Wound healing is an intricate process that requires complex coordination between many cell types and an appropriate extracellular microenvironment. Chronic wounds often suffer from high protease activity, persistent infection, excess inflammation, and hypoxia. While there has been intense investigation to find new methods to improve cutaneous wound care, the management of chronic wounds, burns, and skin wound infection remain challenging clinical problems. Ideally, advanced wound dressings can provide enhanced healing and bridge the gaps in the healing processes that prevent chronic wounds from healing. These technologies have great potential for improving outcomes in patients with poorly healing wounds but face significant barriers in addressing the heterogeneity and clinical complexity of chronic or severe wounds. Active wound dressings aim to enhance the natural healing process and work to counter many aspects that plague poorly healing wounds, including excessive inflammation, ischemia, scarring, and wound infection. This review paper discusses recent advances in the development of biomaterials and nanoparticle therapeutics to enhance wound healing. In particular, this review focuses on the novel cutaneous wound treatments that have undergone significant preclinical development or are currently used in clinical practice. Cutaneous injuries are a universal aspect of medical care, with approximately 300 million chronic and 100 million traumatic wound patients worldwide. Wounds have an immense financial burden on health-care systems worldwide, accounting for over $25 billion every year in the US alone  is the most common hospital-borne infection affecting millions of patients daily   have been shown to have both antibacterial and cell adhesive properties  (Becaplermin), and, while approved by the FDA, it has shown mixed results in clinical trials on chronic ulcers and has only seen limited clinical adoption  were engineered to overexpress the vascular endothelial growth factor (VEGF) gene and encapsulated in a poly(β-amino ester) hydrogel  forms. Glycerin-based wound dressings with high water content are available in sheets, gels, or impregnated gauzes  NPs  accelerates the healing of full-thickness dermal wounds in mice  are the most widely studied among metal NPs. These NPs have been shown to enhance healing in a full-thickness excisional wound model in mice  plays numerous roles in wound healing and can regulate deposition of ECM proteins, cell proliferation, and endothelial function. Incorporation of a functional group of diazeniumdiolate into materials results in the release of biologically active NO when exposed to an aqueous environment  that directly influence the wound healing cascade. Here, we reviewed biomaterials used in the clinic and those under preclinical development. We are excited about the potential of the biomaterials undergoing development, specifically those that encapsulate bioactive compounds or cell therapies. NP therapies on the other hand have not been used widely in clinic barring silver NPs. However, there is a lot of compelling NP therapies that have shown great potential in animal models as we discussed in the paper.With the advent of CRISPR-Cas9 technology, it would be interesting to see how scientists apply this remarkable gene editing technology to engineer the wound microenvironment (Jinek et al., Wound care is a significant economic and social burden on both the patient population and the health-care system at large. In this review, we have discussed the different biomaterial and NP-based wound therapies, which are either in current clinical usage or in preclinical development. Since there is significant variability of presentation of symptoms in the patients, effective wound care therapies need to have a multipronged approach to tackle the complex problems of pain, inflammation, infection caused by resistant bacteria, delayed healing, and associated costs to health systems and populations worldwide. The precipitous rise in multidrug-resistant bacteria is going to be the biggest challenge for wound care professionals all over the world in this decade. Emerging treatments using biomaterials or NPs to target multiple aspects have great promise for enhancing wound care and will add to the clinical armamentarium to address poorly healing wounds.SD and ABB wrote and edited the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Venturia canescens can coexist in southern Europe. We test the hypothesis that both forms are adapted to different habitats within their area of distribution. Sexuals inhabit natural environments that are highly unpredictable, and where density of wasps and their hosts is low and patchily distributed. Asexuals instead are common in anthropic environments  where host outbreaks offer periods when egg-load is the main constraint on reproductive output.Coexistence of sexual and asexual populations remains a key question in evolutionary ecology. We address the question how an asexual and a sexual form of the parasitoid d value; We present a meta-analysis of known adaptations to these habitats. Differences in behavior, physiology and life-history traits between sexual and asexual wasps were standardized in term of effect size (Cohen’s Seeking consilience from the differences between multiple traits, we found that sexuals invest more in longevity at the expense of egg-load, are more mobile, and display higher plasticity in response to thermal variability than asexual counterparts.Thus, each form has consistent multiple adaptations to the ecological circumstances in the contrasting environments. Populations of a species from different localities often are locally adapted in life history traits, behavior and physiology , but indA variety of processes, including “loss of sexuality” mutations, hybridization and endosymbiotic infection, cause the occasional generation of asexual strains from sexually reproducing individuals in a range of eukaryotic taxa . This phTheoretical studies reveal that coexistence of sexual and asexual competitors is only possible if the newly arisen asexual forms have a smaller inhibitory effect on the sexual forms than the sexual strains have on themselves . This maEmpirical tests of the hypothesis of coexistence of sexual and asexual forms being mediated by ecological differentiation are lacking see . Such a Asobara tabida .This work aims, through a meta-analysis of life history traits involved in the above mentioned trade-offs, of foraging behavior and morphology to provide an empirical test of the hypothesis of ecological differentiation outlined above using the parasitoid V. canescens for four reasons. First, both reproductive modes are obligatory  with no known direct benefit of sex such as the formation of resting stages able to resist to harsh environmental conditions  koinobiont  endoparasitoid (eggs are laid inside the hosts). Adults emerge with a stock of mature eggs and continue to produce eggs during their life : males arise from unfertilized eggs and are haploid, while females originate from fertilized eggs and are diploid. Sex ratio manipulation has never been observed in this species  OR (sex* AND asex*)] between 1999  from life-history and behavioral studies (n = 16 encompassing 46 traits compared) and focused our analysis on these 16 studies . We first selected all the papers with the topics “ studies Table A1 studies  are trea studies  , one physiological character (energy level), three behavioral characters  and feeding), and capacity to respond to changes in temperature. In each category, several traits are considered and for each of these traits, we obtained one to six data points from independent studies.d effect size measurements  urements . Cohen . Flight measurements deserve some additional explanations. As recorded, flight bouts were composed of alternate periods of flying and resting. What was observed was that sexual wasps covered similar distances in fewer flights . Sexual wasps also fly faster .A higher investment has been observed in sexuals under experimental conditions (in the field and in lab), as evidenced by the small to large effect sizes of the traits belonging to flight category , but effect size for glycogen reserves are medium to large, with greater glycogen content (at emergence and after flight) in sexual than asexual females (points 51–53). The consumption rates of glycogen (consumption per unit time) do not differ between modes of reproduction (point 27). The results of field experiments are consistent with differences in behavioral and physiological traits found in the laboratory: sexual V. canescens initiate dispersal faster after release (point 37) and are less often recaptured in the vicinity of the release point (point 33). Although this could be attributed to the traps being less attractive to sexuals at distance, the result is also consistent with sexuals being more mobile and leaving earlier the release site.Consistent with their greater dependence on flight, sexual females have higher total metabolic reserves at emergence than asexual ones (point 41). Interestingly, the amount of nutrients not involved in flight show small Differences in initial energy reserves between adults of the two forms can potentially be compensated by feeding on carbohydrates. When experimentally offered food, asexuals have the same feeding behavior as sexual forms (feeding time and number of feeding bouts per unit of observation time) (points 46 and 9).d value in point 55. This point illustrates the higher breadth of performance curve of sexual females  when exposed to different temperature during development.Sexual, but not asexual, individuals respond to a sudden drop in temperature by exploiting each host-patch more thoroughly . This is consistent with the predicted difference in sensitivity to weather cues. The faster recovering of sexuals from chill coma (point 40) is also indicative of sexuals’ higher capability to deal with temperature changes. The lower sensitivity of sexual individuals to temperature changes is also reflected by the large positive d values for other performance curves or reaction norms, quantifying nutrient contents , longevity (point 22), a measure of total fecundity in another study (point 54), fecundity at emergence (point 43) and developmental rate (point 36) according to temperature, indicate a higher tolerance in sexual forms. A measure for longevity yielded a negative value (point 21), but in this case, d did not differ significantly from 0. In contrast with these results, reaction norms for hind tibia length differed between two studies. Either the two forms express similar curves (point 18) or asexuals show a larger breadth of the curve (point 17). This difference in reaction norms for size was mainly due to differential response at low temperature: lower decrease in size for asexuals when temperature decreases  than hosts previously parasitized by sexual females. There was no effect of the reproductive mode of second females on the incidence of superparasitism. Recognizing parasitized hosts allows females to assess the level of exploitation of a patch. When exploiting partly depleted host patches , only asexual females decrease patch time (point 16).Venturia canescens predominate in different ecological scenarios, life-history, anatomical and physiological traits will reflect adaptations to the circumstances of each form. Asexuals proliferate in stores, where hosts are clumped and there is no food for adults, while the hosts of sexual forms tend to be solitary (one per patch), spatially separated, and occur where food for adult wasps is available. These distinct habitats led us to predict that sexuals should show higher investment in flight capacity, longevity, and ability to tolerate thermal changes, while asexuals will aim at the potential maximum reproductive output conferred by a larger egg-load which they, but not the sexuals, have opportunities to deploy.The overarching hypothesis under test is that because sexual and asexual forms of V. canescens to fly and reach host microhabitats.Contrary to host distribution, the potential high food availability in the field  could seV. canescens females mate only once, search for hosts and lay eggs just after emergence even if unmated . In addition to being more plastic, sexual individuals are better able to tolerate extreme temperatures. Only sexual females, which live in variable weather conditions, adjusted their oviposition behavior—increasing their oviposition rate—when experiencing a sudden change in temperature  performance curves and are less sensitive to temperature than those living in stores that are specialized to a narrow range of thermal values. Sexual wasps are less affected by temperature in their energy allocation to different functions  Table A1Venturia canescens are adapted to the different ecological niches in which these forms are typically found. However, the origin of the differences between the two forms and notably, whether the loss of sex is secondary or pre-existing to the invasion of storage sites remains unknown. Nevertheless, the probably rare occurrence of asexuality, the absence of genetic exchange between forms (that can be inferred from the complete separation of the two forms according to the nuclear marker composition) and the low genetic variability of asexual females may impede their adaptability  of tability . For thiVenturia canescens. Our conclusion is congruent with previous studies reporting, in several taxa, differences in habitat preferences and in responses to environmental conditions between closely related sexual and asexual strains: in plants (dandelions, V. canescens, further investigations are needed to experimentally test, as done by Daphnia pulex, whether the relative fitness of the sexual and asexual wasps depends on ecological conditions, that is, whether sexuals outperform asexuals in the field and asexuals outperform sexuals in building conditions.Understanding the paradoxical coexistence of sexuals and asexuals requires quantifying the balance between costs and benefits of sex via a species-specific approach . Three mV. canescens shows that sexual and asexual individuals are each better adapted to the ecological niches which they occupy in a whole suit of characters. This conclusion is strengthened by the consistency between multiple observed differences, which are in accordance with the inferred selective pressures in both habitats. The life history traits that show the strongest relative divergences (high absolute values of d in Our comparison of life history traits between the two modes of reproduction in 10.7717/peerj.3699/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.3699/supp-2Supplemental Information 2Click here for additional data file.


The retinal pigment epithelium (RPE) is a monolayer of highly specialized cells that help maintain the chemical composition of its surrounding subretinal and choroidal extracellular spaces. Retinal cells (photoreceptors in particular), RPE, and choroidal endothelial cells together help ensure a homeostatically stable metabolic environment with exquisitely sensitive functional responses to light. Aging and disease of the RPE impairs its supportive functions contributing to the progressive loss of photoreceptors and vision. The prevalence of RPE associated retinal degenerations has prompted researchers to develop new therapies aimed at replacing the affected RPE with induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) derived RPE. Despite recent attempts to characterize stem cell derived RPE and to truly authenticate RPE for clinical applications, there remains a significant unmet need to explore the heterogeneity resulting from donor to donor variation as well as the variations inherent in the current processes of cell manufacture. Additionally, it remains unknown whether the starting cell type influences the resulting RPE phenotype following reprogramming and differentiation. To address these questions, we performed a comprehensive evaluation  of 15 iPSC derived RPE originating from different donors and tissues and compiled a reference data set for the authentication of iPSC-derived RPE and RPE derived from other stem cell sources. The RPE is a monolayer of pigmented cells located at the back of the eye and forms the outer blood retina barrier. It maintains the volume and chemical composition of the subretinal space (SRS), and regulates nutrient and metabolite flow to and from the light sensitive photoreceptors of the retina. Several degenerative eye diseases including age-related macular degeneration (AMD) result from a disruption of structural and functional integrity of the RPE monolayer that subsequently leads to retinal degeneration.A worldwide trend towards increasing prevalence of blinding retinal degenerative diseases affecting the retinal pigment epithelium (RPE) imposes a substantial personal and economic burden on individuals and society o FDA-approved treatments exist for an advanced AMD stage called “dry” AMD where RPE cells atrophy leads to photoreceptor cell death. Multiple ongoing efforts utilize pluripotent or adult stem cells to generate healthy RPE cells as potential replacement for damaged/atrophied RPE monolayer with the goal to prevent photoreceptors loss –6. These efforts are founded on successful earlier studies which demonstrated that autologous RPE-choroid graft translocated from an unaffected peripheral area to the macula could lead to improved vision in AMD patients , 8. However, there is currently no acknowledged gold standard for what constitutes the defining characteristics of an authentically derived RPE or agreement as to how those cells can best be evaluated and selected prior to transplantation.Currently nThe Lancet revealed limited functional validation of RPE cells prior to injection in patients . The results in patients suggested that the injected cells were well tolerated with systemic immune suppression, but functional gains in vision remained unclear for this phase I/IIa trial , 10. Two other groups are following a similar approach. Cell Cure Neurosciences Ltd., based in Jerusalem is injecting a bolus of ESC-RPE in AMD patients while the Neural Stem Cell Institute, in New York is planning to inject adult RPE stem cell derived RPE cells in AMD patients. Although the transplantation approach is similar across these three potential therapies, they are markedly different in starting cells and in their manufacturing processes.Pioneering work in stem cell derived RPE replacement therapy was carried out by Schwartz and colleagues at Advanced Cell Technology (ACT) - now called Astellas Institute of Regenerative Medicine. ACT initiated a clinical trial to assess the safety of a bolus injection of human embryonic stem cell (ESC)-derived RPE cells into the subretinal space of patients with Stargardt’s macular dystrophy or dry age-related macular degeneration. A preliminary report in the journal , 11–19. The London and the California projects use ESC-derived RPE monolayers on plastic non-degradable scaffolds (polyester and parylene-c respectively) as the transplant support , 20–22. In comparison, the RIKEN initiative (stopped after the first patient transplantation due to transplant manufacturing concerns) and the NEI ongoing phase I trial are planning to use autologous iPS cells. RIKEN used a collagen-based scaffold and NEI is using a biodegradable scaffold, both with the goal of having the RPE monolayer supported by their own extracellular matrix (ECM) , 23. Going forward, RIKEN institute and other groups  have announced HLA-matched iPS cell lines for RPE-based trials for AMD and other retinal degenerative diseases. . HLA-matched iPSC work is based on the hypothesis that iPSC-RPE manufactured from individuals homozygous for class I MHC HLA alleles will have a higher probability of immune acceptance in patients , 29. Some of the preliminary work published recently from transplantation of HLA-matched monkey iPSC-RPE showed promising results . The transplants were able to integrate in the back of the eye and were accompanied by minimal adaptive immune response as compared to completely allogeneic transplants that resulted in a major immune response against iPSC-RPE transplants.With the continued evolution in technology, other groups have altered the manufacturing process and created RPE monolayer transplants instead of cell suspension. These include the London Project to Cure Blindness, the California Project to Cure Blindness, the RIKEN, Japan initiative, and the National Eye Institute project –33. Currently not known is the extent to which variability amongst donor tissues, starting stem cells, the manufacturing process, or the mode of transplantation would affect RPE cell characteristics . Whether several of these variables affect epigenetics of the cells while they are differentiating into the RPE lineage, thus influencing RPE cell fate and ultimately cell function is also unclear . Similarly it remains unknown whether the phenomenon of X-inactivation in cells of female origin , resulting in mosaic expression at the cellular level, would lead to variability among cells from a given female donor.Several of these ongoing and planned clinical studies are based on acceptable characterization of RPE cells including gene expression profile, ability of cells to form tight junctions, polarized cytokine secretion, and ability to phagocytose photoreceptor outer segments. It is, however, worth noting that in many cases this work was done as part of a preclinical analysis to characterize cells grown under laboratory research-grade conditions. Most of these procedures have not been validated or benchmarked for use as “release criteria” and are not validated for comparative use across multiple different studies. Several groups have urged the need for standardize practices and assays to assess adult stem cell, ESC or iPSC-derived RPE for clinical applications .We addressed these questions by evaluating the structure, molecular, and physiological differences arising from 15 iPSC derived RPE generated from distinct tissues of several different donors. In addition to the well-established practice of verifying the typical RPE markers, gene expression profile, tight junction formation, and phagocytic ability, we employed several key functional assays  that utilize a purinergic signaling pathway with critical physiological implications to verify the structure, functional intactness and integrity of whole RPE monolayer rather than single RPE cells . However, we found that the functional analysis of the ATP-mediated purinergic signaling pathway provides the most sensitive readout of RPE authenticity highlighting variation among RPE derived from different tissues and donors  . We demonstrate that the basis for functional variation stems from manufacturing/technical heterogeneity, which exceeds epigenetic influences of the starting tissue. Additionally, we found that clonal variability among iPSC-RPE derived from female donors is relatively low suggesting that X-chromosome inactivation does not strongly influence RPE function In summary, we have compiled a reference data set that encompasses genomic, structural, and functional variation and can be used to compare and authenticate any stem cell derived-RPE derived for therapeutic or research purposes. This data set will allow groups to select one release criterion that can be used to compare clinical RPE product across different trials. Our work provides a range of values expected for most RPE functions within which RPE cells can be deemed authentic. We believe that this data will have significant practical benefit and suggest helpful guidelines as a basis for further improvements to the scientific community and will help translate RPE into commercially successful clinical applications.


Epigenetic modification provides a stable mechanism by which cells with the same genotype can modulate their gene expression and exhibit different phenotypes. In the past two decades, excellent progress has been made to profile these modifications and our understanding of epigenetic marks has surpassed beyond the basic phenomenon of cellular heterogeneity. It is now established that epigenetic marks are altered in almost all common human diseases. The Epigenomics of Common Diseases meeting, 1–4 November 2016, provided an account of the progress made in this area and also indicated future areas that are yet to be addressed. Although disease focussed, several other aspects were discussed that are relevant to epigenetics as a field, including cellular heterogeneity, epigenomic association studies, emerging concepts in cancer epigenetics and new innovative techniques of broad application . Here, we provide a brief report of some of the key ideas and themes discussed in this meeting and based on these, we speculate on future research directions.Recent advances in high-throughput DNA analysis now enable researchers to examine epigenetic modifications across the genome, primarily DNA methylation marks, for association with numerous disease phenotypes. As such, epigenome-wide association studies (EWASs) have been fruitful in their findings but they also harbour their own unique challenges. EWASs provide an opportunity to investigate a large number of CpGs across large numbers of patients and controls to detect aberrant methylation signals at a population level . Furtherhttp://eforge.cs.ucl.ac.uk), which identifies tissue or cell-type specific signals from Illumina 450K methylation array data [https://github.com/sjczheng/EpiDISH), which can be used for epigenetic dissection of heterogeneity within a sample, and CORALINA (comprehensive guide RNA library generation through controlled nuclease activity), a universal method for generating guide RNA libraries for large-scale CRISPR-based genomic and epigenomic screening [Several speakers presented vignettes of many interesting EWAS findings, including Stephan Beck  who shared some “new twists” in EWAS analytics and started off with a plea for authors and editors alike to ensure EWAS papers include CpG numbers. This oversight, he suggested, was akin to publishing a genome-wide association study without including rs numbers for single-nucleotide polymorphisms. Methylation of a CpG site and its relationship to the expression of a corresponding or nearby gene is very context specific. For example, the methylation of a particular gene promoter is likely to be very different to the methylation of the gene body. Methylation in either of these elements could have a different function for the specificity of the corresponding transcription . Thereforay data , EpiDISHcreening .IL5RA), which is consistent with the presence of activated eosinophils in atopic subjects. Notably, a monoclonal antibody targeted to IL5RA is currently in phase 3 trials for the treatment of severe asthma. Cookson reiterated the point that cell heterogeneity and genetic factors need to be accounted for in EWASs. The WCGNA package (freely available from https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/) that was used to account for cellular heterogeneity in this study was suggested as an alternative to other more established methods that adjust for cell mixtures in the analysis of DNA methylation data.Bill Cookson  presented an EWAS of total serum immunoglobulin E (IgE), which is a central mediator in asthma and atopy, in peripheral blood leukocyte methylomes . Of the TXNIP, ABCG1, and SREBF1) have biological plausibility in the context of type 2 diabetes. In an EWAS investigating adiposity, methylation markers at 187 loci were found to be associated with body mass index (BMI) [Also from Imperial College London, UK, John Chambers reported findings from the EpiMigrant study, which investigated changes in DNA methylation patterns associated with high risk of type 2 diabetes in South Asians . Methylaex (BMI) . These lThe mechanisms involved in the hallmark properties of a cancer cell are well described and continue to be heavily investigated . Until rIn her talk on epigenetics of the cancer microenvironment, Susan Clark  began by quoting Stephen Paget’s 1889 “soil and seed hypothesis”—“When a plant goes to seed, its seeds are carried in all directions; but they can only live and grow if they fall on congenial soil”. The focus of Clark’s investigation was on pro-tumorigenic cancer-associated fibroblasts (CAFs), which in vitro studies have shown, were not just transiently activated by signalling from tumour cells but they retain their phenotype even when tumour cell stimuli were removed. Whole-genome bisulfite sequencing (WGBS) and RNA-Seq were used on CAFs from prostate cancer and compared to matched non-malignant prostate fibroblasts (NPFs). This comparison showed a large number of discrete differentially methylated regions (DMRs), mainly hypomethylated, that were enriched at promoters and enhancers. A subset of DMRs shared methylation changes with tumour epithelial cells, suggesting convergent epigenetic programming. These robust biomarkers show promise for improved early prostate cancer diagnosis.Peter Jones  shared some compelling findings that support treating haematological and other cancers with a combination of epigenetic therapy, e.g. the DNA methylation inhibitor 5-aza-2′-deoxycytidine, and physiological levels of vitamin C. From in vitro studies of cancer cells, this combination treatment was shown to synergistically inhibit proliferation and increase apoptosis by enhanced demethylation of endogenous retroviruses (ERVs). The subsequent increased expression of ERVs stimulated immune signalling leading to apoptosis. Interestingly, it has been recently shown that treatment of ovarian cancer cell lines with a DNA methylation inhibitor triggered double-stranded RNA (dsRNA) sensing in the cytoplasm causing a type I interferon response and apoptosis. This response was associated with upregulation of hypermethylated ERV elements. They also observed a similar event in melanoma . Taken tTBC1D16 promotes melanoma growth and metastasis and in a clinical setting is associated with poor prognosis [Manel Esteller  was able to showcase a large amount of impressive work to come out of his lab recently. These included a genome-wide DNA methylation analysis of cell lines derived from the primary melanoma tumour and matched lymph node metastasis from the same individual. From this analysis, Esteller and colleagues discovered that hypomethylation-associated reactivation of a cryptic 47kDa transcript of rognosis . In an erognosis . Of interognosis . By mapprognosis .Duncan Sproul  described a work that used WGBS in oestrogen receptor-positive breast tumours to explore causes of widespread DNA hypomethylation in cancer. These tumours were specifically hypomethylated at large partially methylated domains (PMDs) that replicated in late S-phase and were associated with dysfunctional DNA methyltransferases. Loss of the de novo DNA methyltransferase DNMT3B resulted in preferential hypomethylation of PMDs, whereas loss of the maintenance DNA methyltransferase DNMT1 resulted in genome-wide hypomethylation. Furthermore, hypomethylation of PMDs was correlated with an increase in copy number variations that would contribute to chromosome instability in these tumours. Although the existence of PMDs in cancer cells and somatic cells has been described and analysed in several studies using different methods –23, theiOne technical aspect of genome-wide methylation profiling that was evident from both Susan Clark’s and Duncan Sproul’s talks was the power of using WGBS. Although 450K or 850K are very reproducible and are feasible platforms for large-scale EWAS, it was clear that large genomic methylation changes could escape detection of these array platforms, which have representative CpG probes for specified regions. For example, the use of WGBS enabled the detection of PMDs in breast cancer as discussed by Sproul. Susan Clark showed examples of some large regions showing differential methylation in prostate cancer, and this detection would not be possible with the existing array platforms. Sequencing-based genome-wide methods such as reduced representation bisulfite sequencing (RRBS) and WGBS are also more likely to detect methylation aberrations in regions distant from coding genes. Although the cost of WGBS is still substantially very high to be applied for large-scale studies at the moment, these techniques can be used for more niche questions on limited samples to gain biological insight for further exploration.The field of epigenomics continues to rapidly expand, which is aided by researchers embracing or even developing new techniques and technologies to decipher unanswered questions. Two of the most exciting concepts to emerge recently are the use of single-cell profiling and targeted epigenetic genome editing using the CRISPR/Cas9 system.Wolf Reik  has used single-cell epigenomic profiling of embryonic stem cells (ESCs) to explore the relationship of epigenetic heterogeneity and cell fate decisions in mammalian development. DNA methylation analysis of naïve compared to primed ESCs showed the greatest methylation heterogeneity in enhancer elements. By modelling DNA methylation in primed ESCs, Reik suggested that oscillations of methylation and demethylation by the DNMT3 enzymes and the TET enzymes could explain the transcriptional heterogeneity observed in primed ESCs. Single-cell sequencing revealed global regulation of heterogeneity at gastrulation. The absence of “translational substructure” in the primed states indicated transcriptional noise was at a peak prior to major cell fate decisions. Reik concluded by briefly introducing a new single-cell method based on NOMe-Seq  that incContinuing on in a similar vein, John Marioni  used single-cell RNA-Seq to explore cell fate decisions in early embryo development. Analysis of mouse embyros captured from early gastrulation to primitive erythrocyte formation identified ~2000 highly variable genes that were allocated to ten distinct clusters. Repeating this approach in Tal1 knockouts allowed sharper insight into whether this cell fate decision followed a step-wise restriction model . In anotEmily Saunderson  from the laboratory of Gabriella Ficz presented a study that targeted a CRISPR/Cas9 DNMT 3A/3L fusion to a panel of genes in primary breast myoepithelial cells. The hypermethylation-associated repression of RASSF1 and CDKN2A resulted in increased cell proliferation. Outgrowing cells were not immortalized, but they escaped senescence. Interestingly, targeting the CpG islands of both CDKN2A gene products, p16 and p14, but not individually allowed the cells to escape senescence.Sox9 locus that in humans caused female to male sex reversal when the duplications were contained within a non-coding chromatin-partitioning unit called a topologically associated domain (TAD). Capture Hi-C and 4C-seq analysis showed that intra-TAD duplications resulted in sex reversal and no overall change in TAD conformation. However, in contrast, inter-TAD duplications across TAD-boundaries were associated with a normal phenotype due to an insulation effect and new chromatin domains called neo-TADs were formed. However, when inter-TAD duplications included an adjacent potassium channel, Kcnj2, the duplicated gene was now located inside the neo-TAD and misexpressed by the Sox9 regulatory landscape which caused a different disease, Cooks Syndrome [Daniel Ibrahim  and colleagues used CRISPR/Cas9 in mice to recapitulate genomic duplications at the Syndrome .Rieke Kempfer  from the Epigenetic Regulation and Chromatin Architecture laboratory of Ana Pombo introduced a novel cryosectioning-based method, genome architecture mapping (GAM), that can be used to explore relationships between 3D interactions and gene expression. This method has been applied to mouse ESCs, where specific domains have shown enrichment for interactions between distant enhancers and actively expressed genes. It is hoped that this method, which achieves 30 kbp resolution using as little as 400 cells, can be applied to a clinical setting in the future .In conclusion, this meeting covered many different concepts and new developments in the area of disease epigenetics. One aspect that generated some discussion was the correction for cellular composition in epignenetic profiling. Although sophisticated algorithms have been developed in the last few years to correct for methylation bias due to cell composition in the blood, it was clear from the discussion that the existing methods are far from perfect. It is important to note this potential bias as a result of mixed cell type, rather than assuming that the algorithms will provide the ideal results. This is particularly important as the blood continues to be the most widely used tissue for any epigenomic study . For other cell types , the methods are yet to be developed for cell composition correction. In summary, although hundreds of reference methylomes are now available, correcting for methylation biases due to cell composition continue to pose substantial challenges.DNA methylation is generally perceived as a suppressive mechanism, and this perception was developed with the repeated observation of gene silencing due to promoter methylation. However, as we are now able to profile methylation at a genome-scale , it is becoming clear that the relationship of methylation with gene expression is more complex than the simple assumption of negative regulation of expression by promoter methylation. In fact, from several talks, it became clear that methylation changes alter distal regulatory elements, particularly enhancers, which could result in large changes to the transcriptional program. Talks from cancer epigenetics also highlighted the importance of investigating the relationship between DNA methylation and repeat elements. Currently, little is known about methylation and the regulation of distant elements and future research is likely to reveal more on the nature of these relationships.It was also evident from the meeting that the development of new tools and approaches is an active area of research in this field. Especially with the release of many publicly available datasets, it is important to develop new tools that are accessible to biologists as eventually it has to be useful for them to address their research questions. From this meeting, it was evident that exciting research on understanding the basis of epigenomics of common disease will continue to take place in the future.


In modern toxicology, substantial efforts are undertaken to develop alternative solutions for in vivo toxicity testing. The adverse outcome pathway (AOP) concept could facilitate knowledge-based safety assessment of chemicals that does not rely exclusively on in vivo toxicity testing. The construction of an AOP is based on understanding toxicological processes at different levels of biological organisation. Here, we present the developed AOP for liver fibrosis and demonstrate a linkage between hepatic injury caused by chemical protein alkylation and the formation of liver fibrosis, supported by coherent and consistent scientific data. This long-term process, in which inflammation, tissue destruction, and repair occur simultaneously, results from the complex interplay between various hepatic cell types, receptors, and signalling pathways. Due to the complexity of the process, an adequate liver fibrosis cell model for in vitro evaluation of a chemical’s fibrogenic potential is not yet available. Liver fibrosis poses an important human health issue that is also relevant for regulatory purposes. An AOP described with enough mechanistic detail might support chemical risk assessment by indicating early markers for downstream events and thus facilitating the development of an in vitro testing strategy. With this work, we demonstrate how the AOP framework can support the assembly and coherent display of distributed mechanistic information from the literature to support the use of alternative approaches for prediction of toxicity. This AOP was developed according to the guidance document on developing and assessing AOPs and its supplement, the users’ handbook, issued by the Organisation for Economic Co-operation and Development. Liver fibrosis typically results from repeated-dose toxic injury and is an important human health issue associated with chemical exposure, which disrupts the normal liver architecture, alters organ function and may further develop to cirrhosis and liver cancer with considerable mortality attributable to these end stages —and progressing through a dependent series of intermediate KEs at different levels of biological organisation, finally culminating in an adverse outcome (AO). AOPs are typically represented sequentially, moving from one KE to another, as compensatory mechanisms and feedback loops are overcome. These KEs are a limited number of measurable and toxicologically relevant molecular occurrences that are essential for progression towards the AO. It is important to keep in mind that an AOP does not provide a comprehensive molecular description of every aspect of the biology involved (the mechanism of action), but focuses on the critical steps in the pathway Fig.  Ankley . The strSince liver fibrosis results from a complex interplay between various hepatic cell types, receptors and signalling pathways  research project, which aimed at developing alternative models for safety assessment based on mechanistic knowledge. To this aim, AOPs for chronic liver injury were developed in order to facilitate design of studies for predicting selected types of repeated-dose toxicity , a central AOP development platform and repository within the OECD AOP Development Work Plan, managed by the Extended Advisory Group on Molecular Screening and Toxicogenomics . The AOP Wiki is one component of the AOP Knowledge Base (https://aopkb.org) that has been created to enable the scientific community, in one central location, to share, develop, and discuss their AOP-related knowledge.Here, we describe the AOP developed for liver fibrosis, from hepatic injury caused by protein alkylation to the formation of liver fibrosis. It is among the first AOPs that were developed according to the guidance document on developing and assessing AOPs and its supplement, the users’ handbook, issued by the Organisation for Economic Co-operation and Development (OECD). The AOP development activity was started in the context of the Safety Evaluation Ultimately Replacing Animal Testing  and allyl alcohol  to a protein amino acid. Alkylating agents are highly reactive chemicals that introduce alkyl radicals into biologically active molecules and as a consequence can impede or alter their biological function Liebler . CovalenEven though protein alkylation is a generic process having an impact on multiple physiological processes in the cell, there is experimental evidence that covalent protein alkylation does lead to cell injury  in mitochondria, cytoskeletal components, endoplasmic reticulum and nucleus, or indirectly through activation or inhibition of signalling cascades and transcription factors as well as changes in transcriptional activity. The outcome may be either triggering of necrotic or apoptotic processes or sensitisation for the action of cytokines (Kaplowitz 2O3) , cytokines, including transforming growth factor beta 1 (TGF-β1) and tumor necrosis factor alpha (TNF-α), and chemokines which all contribute to oxidative stress, inflammatory signalling and finally activation of KCs . Differentiated (i.e. fenestrated) LSECs prevent HSC activation and promote reversal of activated HSC to quiescence. However, upon liver injury, they lose this role. Preclinical studies have demonstrated that LSECs undergo defenestration as an early event that not only precedes liver fibrosis, but on its own may also promote it, proving that changes in LSEC differentiation might be an integral part in the development of fibrosis. In addition, during fibrogenesis, LSECs become highly pro-inflammatory and secrete an array of cytokines and chemokines  and latent TGF-β binding protein (LTBP) and undergoes proteolytic cleavage by the endopeptidase furin to generate the mature TGF-β dimer. Three TGF-β isoforms  have been identified, but only TGF-β1 was linked to liver fibrogenesis and is the most potent fibrogenic factor for HSCs  mRNA increases 20-fold in activated HSCs compared to quiescent HSCs 3). HenceDevelopment of liver fibrosis is also driven by chronic inflammation in response to injury affecting all cell types involved in the pathogenesis. The fibrogenic cascade is maintained by mediators secreted by inflammatory and pro-fibrogenic cells that stimulate each other in amplifying the pathogenic process Fig. . DamagedH1) and pro-fibrotic (TH2) T helper cells could influence the outcome of the fibrotic response, as observed in mouse models for fibrosis  that have exhibited both anti-fibrogenic  that could serve a pro-fibrogenic role , which drives increased (cannabinoid-receptor) CB1 signalling. Stimulation of the CB1 receptor is pro-fibrogenic, whereas the CB2 receptor is anti-fibrotic and hepatoprotective. Opioid signalling increases proliferation and collagen production in HSCs. Serotonin has a pro-fibrotic effect that synergises with PDGF signalling. Also thyroid hormones enhance activation of HSC (through increased p75 neurotrophin receptor (p75NTR) and activation of Rho), thereby accelerating the development of liver fibrosis Friedman .Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine that stimulates DNA synthesis, cell migration, procollagen α1(I) mRNA expression, and secretion of TGF-β1 and inflammatory cytokines. These fibrogenic actions are mediated by NOX , temporality (KEup occurring at earlier time point than KEdown), and incidence (KEup with higher incidence than KEdown). Due to the limited availability of adequate cell models, dose–response data on KERs are not available. Only in the case of the last KER (collagen accumulation-liver fibrosis), empirical support was considered as “high” due to sufficient amount of empirical and clinical evidence.Biological plausibility for the first KER  was rated as “moderate”, whereas all others were rated as “high”, because there is good scientific understanding of these relationships. On the other hand, empirical support for KERs was rated as “moderate”, because there is only limited empirical evidence that a change in the upstream KE leads to an appropriate change in the respective downstream KE, especially in respect to dose–response  for which liver fibrosis has not been observed. The difference in progression to liver fibrosis might lie in various cellular responses such as apoptosis, necrosis, transdifferentiation, or repair and regeneration. There is increasing evidence for apoptosis being a main fibrogenic trigger  and rarely describe mechanistic sequences in detail. Single cell cultures of various liver cell types allow studying the individual cell responses to injury and provide the opportunity to understand the roles that different liver cell types play in fibrogenic processes. Several co-culture models allow the investigation of interactions between some individual actors in vitro. But in general, these studies were not designed to investigate the linkages between various actors and on the whole it is rather difficult to find sound empirical evidence to support KERs. Due to the pathogenic complexity of liver fibrosis with the involvement of many different cell types, there is currently no suitable cell model available to challenge the reliability and robustness of the AOP and to mimic and further explore the sequence of events, especially in quantitative terms. The investigation of quantitative aspects regarding how much change for how long in an upstream KE is needed to cause a corresponding change in the next downstream KE remains an ambitious goal.Two actors have been highlighted as essential contributors to fibrogenesis without being labelled KEs, namely chronic inflammation and oxidative stress, which are ongoing processes throughout the pathway and mutually interconnected with most of the KEs  markers for downstream events can be identified to facilitate a testing strategy for chemical risk assessment. In addition, the KEs can be used for hazard identification and read-across to assess the toxic potential of an untested substance. Therefore, AOPs have the potential to become a powerful tool to support alternative methods for chemical risk assessment which may be predictive of the AO in vivo without the need to actually demonstrate the AO. Further AOP development will be facilitated by the AOP Wiki, which provides a platform for interdisciplinary collaboration between the scientists and the regulators. It is essential that many researchers from various disciplines like toxicology, biology, chemistry, clinical medicine, and computer modelling engage in this activity and contribute to AOP development. AOP developers need to share and connect their AOPs through common KEs to build AOP networks that eventually will better represent the complex biological processes and interactions in response to various chemical exposures.This AOP description demonstrates how detailed and distributed mechanistic information from the literature can be assembled and coherently displayed to support the use of alternative data. Together with other AOP descriptions leading to chronic liver injury, it provided the basis for the design of feasibility studies for predicting selected types of liver toxicity within the SEURAT-1 research project. The addition of quantitative data on dose–response relationships, threshold values, and temporal sequences, the acquisition of which is dependent on the availability of suitable cell models, would substantially improve the applicability of this AOP.


Patterns of woody-plant mortality often reflect tradeoffs associated with resource allocation. Plants that allocate a high proportion of carbon acquired from photosynthesis to non-structural carbohydrate storage may be buffered from the synergistic effects of climate change and episodic disturbance. Tamarix spp. is a dominant, non-native riparian tree in western North America that is experiencing dieback in some areas of its range due to episodic herbivory by the recently introduced northern tamarisk leaf beetle (Diorhabda carinulata). We propose that genotype × environment interactions largely underpin current and future patterns of Tamarix mortality. We anticipate that (i) despite its recent introduction, and the potential for significant gene flow, Tamarix in western North America is generally adapted to local environmental conditions across its current range in part due to hybridization of two species; (ii) local adaptation to specific climate, soil and resource availability will yield predictable responses to episodic herbivory; and (iii) the ability to cope with a combination of episodic herbivory and increased aridity associated with climate change will be largely based on functional tradeoffs in resource allocation. This review focusses on the potential heritability of plant carbon allocation patterns in Tamarix, focussing on the relative contribution of acquired carbon to non-structural carbohydrate (NSC) pools versus other sinks as the basis for surviving episodic disturbance. Where high aridity and/or poor edaphic position lead to chronic stress, NSC pools may fall below a minimum threshold because of an imbalance between the supply of carbon and its demand by various sinks. Identifying patterns of local adaptation of traits related to resource allocation will improve forecasting of Tamarix population susceptibility to episodic herbivory.Patterns of woody-plant mortality have been linked to global-scale environmental changes, such as extreme drought, heat stress, more frequent and intense fires, and episodic outbreaks of insects and pathogens. Although many studies have focussed on survival and mortality in response to specific physiological stresses, little attention has been paid to the role of genetic heritability of traits and local adaptation in influencing patterns of plant mortality, especially in non-native species.  Plant ecologists have recently paid considerable attention to woody-plant mortality because of continental-scale die-offs of woody plants across the globe , biomass allocation, water use efficiency, nutrient fertilization impacts on NPP and hydrologic processes , released for biological control of this genus  are there fundamental plant tradeoffs in carbon allocation patterns associated with herbivory tolerance? and (ii) is herbivory tolerance/sensitivity tied to adaptation to local environmental conditions? Although considerable research has been undertaken on Tamarix invasion into arid riparian ecosystems of North America, including identification of the hybrid nature of the genus in the western USA  despite its recent introduction, and the potential for significant gene flow, Tamarix in western North America is generally adapted to local environmental conditions across its current range in part due to hybridization of two species, (ii) local adaptation to specific climate, soil and resource availability conditions will yield predictable responses to episodic herbivory and (iii) the ability to cope with a combination of episodic herbivory and increased aridity associated with climate change will be largely based on functional tradeoffs in resource allocation that fall along a predictable trait spectrum.This paper synthesizes ongoing research on the patterns and mechanisms of Tamarix has become one of the most successful non-native woody plants in the western USA, covering nearly 500 000 hectares  are now recognized to dominate desert riparian habitats , but these are less common species and even rarer as parents of hybrids  to determine its suitability as a biocontrol agent , but not in Eurasia in part because of the allopatric distributions in their native ranges , and there has been extensive backcrossing, it is likely that hybridization occurred soon after introductions into western North America , and Tamarix across a latitudinal gradient to investigate variations in cold hardiness and phenology  involves 18 populations derived from an elevation gradient from 15 to 1940 m and a latitudinal range of 32.0° to 37.3°N. Beginning in January 2016, bimonthly phenological observations of floral characteristics were made on at least 36 individual plants from each population. Each individual was assigned a score for foliage greenness on a scale from 0 to 5, with a score of 0 indicating that the plants were still dormant, and a 5 indicating that the leaves were fully flushed out. Low elevation populations broke dormancy and were fully leafed out earlier than those that originated from mid- and high-elevation sites  than genotypes collected from higher elevations with cooler climate means than in Yuma  when compared with high-elevation populations , then one plant demonstrating greater tissue synthesis and subsequent growth will do so at the expense of having less photosynthate to allocate to other sinks including NSC storage  or cottonwoods (Populus spp.)  tolus spp.) . More imt glands . Howevert glands . To be m sucrose . The higThe biocontrol agent, 8 genera . This ples Table . These ses Table , all at Tamarix, allocation to NSC storage may be selected for in populations that experience freezing temperatures during the growing season. A recent common garden experiment indicated that Tamarix genotypes from higher latitudes, where freezing temperatures during the growing season are common, showed greater tolerance to herbivory by D. carinulata (i.e. expressed higher canopy regrowth following herbivory) than lower latitude genotypes  the construction and maintenance of common gardens, such as the one described earlier and currently in operation in Yuma, AZ; (ii) expanded efforts to quantify plant carbon budgets over time, particularly better seasonal accounting of changes in whole-plant NSC pools across genotypes and populations; (iii) genomics and epigenetics research to link phenotypic traits such as resource allocation, tolerance to salinity and drought to specific molecular markers and gene expression, and (iv) ground and remote sensing-based monitoring of Diorhabda spp. distributions and subsequent impact on Tamarix fitness, recruitment and survival.This review highlights three main points. The first is that despite its recent introduction into North America, rix Fig. . As the sues Box . To bettTamarix. These projects could target a wide range of specific objectives depending on the projected impacts of foliage herbivory on Tamarix including fire prevention, native plant restoration and habitat restoration. For example, Tamarix populations that are predicted to experience the highest levels of mortality and canopy dieback could be areas that are given the highest priority for restoration efforts. Knowing these patterns may be particularly important for guiding habitat restoration of threatened and endangered avian species such as the southwestern willow flycatcher (Empidomax traillii extimus) that under some circumstances rely on Tamarix canopies for nesting (Tamarix will impact riparian habitat for E. trallii extimus. However, the combination of common garden studies of plant resource allocation and stress tolerance with technological advances in molecular genomics will provide critical information on how Tamarix populations will likely respond to multiple stressors going into the future.Information acquired from current research activities, and research approaches advocated here would provide critical information for targeted restoration and conservation of valued riparian ecosystems. Data on plant genetics and phenotypic trait expression could potentially guide restoration ecologists and land managers on how to best use limited resources for riparian restoration by improving predictive ability based on phenotype/genotype screening on when and where climate change by defoliation interactions will most impact


The affordability of smartphones and improved mobile networks globally has increased the popularity of geosocial networking (GSN) apps  as a method for men who have sex with men (MSM) to seek causal sex partners and engage with the queer community. As mobile penetration continues to grow in India, it is important to understand how self-presentation on GSN app is relevant because it offers insight into a population that has not been largely studied. There is very little information about how Indian MSM discuss their sexual preferences and condom preferences and disclose their human immunodeficiency virus (HIV) status with potential sex partners on Web-based platforms.The objective of this study was to describe how self-presentation by Indian MSM on GSN apps contributes to sexual preferences, HIV or sexually transmitted infection (STI) disclosure, and if the presentation differs due to proximity to the Greater Mumbai or Thane region.Between September 2013 and May 2014, participants were recruited through banner advertisements on gay websites, social media advertisements and posts, and distribution of print materials at outreach events hosted by lesbian, gay, bisexual, transgender (LGBT) and HIV service organizations in Maharashtra, India. Eligible participants self-identified as being MSM or hijra (transgender) women, living in Maharashtra, aged above 18 years, having regular Internet access, and having at least one male sex partner in the previous 90 days.Indian MSM living inside and outside the Greater Mumbai or Thane region reported an average of 6.7 (SD 11.8) male sex partners in the last 3 months; on average HIV status of the sex partners was disclosed to 2.9 (SD 8.9). The most commonly used websites and GSN apps by MSM living inside Greater Mumbai or Thane region were Planetromeo, Grindr, and Gaydar. Results demonstrated that MSM used smartphones to access GSN apps and stated a preference for both condomless and protected anal sex but did not disclose their HIV status. This low level of HIV disclosure potentially increases risk of HIV or STI transmission; therefore, trends in use should be monitored.Our data helps to fill the gap in understanding how Indian MSM use technology to find casual sex partners, disclose their sexual preference, and their HIV status on Web-based platforms. As mobile penetration in India continues to grow and smartphone use increases, the use of GSN sex-seeking apps by MSM should also increase, potentially increasing the risk of HIV or STI transmission within the app’s closed sexual networks. The affordability of smartphones and improved mobile networks globally has likely contributed to the increased popularity of geosocial networking (GSN) apps  as a method for men who have sex with men (MSM) to seek causal sex partners and engage with the queer community -3. The gTill date, research on MSM’s use of GSN apps has focused on urban and rural Western MSM populations, examining sexual risk behaviors, partner preferences, condom use, and human immunodeficiency virus (HIV) or sexually transmitted infection (STI) disclosure . WesternThe use of GSN apps as a tool for seeking sexual partners raises the questions of how urban and rural MSM present themselves on Web-based platforms and how they disclose their sexual and condom preferences and HIV or STI status ,15. SuleTo our knowledge, there is no published data on how Indian MSM use GSN apps to seek sex partners. It is particularly important to understand this gap in the literature as the Telecom Regulatory Authority of India reports that India has 979 million wireless phone users . As InteAs a first step to understanding to what extent the use of GSN apps by Indian MSM mirror the behavioral risks contributing to HIV or STI transmission among Western MSM, descriptive data is needed about which apps Indian MSM use and how they construct an online identity when using these apps. Presentation of self and disclosure of HIV or STI status is important in this context because virtual communities, such as the sexual communities created by GSN apps, provide a space where individuals can explore the boundaries of their sexual attraction and potentially identify persons willing to engage in casual sex. This has been supported in the literature when looking at unidentified gay men, who create multiple profiles on GSN apps to have same-sex encounters ,16. AddiThe recruitment procedure is described in detail elsewhere . BroadlyParticipants were asked about which websites or GSN apps they used to meet sex partners, the number of profiles on each , and if they disclosed their preferred sexual position and HIV status on their profile; participants were provided a write-in option. To identify all possible websites or GSN apps, participants were provided with a list that was generated by the authors who work for a nongovernmental organization serving MSM in the Greater Mumbai or Thane region; a write-in option was provided, so that websites and apps not included in the provided list could be captured. To minimize data-entry errors by participants, an algorithm was employed that reminded participants how many profiles they identified; as participants’ wrotein responses, the algorithm subtracted the responses from the total number of profiles identified.Participants were asked in which district of Maharashtra they lived in. Persons who indicated living in Greater Mumbai or Thane were placed in one category and all other participants were placed in another.Participants were asked to identify their gender, age, educational degrees obtained, employment status, sexual orientation, openness (outness) about their same-sex attraction, HIV status, and number of female, hijra (transgender) women and male sex partners they had in the last 3 months. Participants self-reported how they stated a preference for condoms and sexual position of their GSN apps.2) of independence were used to examine the relation between MSM living within or outside of the Greater Mumbai or Thane region, demographic characteristics, Internet-enabled device, and social media platforms. Chi-square tests of independence were also used to examine the differences between disclosure of sexual preferences and HIV status on websites and GSN apps. Fisher’s exact tests were used to identify differences when counts were small.Bivariate analyses were used to identify which websites and GSN apps Indian MSM access and the frequency of disclosure of their sexual preferences and HIV status. Chi-square tests  identified as cisgender males . Most participants had completed college and were employed. Over three-quarters of participants self-identified as being gay or bisexual, but only 19% (86/445) reported being open about their same-sex attraction (out) to most or everyone in their life. Participants inside and outside the Greater Mumbai or Thane region reported an average of 6.7 male sex partners (SD 11.8) in the last 3 months; on average HIV status was disclosed to 2.9 (SD 8.9) of the sex partners. Participant’s demographics are presented in Indian MSM in our sample had GSN apps running in the background for much of the day, reporting an average of 8 hours per day looking for potential sex partners. The most commonly used Internet-enabled devices for sex-seeking were smartphones , personal desktop , and laptop computers . The most commonly used websites and GSN apps by MSM living inside Greater Mumbai or Thane region were Planetromeo, Grindr, and Gaydar. Additionally, MSM living in the Greater Mumbai or Thane region had created more profiles on GSN apps and websites than MSM living outside the Greater Mumbai or Thane region. Despite the difference in websites and GSN apps between groups, a majority of participants had multiple profiles on websites and GSN app see .P<.001 and SD 5.34, P=.02, respectively) on GSN apps and websites compared with MSM living outside Mumbai or Thane region. MSM living in Greater Mumbai or Thane region also showed significant difference in preference for more condomless insertive , condomless receptive  and condomless versatile anal sex  than MSM living outside Greater Mumbai or Thane region. There were no differences between the MSM within and outside of Greater Mumbai or Thane region in stated preference for versatile anal sex with condoms  and disclosing their HIV status on websites or GSN apps .MSM living within Greater Mumbai or Thane region stated a greater preference for protected insertive and receptive anal sex  as their primary means to access sex-seeking GSN apps to search for potential sex partners. Most men in the study had multiple active accounts on sex-seeking GSN apps and allowed these apps to run much of the day. These findings are consistent with Western MSM use of GSN apps to connect with potential sex partners .That Indian MSM have multiple profiles potentially allowing them to present themselves as someone who prefers to be insertive, receptive, or versatile and someone who does or does not endorse condom use during sex complicates how we understand the presentation of self on GSN apps. That MSM in the Greater Mumbai or Thane region endorsed a preference for both using condoms for anal sex and engaging in condomless anal sex further complicates how we understand the way in which Indian MSM present themselves in an online space. Regarding the use of multiple profiles, Ross and colleagues ,18 and oMen living inside the Greater Mumbai or Thane region were more likely to endorse using condoms than MSM living outside the MSA. The difference between groups in stated condom preference could be the result of HIV prevention services and outreach being concentrated within the physical cruising sites of Greater Mumbai or Thane region, suggesting a possible overlap between populations on physical and virtual cruising platforms. HIV prevention services should now also be targeted toward encouraging Internet-using MSM to disclose their condom use preferences on GSN apps to facilitate conversations between potential sex partners about condom use before meeting for a sexual encounter.The results of this study should be considered within the limitations of this understudied population. Due to the rapid changes in technology and rapid creation of mobile apps, the GSN apps reported in this paper may increase or decrease in use overtime. On average, the participants were highly educated, employed, and self-identified as either bisexual or gay, but were not out or open about their sexual identity to most people in their lives. Because the study was cross-sectional, we could not determine if disclosing a preference for condomless anal sex on GSN apps leads to an increased risk for HIV or STIs among Indian MSM or vice versa. There is robust evidence supporting the connection between condomless anal sex and increased risk for HIV transmission . HoweverThe purpose of this study was to describe how Indian MSM present themselves on GSN apps, how their presentation contributes to sexual preferences, and if the presentation differs due to proximity to the Greater Mumbai or Thane region. Our data begins to fill the gap in understanding these aspects. As mobile penetration in India continues to grow and smartphone use increases, the use of GSN sex-seeking apps by MSM should also increase, potentially increasing the risk of HIV or STI transmission within the app’s closed sexual networks. Considering that HIV interventions are solely focused on physical cruising sites in India, our findings highlight the need for Indian HIV Interventions to look beyond current scopes and expand the ambit of HIV interventions to virtual platforms as well. Interested researchers, including mobile interventions, should continue to monitor how MSM use mobile technology to meet male sex partners.


Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Diabodies are engineered antibody fragments that are composed of two Fv domains connected by short peptide linkers. They are attractive candidates for mediators in assembling protein nano-structures because they can simultaneously bind to two different proteins and are rigid enough to be crystallized. However, comparison of previous crystal structures demonstrates that there is substantial structural diversity in the Fv interface region of diabodies and, therefore, reliable prediction of its structure is not trivial. Here, we present the crystal structures of ten mono- and bi-specific diabodies. We found that changing an arginine residue in the Fv interface to threonine greatly reduced the structural diversity of diabodies. We also found that one of the bispecific diabodies underwent an unexpected process of chain swapping yielding a non-functional monospecific diabody. In order to further reduce structural flexibility and prevent chain shuffling, we introduced disulfide bridges in the Fv interface regions. The disulfide-bridged diabodies have rigid and predictable structures and may have applications in crystallizing proteins, analyzing cryo-electron microscopic images and building protein nano-assemblies. Linking protein components to form a predictable and rigid structure is a prerequisite for generating complex protein assemblies in a predetermined fashion. Most of the chemical cross linkers available have long and flexible spacers. Because of this the linked proteins have significant structural flexibility, and the relative orientation and distance between their protein components is largely unpredictable. This is the case even when the chemical cross linkers themselves have rigid structures since they are attached to flexible side chains such as cysteines or lysines. Recently several research groups have reported novel methods that can produce artificial protein assemblies with predictable and stable structures1234H) and light-chain variable fragments (VL) with short “GGGGS” peptide linkersH C terminus to the VL N terminus. Therefore, the two VH-linker-VL chains associate to form a dimer with two antigen binding sites, each consisting of the VH and VL fragments from the different chains.Recombinant bispecific antibodies are artificial proteins containing the antigen- binding domains of two different antibodiesWhen the two Fv domains in the diabody are identical, the structure is called a monospecific diabody. Such diabodies induce homo-dimerization of target proteins because they can simultaneously bind two identical protein molecules. On the other hand, bispecific diabodies have Fv domains originating from two different antibodies and so can bridge different proteins leading to their hetero-dimerization. They can be used to connect a wide range of molecules because antibodies can be generated against almost any kind of molecule. Hundreds of thousands of antibody clones are developed each year. Furthermore, more than 2,000 antibody structures with and without bound antigen have already been deposited in the Protein Data Bank (PDB). Diabodies can easily be produced from this huge array of antibodies and can be used as building blocks to construct artificial protein assemblies for a variety of applications.H, and light chains, VL, are genetically fused in the order VH-VL. However, Carmichael et al. have reported the structure of an unusual diabody in which VL precedes VHThe crystal structures of several monospecific diabodies have been reported8In order to use diabodies as general and reliable mediators of artificial protein assemblies, one must be able to predict the orientation and distance between the two bound proteins with great accuracy. Therefore, the structures of diabodies must be rigid and predictable. The individual Fv domains of diabodies do have rigid and predictable structures as shown in the thousands of antibody crystal structures deposited in the PDB. However, the interfaces between the two Fv domains in the diabodies differ substantially in sequence and appear to be too small to have stable structures8H-VL protein chains form a homo dimer .


This ventilatory  is a chronic, progressive condition that manifests not only in cardiac dysfunction but also in respiratory dysfunction. Diaphragmatic myopathy is common and contributes to dyspnea and exercise intolerance in advancing stages of HF  during a normal tidal breath reflects the ventilatory drive , which was calculated from esophageal pressure measured during normal eupneic breathing in the anesthetized mouse.Foster et al. use the term “ventilatory drive” to describe the drive to breathe but there are several other synonyms including central drive, neural drive, and respiratory drive. Because measurement of phrenic nerve activity, the most direct measure of ventilatory drive, is not feasible in humans and difficult in animals, minute ventilation can be used as a surrogate. However, in pathological conditions minute ventilation may not accurately reflect the drive to breathe  or inspiratory pressure (PI) reflects increased activation of the phrenic nerve and concomitant increased contraction of the diaphragm.Elevated mouth occlusion pressure (PImax) . In summary, inspiratory pressure is directly proportional to ventilatory drive, maximal inspiratory pressure is inversely proportional to diaphragm weakness and, although the mechanisms are not well understood, chronic elevated ventilatory drive is associated with diaphragmatic weakness.Diaphragmatic weakness is assessed in humans by voluntarily generating a maximal inspiratory pressure  arm and a protective AT2R/ACE2 arm. Increasing evidence suggests that in HF there is an imbalance to favor the excitatory arm, leading to sympathoadrenergic activation and that this imbalance is modulated in part in the cardiovascular-control regions of the brain  and the sympathetic nervous system ] employed in this study may have initiated changes in the heterodimer ratio of AT1 receptors to βARs that resulted in signaling changes at the diaphragm muscle cells themselves, or myopathy secondary to activation of ventilatory overdrive.However, Foster et al., did not measure systemic or brain levels of ANGII, nor did they speculate about the specific central location at which ANGII acts to stimulate ventilation. In fact, they state that the ability of ANGII/catecholamines to cross the BBB is controversial. In conscious dogs, systemic-derived ANGII cannot cross the BBB; however, it is still able to centrally stimulate ventilation by activating circumventricular organ neurons which effectively lack a BBB but project to medullary cardiorespiratory control centers behind the BBB . GPCRs, the largest family of cell surface receptors, are implicated in numerous diseases and thus their signaling pathways are prime targets for therapeutic intervention , a βarrestin-biased angiotensin receptor agonist, inhibits/dampens bradykinin 2 receptor signaling , that these signaling pathways are not necessarily equally activated  and that signaling at one receptor can modulate the signaling at a second receptor family (heterodimer transmodulation). Regulation of GPCR signaling is extremely complex and our current understanding of how most pharmacologic drugs, including those used in this study, impact βarrestin-dependent-, biased- and heterodimer-signaling is incomplete, as is the cell specificity of the drug effects. Moreover, how the ratio of monomer to heterodimer receptors may impact pathology (or vice-versa) is largely unexplored in the context of drug design. Although a daunting task, the design of novel molecules that have pluridimensional efficacies holds great promise for treatment of multiple morbidities associated with complex diseases such as HF and diaphragmatic myopathy.The Foster et al., study significantly advances our understanding of diaphragmatic myopathy and helps to reconcile why various drugs with comparable cardiovascular effects can have differing benefits on symptomatology and mortality and reveal potential pathways for early intervention and future translational investigations.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We previously demonstrated such behaviors in devices operated in the unipolar mode, opening up the question of whether we could add plasticity to the list of features exhibited by our devices. Here we demonstrate clear STDP in unipolar devices. Significantly, we show that the response of our devices is broadly similar to that of biological synapses. This work further reinforces the potential of simple two-terminal RRAM devices to mimic neuronal functionality in hardware spiking neural networks.Resistance switching, or Resistive RAM (RRAM) devices show considerable potential for application in hardware spiking neural networks (neuro-inspired computing) by mimicking some of the behavior of biological synapses, and hence enabling non-von Neumann computer architectures. Spike-timing dependent plasticity (STDP) is one such behavior, and one example of several classes of plasticity that are being examined with the aim of finding suitable algorithms for application in many computing tasks such as coincidence detection, classification and image recognition. In previous work we have demonstrated that the neuromorphic capabilities of silicon-rich silicon oxide (SiO Non-von Neumann computing architecture, inspired by the neuronal architecture of the brain, is receiving considerable interest as a more efficient way to tackle a range of machine learning tasks such as classification, sorting, and image recognition  dissolution. Adjusting the bias correctly enables us to generate voltage spikes with a current-controlled frequency, removing the need for much of the CMOS electronics used in conventional systems to model neuronal function. While much work remains to be done in this field, the reduction of CMOS circuitry footprint in neuromorphic systems could yield savings in valuable silicon chip real-estate. Other benefits such as power reduction are more uncertain, and remain so until further work has been performed on algorithm development and a fuller understanding of how devices can be designed to operate at very low current and/or duty cycle. Nevertheless, a reduction in circuit complexity by exploiting simple two-terminal devices is an attractive goal. Building on our previous work, here we report, for the first time, spike timing dependent plasticity in such unipolar devices—a result that, when combined with neuronal functionality, promises a greatly reduced component count for RRAM hardware spiking neural networks. Further, we present a statistical analysis of STDP conductance modification mimicking the response of a biological synapse. RRAM devices in our experiments are based on SiOx switching layers; this ensures full compatibility with CMOS technology  electrodes sandwich a 37 nm thick SiOx resistance switching layer. All layers were sputter deposited (the SiOx layer was co-sputtered from Si and SiO2 targets). The SiOx active switching layer was amorphous and sub-stoichiometric (x ≈ 1.3). The top TiN layer was patterned using conventional lithography techniques into square electrode structures with edges ranging from 100 to 400 μm  systems  to create custom pulse shapes. The PMU does not allow manual control of current compliance, which we implemented instead by carefully controlling pulse voltage and duration. We used custom Matlab scripts to control the system either directly or through custom C drivers. In all cases we contacted devices with tungsten needle probes on a Signatone probe station. Devices were tested in ambient conditions in an isolated low-light environment.Details of resistance switching in intrinsic oxide RRAM devices may be found elsewhere . These pulses were applied in between each of the ascending or descending pulses that make up the sawtooth voltage ramp  that do not induce any switching . The results in Figure x RRAM devices. Furthermore, we have demonstrated that STDP behavior is a statistically favorable occurrence, even in non-optimized devices. This extends and adds to our previous work showing spiking, integration and thresholding in unipolar SiOx RRAM devices (Yu et al., We have shown that it is possible to achieve an STDP response from unipolar SiOKZ: Performed the STDP experiments, designed the study, interpreted the data, and wrote the first draft of the manuscript; AM: Assisted in designing the study, interpreted the data, and assisted with drafting the manuscript; LM and MB: Assisted with experimental design and measurement; SH: Assisted with experiments; AK: Oversaw the work, co-designed the study, and assisted with interpreting the results; All authors contributed to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


By the end of 2014, a total of 955,081 persons in the United States  had received a diagnosis of human immunodeficiency virus type 1 (HIV-1) infection   who want to conceive, considerations in choosing the optimal method to achieve pregnancy include transmission risk, treatment efficacy, and affordability. Use of HIV-negative donor sperm that meets Food and Drug Administration donor eligibility criteria remains the safest option for avoiding HIV infection of the female partner  who wish to conceive a biological child, strategies to minimize the risk for sexual transmission are needed. In 1990, CDC recommended against insemination with semen from HIV-infected men.Recent data regarding the safety of semen processing suggest that such processing is a viable option for HIV discordant couples attempting conception. The risk for transmission from an HIV-infected man to an HIV-negative woman is low if appropriate risk-reduction strategies, such as the use of highly active antiretroviral therapy, antiretroviral preexposure prophylaxis, and sperm washing are implemented. Recent evidence suggests that discordant couples might consider condomless intercourse timed to coincide with ovulation or intrauterine insemination of the woman or in vitro fertilization in combination with sperm washing after discussing the risks and benefits of each option with a medical provider.As further data emerge, the risk profile for each treatment option will be further defined. HIV-discordant couples who desire to conceive might wish to discuss treatment options with a medical provider who can explain the risks and benefits of different treatment modalities as they apply to the couple’s specific situation before attempting conception.