.5.4   , iRep waequence)  were useequence) .Ca. S. sediminis-like anammox in natural environments was assessed using IMNGS (The occurrence of ng IMNGS  against Supplementary FileSupplementary FileSupplementary File


Cold atmospheric plasma (CAP) has been widely used in biomedicine during the last two decades. While direct plasma treatment has been reported to promote wound healing, its application can be uneven and inconvenient. In this study, we first activated water with a portable dielectric barrier discharge plasma device and evaluated the inactivation effect of plasma-activated water (PAW) on several kinds of bacteria that commonly infect wounds. The results show that PAW can effectively inactivate these bacteria. Then, we activated tap water and examined the efficacy of PAW on wound healing in a mouse model of full-thickness skin wounds. We found that wound healing in mice treated with PAW was significantly faster compared with the control group. Histological analysis of the skin tissue of mice wounds showed a significant reduction in the number of inflammatory cells in the PAW treatment group. To identify the possible mechanism by which PAW promotes wound healing, we analyzed changes in the profiles of wound bacteria after PAW treatment. The results show that PAW can significantly reduce the abundance of wound bacteria in the treatment group. The results of biochemical blood tests and histological analysis of major internal organs in the mice show that PAW had no obvious side effects. Taken together, these results indicate that PAW may be a new and effective method for promoting wound healing without side effects. Wounds can be caused in various ways, such as through blunt instrument injuries, scrapes, falls, and knife injuries. Wound healing in humans is an extremely complex process that is triggered immediately after injury through the activation of numerous biological pathways in the body ,2. In mo2O2, NO2−, NO3−, O3, ONOO−, •OH, and O2•− [Cold atmospheric plasma (CAP) is a partly ionized gas composed of a large number of charged particles (such as electrons and ions), free radicals, metastable molecules, electrically neutral atoms, and ultraviolet radiation . CAP canand O2•− ,12,13,14and O2•− ,16, suchand O2•− ,18,19, hand O2•− , wound hand O2•− ,22,23, aand O2•− . CAP is and O2•− ,26, as wand O2•− ,28, whicand O2•− ; thus, tand O2•− ,31,32. Iand O2•− ,34,35. Tand O2•− .Currently, most plasma jet devices and dielectric barrier discharge (DBD) plasma devices are supplied by a large-sized high-voltage power supply and a large gas cylinder ,37,38,396/mL) suspension of Pseudomonas aeruginosa was added to the wound, and a piece of sterile gauze was then fixed to the wound. After the wounds had been infected (approximately 3 days after wound cutting), 10 mice were randomly divided into a control group and a treatment group.Male Institute of Cancer Research (ICR) mice  were purchased from and raised in the standard breeding environment in the Animal Experiment Center of Xi’an Jiaotong University . In this study, all experiments were designed and operated in accordance with animal welfare guidelines with Ethic Committee approval (No. xjtu2018-215). During the construction of the wound model, each mouse was injected with an appropriate amount of 1% pentobarbital sodium (an anesthetic) according to body weight. After the mice were anesthetized, the hair on the back of each mouse was removed using depilatory cream, and the hair removal site was wiped with 75% alcohol. We then used sterile medical scissors to cut a round wound with a diameter of 2 cm on the back of each mouse, effectively removing the skin down to the muscular layer including the dermis and epidermis. Then, a 200 μL  of each group was obtained by biopsy and fixed with 4% polyphenol formaldehyde. The samples of wound skin tissue were stained and photographed by Wuhan Seville Biotechnology Company .Pseudomonas aeruginosa was used as it is the most common bacteria found in infected wounds. For inactivation experiments, 0.5 mL of 5 × 107/mL Pseudomonas aeruginosa was centrifuged at 4600 rpm for 4 min and the supernatant discarded. Then, 1 mL PAW (with different activating times) was incubated for a set period of time in the PAW treatment group, with nonactivated water being used instead in the control group. After treatment, the bacteria were resuspended in 5 mL culture medium (Luria-Bertani Medium) for proliferation in a shaking machine for 1–2 h. When the concentration in the control group reached approximately 5 × 107/mL (as detected by flow cytometry), the bacteria from both groups were obtained and analyzed using an ATP detector and flow cytometry for ATP activity and relative bacteria number, respectively. We also investigated the in vitro antibacterial effects of PAW on other bacteria , in addition to Pseudomonas aeruginosa, using a colony counting assay as conducted by a third-party organization . Briefly, samples were serially diluted, and 10 µL of each dilution was spotted onto different agar plates and incubated overnight at 37 °C. Among them, Luria-Bertani agar was used to culture Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Buffer Peptone Water agar was used to culture Salmonella paratyphi-B. The numbers of surviving bacteria were determined by counting the colonies (marked in blue dots).The antibacterial efficiency of PAW was evaluated by total bacterial Adenosine Triphosphate (ATP) measurement using an ATP detector  according to the manufacturer’s instructions, and the relative bacterial number as detected with a high-throughput flow cytometry . On day 10, bacterial samples from wounds were obtained by repeatedly wiping the wounds of mice in the control group and the PAW treatment group with wet cotton swabs. The bacterial samples on the wound were sent to Shanghai Biotree Biotechnology Company  for DNA sequencing. The obtained sequence information was used for flora composition analysis toward systematically analyzing the type and relative abundance of bacteria in the wound after PAW treatment to allow assessment of bacterial inactivation in the wound after plasma treatment.On day 23, the mice from both groups were injected with 1% pentobarbital sodium. Blood was collected from the hearts of comatose mice using a syringe, and placed in a 1.5 mL centrifuge tube at room temperature for 2 h prior to centrifugation at 3000 rpm for 15 min to collect the serum for detection of biochemical indicators, including liver function, kidney function, blood lipids, blood glucose, inorganic ions, or antioxidants. The mice were then sacrificed, and the main internal organs  were dissected and fixed with 4% paraformaldehyde. The serum samples and the visceral tissue samples were respectively tested and stained by Wuhan Seville Biotechnology Company .t-test was applied to evaluate statistical significance. Values of p < 0.05 between two independent groups was considered to be statistically significant.The data were processed using GraphPad Prism 5.01  statistical software. All experimental results are presented as the mean ± standard deviation (SD) of at least three independent experiments. Student’s Pseudomonas aeruginosa, which is one of the most common bacteria found in infected wounds. The results of this experiment used to assess the effect of PAW in inactivating Pseudomonas aeruginosa are shown in Pseudomonas aeruginosa. The sterilization effect of PAW on bacteria was also evaluated by measuring the total ATP content in bacteria and the relative total amount of bacteria. Pseudomonas aeruginosa. The sterilization effect of PAW on bacteria was evaluated by measuring the total bacterial ATP content using an ATP detector and the relative total amount of bacteria using flow cytometry. Pseudomonas aeruginosa within 5 min. Pseudomonas aeruginosa within 3 min, and the sterilization effect was found to increase as incubation time increased.An important factor that hinders wound healing is the susceptibility of wounds to bacterial infection. We evaluated the inactivation effect of PAW on Pseudomonas aeruginosa. This antibacterial effect was plasma-dose-dependent. The sterilization effect of PAW was incomplete when activating water for 1 or 3 min, but was effective with 5 min , Enterococcus ; TM7_3 , and Spirochaetes  were significantly lower in abundance after PAW treatment. We further analyzed the in vivo bacterial inactivation using DNA sequencing on the wound site after PAW treatment. Although the wound was infected using Because PAW must be in direct contact with wounds to promote healing, biosafety is an important factor to consider. In a previous experiment, we demonstrated the effect of PAW on the healing of mice skin wounds. Here, we evaluated the safety of using PAW on the skin tissues of mice using blood biochemical indicators and a histological analysis of major internal organs. The results are shown in According to the results in Pseudomonas aeruginosa. For the treatment of skin wounds, we used a portable DBD plasma device to activate tap water and generate PAW. We demonstrated that immediate preparation of PAW using our portable DBD device can effectively inactivate a variety of bacteria  as demonstrated in vitro. In vivo experiments of wound infection based on Pseudomonas aeruginosa revealed that PAW treatment could accelerate the healing of skin wounds in mice, with a faster healing time observed when compared to the control. By in vivo detection of the bacterial burden on the wound site, we found that PAW treatment significantly reduces the bacteria abundance , which may play a critical role in promoting wound healing by PAW treatment. In addition, PAW treatment was safe for mice, with no significant changes in major organs, tissue structure, and some important blood biochemical indexes. Our results indicated that immediate PAW treatment with a portable device could be a simple, effective, and cheap method for wound healing in the future.In this study, we evaluated the use of PAW in treatment of skin wounds in male ICR mice, in which we created full-thickness skin wounds that were mainly infected with


G. barbatus populations using a panel of 14 microsatellite loci.The bearded vulture is sparsely distributed across a wide geographic range that extends over three continents . Restriction to high-altitude mountainous habitats, low breeding rates, lack of food and a heightened level of persecution have left many local populations severely diminished or extinct. Understanding the genetic connectivity and population structure of this threatened vulture species is critical for accurately assessing their conservation status, and for appropriately managing local populations through captive breeding programmes or translocations. Previous genetic assessments of the species were mainly focused on the European and Asian populations and included limited representation of the geographically isolated southern African population. A single mitochondrial study, which focused on the African populations of the bearded vulture, detected limited genetic differentiation between populations in Ethiopia and southern Africa, with reduced haplotype diversity in the southern Africa population. In this study, we extend the previous genetic assessments of the species by examining the phylogeography and genetic connectivity of global Analyses revealed spatially correlated genetic differentiation between regional populations and low levels of gene flow between these population fragments. In contrast to the mitochondrial data, the microsatellite data support the management of genetically different populations as separate entities.G. barbatus and, to a lesser extent, the northern African populations highlights the need for conservation programmes to effectively manage populations of this species and maintain extant genetic diversity.Low genetic diversity and geographic isolation are known to adversely affect the evolutionary potential of a species in the long-term. The high inbreeding found in the southern African The online version contains supplementary material available at 10.1186/s12862-021-01760-6. Gypaetus barbatus, were considerably more abundant, albeit always sparsely distributed across a wide area ranging from the Palearctic, through Afrotropic and into Indomalay regions [Historically, bearded vultures,  regions . In the  regions –4. A pop regions , 2.In Europe, the population declined in the nineteenth century due to food shortage and direct persecution. This led to local extinction in the Alps by the early twentieth century . The PyrIn Africa, habitat loss, electrocution and collision with energy infrastructure, unintentional poisoning through veterinary drug-use, feeding on poison baits aimed at predator control and intentional poisoning for the use of vulture body parts in the illegal wildlife and the traditional medicine trade, are the primary culprits for the population decline in many vulture species –16. ThesMorocco is home to fewer than 20 individuals and the species is considered extinct in Tunisia . EthiopiDue to the reduced effective population size, small disjunct populations experience amplified genetic drift, inbreeding and a greater loss of genetic diversity –23. LossGypaetus barbatus barbatus is distributed across Europe and Asia, extending into north-west Africa , and Gypaetus barbatus meridionalis is found in north-east Africa, east Africa and extends into southern Africa. Studies based on maternally inherited mitochondrial data found evidence for two distinct lineages of bearded vultures which did not correspond geographically to the morphologically described subspecies. The first lineage described by Godoy et al. (2004) occurs predominantly in western Europe, and the other lineage extends over Africa, eastern Europe and central Asia. This study detected high levels of inter-population differentiation, with admixture between the two lineages occurring in Greece and the Alps [Two subspecies of bearded vulture have been recognized based on plumage characteristics and geography —Gypaetusthe Alps . Godoy ethe Alps . These cthe Alps . Most gethe Alps , 29, 30.The genetic results have implications for captive breeding programmes and open the possibility for the translocation of individuals into shrinking populations. Unfortunately, previous studies have focussed their analyses almost entirely on the European population (with limited representation from southern Africa). The single study that included good representation of the isolated southern African population was based on mitochondrial DNA . In thisST values , suggesting that null alleles have a very limited effect on the genetic structuring analyses in these samples of bearded vultures. In addition, many of the analyses we conducted are not strongly influenced by the presence of null alleles [A total of 236 individuals were genotyped with the 14 microsatellite markers. All loci were polymorphic with the number of alleles per individual ranging from 6 (Gf11A4) to 17 . A summary of locus-by-locus measures of genetic diversity are provided in Additional file  alleles . LinkageSignificant departures (p < 0.05) from HWE were obtained for all loci in the southern African population. Loci 1, 3 and 4 were in HWE for the European, northern African and Eastern population complexes, respectively. The remaining loci did not conform to HWE predictions. This was not unexpected as these small isolated populations naturally deviate from the assumptions of HWE.IS > 1, Table O < HE; Table The average number of alleles and effective alleles ranged from 5.93–7.00 and 2.81–3.23 respectively , followed by the southern African population Q = 0.92) and the Asian populations (Q = 0.88). The highest admixture was detected in the northern African populations .In all analyses, the isolated southern African bearded vulture population is genetically distinct from all other bearded vulture populations. Genotypes collected from this region appear to belong to unique genetic clusters and are characterized by unique allele frequencies. The genetic distinctiveness of the southern African population is also reflected in the number of unique alleles found in this population . The European population complex contains only 18 unique alleles and the Asian and northern African complexes have fewer than 10 unique alleles each. Only 31 alleles are shared by all populations. Results from the Mantel test indicate that there was no positive correlation between genetic and geographical distance  of variation was attributed to within individual differences, while differences among individuals and among populations explained 39% (p < 0.001) and 12% (p < 0.001) of the diversity respectively.The BayesAss 3.0 analysis indicates that the highest migration seems to have occurred from the European populations to the Asian populations . This supports the hypothesis that, before the extinction of bearded vultures in the Alps, there was an interconnected European population from Spain to the Alps, Balkans to Turkey and Asia.Minimal migration has occurred within Africa in the recent past. Less migration took place from northern Africa to southern Africa, than the reverse . There is debate over the splitting of the northern lineage into either one, Gypaetus barbatus barbatus [Gypaetus barbatus aureus , Gypaetus barbatus hemachalanus  and Gypaetus barbatus barbatus  [G. b. barbatus in Eurasia and northern Africa and G. b. meridionalis in eastern and southern Africa. The diversification of the northern and southern bearded vulture lineages is hypothesised to be linked to the expanding Sahara Desert which acted as a physical barrier during the last glacial maximum [The pattern of population differentiation found here provides clarification of the population structure and migration of bearded vultures. Although there is debate over the number of subspecies of bearded vultures –43, our barbatus , or 3 sus, 1758) , 42. Bas maximum . When thBased on this species’ philopatric dispersal behaviour, genetic differentiation between regional populations is expected to increase with geographic distance following an isolation-by-distance model , 47, espApart from past persecution, the southern African population of bearded vultures continues to suffer threats from several sources, adding to their continued decline , 48. PasThis study highlights the utility of integrating genetic data to inform conservation strategies as it elucidates information that is not readily evident in the field. Our results suggest that the southern African bearded vulture population, being distinct from other such populations, may represent a reservoir of genetic variation that should be given separate conservation status.A total of 236 bearded vulture specimens were analysed in this study. These include samples from across the global distribution of the species Fig. , and incThe NucleoSpin® Tissue kit (Macherey–Nagel) was used for all DNA extractions. The standard protocol for extracting genetic material from blood was followed for blood samples. The feather and archival tissue extractions were done using the standard protocol for muscle tissue, with the following modifications to increase DNA yield: incubation of the ~ 5 mm feather tip/skin snip and proteinase K for 48 h, lysate was then incubated in B3 (buffer) for 45 min (70 °C), the final volume of pre-warmed Buffer BE was decreased to 80 μl, incubation at 70 °C for 20 min followed by centrifuging and then reapplication of the solution onto the membrane, followed by incubation at 70 °C for an additional 5 min and a final centrifugation step. DNA concentrations were determined using the ThermoScientific NanoDrop 2000 spectrophotometer . All DNA extracts were stored at -20 °C.Gyps species [Fourteen microsatellite loci Table  were amp species . MicrosaST values [ST values were compared to FST values corrected using the excluding null alleles (ENA) method using a paired t-test. The presence of scoring errors, such as incorrect assigning of genotypes and fractions in genotypes, was assessed using GenAlEx v6.5 [A), allelic richness (AR), inbreeding co-efficient (FIS) and gene diversity. Effective alleles at each locus (NE), observed (HO) and expected (HE) heterozygosity were calculated in GenAlEx. FSTAT was used to determine allelic richness and gene diversity. Tests for deviation from Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium were performed in Genepop v4.2 [IS) and population differentiation (FST) [IS and FST explain the deviations seen from HWE. Positive FIS values suggest higher inbreeding values (due to higher observed homozygosity) than would be expected under HWE, and vice versa. FST shows the genetic differentiation between two populations which no longer conform to the HWE assumptions.Null alleles in data can be problematic for calculations of genetic variability as they can decrease within-population variance and so inflate FT values . The preT values . UncorrelEx v6.5  and FSTAlEx v6.5 . These ppop v4.2  and FSTAon (FST) , 57 wereon (FST) . FIS andPopulation structure across the global distribution of the bearded vulture was assessed using a Bayesian clustering method implemented in the software program STRUCTURE version 2.3.4 , 60. TheAdmixture and No Admixture model parameters. If there is reason to consider each population as completely discrete, the No Admixture model is appropriate. In contrast, the Admixture model allows for a large proportion of sampled individuals to have ancestry from multiple lineages, which implies a shallower population structure. The historically wide geographic distribution of the bearded vulture favours the Admixture model, and previous molecular work suggests that the African, European and Asian populations are genetically closely linked [Analyses were run using both the y linked , 29. TheAll STRUCTURE analyses were performed with 100 000 Markov-Chain Monte Carlo (MCMC) replicates with a burnin of 10 000. The number of iterations for all assignments was 10 with K ranging from 1 to 10. Once all the simulations were completed, STRUCTURE Selector  was usedST) values were calculated in FreeNA using the ENA-corrected method. Population assignment analysis was conducted in GenAlEx. Pairwise FST values were calculated using two grouping schemes. Individuals were grouped by country of origin, and then because some countries have very low sample sizes, individuals were also grouped by region  was estimated using the Bayesian algorithm implemented in BayesAss 3.0 . The anaAdditional file 1. Supplementary data for Bearded Vulture analysis.