HCV antiviral therapy, global control of HCV infection becomes feasible but depends on the capacity of countries to identify infected people and to offer them treatment. To achieve the WHO goal which targets a diagnosis rate of 90% by 2030, simplification of screening and diagnosis will be crucial.In the light of the advances in HCV core Ag and dried blood spots; starting from 2008 until November 2017. In addition, a manual search was undertaken to detect relevant papers or websites related to specific data from countries which underwent or are planning a programme of HCV elimination.Published literature, unpublished data and expert consensus were used to determine key parameters, including point‐of‐care, rapid diagnostic testing, screening, the use of HCV core antigen tests and new nucleic acid amplification technologies for use in decentralized settings. In high prevalence settings, a one‐step screening and diagnosis strategy could simplify diagnostic algorithms provided the cost is reduced. Finally, genotyping may no longer be required in the context of availability of pangenotypic antiviral therapy.Several strategies have been developed and evaluated these last years to simplify and facilitate access to screening and diagnosis, the development of reliable Despite relevant advances in HCV screening and diagnosis, the overall diagnosis package is still too expensive today and efforts must be made to allow generalized implementation of reliable tests in low and middle income countries. These efforts will be key factors to foster a real public health approach to HCV elimination. Nowadays, the advent of highly effective direct antiviral agents (DAAs) and the availability of generic versions of these agents have changed the paradigm for the management of HCV infection. With the excellent safety and tolerability and high cure rates of DAAs (>95%), the major remaining barrier to treatment is the under‐diagnosis of HCV and limited access to treatment in the diagnosed population. DAA prices are starting to fall due to innovative approaches, to negotiations with industry, as well as continued development of new agents bringing competition to the marketplace.Current screening of HCV infection is based on anti‐HCV antibody detection using a third‐generation enzyme immunoassay (EIA). Third‐generation EIA use a multiantigen format including antigens from the core, NS3, NS4 and NS5 regions; these tests show excellent sensitivity and specificity (>99%) and are considered reference standards to detect anti‐HCV antibodies The primary goals of simplifying HCV screening and management are to better identify infected individuals, increase rates of retention and linkage to care and treatment, reduce the costs of diagnosis for patients and the healthcare system with the ultimate goal of reducing viral transmission at a population level, and progression of liver disease and hepatitis‐related mortality at an individual level. Dynamic HCV transmission models demonstrate that screening and treatment of HCV‐infected individuals in high prevalence settings can reduce the incidence of HCV by reducing HCV transmission (termed ‘‘cure as prevention”) The aim of this review is to provide a comprehensive overview of new simplified approaches for screening, diagnosis and monitoring HCV infection within different country‐specific settings and to describe key promising tools in future diagnostics.2Published literature, unpublished data and expert consensus were used to determine key parameters consisting of the following terms: sample type , point‐of‐care, rapid diagnostic testing, screening, the use of HCV cAg and dried blood spots. In addition, a manual search was undertaken to detect relevant papers or websites related to specific data from countries, which planned or are planning a programme for HCV elimination. The literature search was limited to English language, available from 2008 until November 2017.The study population was analysed in each study . The high risk population groups include men who have sex with men, sex workers, PWID and prisoners. Studies that only reported sensitivity or specificity, or those that only used reference assays for positive samples were excluded from the review.3With the emergence of highly efficacious antiviral treatment, the possibility of curing the vast majority of patients is now a realistic goal Much of the challenge lies in the fact that many populations with a high prevalence of HCV, such as people who inject drugs (PWID), sex workers, migrants, those with mental health issues and incarcerated individuals often have reduced access to care in traditional medical settings for many reasons. As such, even if screening occurs, linkage to and retention in care is often limited, greatly reducing the overall impact of screening efforts. In LMICs, the situation is compounded by lack of access to high quality medical facilities, particularly for non‐acute medical issues. As such, decentralized rapid test‐based screening will be crucial to improve screening and linkage in both high and low/middle income countries.3.1Facilitating access to screening can be achieved by improving serological tests in terms of rapidity and simplicity of performance such as using easy‐to‐access samples like fingerstick capillary whole blood or oral fluid. Simpler tests are more appealing to those being tested and potentially allow testing to be done by less skilled individuals, greatly increasing testing capacity both in numbers and the locations in which it can be performed. Rapid diagnostic tests (RDTs) have been already been developed for HCV antibody screening, with significant advantages over classical enzyme immunoassays (EIA).® HCV rapid antibody test  was excellent in finger stick whole blood (99.4%) as well as in oral fluid (97.6%) while the Labmen HCV test  showed poor sensitivity in fingerstick whole blood (63.1%) Technically, classical enzyme immunoassays (EIA) require laboratory infrastructure and expertise in their operation. In contrast, RDTs do not require investment in laboratory equipment with minimal maintenance costs and reagents. Therefore, RDTs are suited for decentralized settings to reach individuals at highest risk for HCV who may remain outside the traditional medical system. To be acceptable for screening, RDTs need to meet the high standard of traditional testing tools in terms of analytical performance for accuracy, reproducibility, sensitivity and specificity and in terms of clinical performances. Clinical performances of individual tests are heterogeneous and vary widely 3.2In LMICs, the use of classical serological enzyme immunoassays (EIA) for screening may be limited for many reasons including poor laboratory infrastructure, insufficient staff and/or insufficiently trained staff, and poor‐quality management systems for relaying results, all of which hamper the access to accurate and timely screening. In these contexts, point‐of‐care RDTs appear promising Some countries  have already engaged partners to develop an efficacious prevention and control plan, which has led to an improved access to diagnostics and treatment for HCV‐infected individuals with severe disease. In Georgia, an estimated 5.4% of the adult population  has chronic HCV infection, and most of them were unaware of their infection in 2015  In Egypt, where the prevalence of hepatitis C virus (HCV) infection is the highest in the world As each region/country needs to plan its own approach by implementing adequate HCV testing, the experience from those countries who have taken the first step towards eliminating HCV can help pave the way for other countries experiencing high rates of HCV infection to undertake similar initiatives, and help curb the global epidemic of viral hepatitis.3.3Dried blood spot sampling is an interesting alternative collection method to collect whole blood specimens (either by capillary fingerstick or by venipuncture). The sample is easily transferred onto filter paper and can then be easily transported to a centralized laboratory where testing can take place. The two main advantages of this sample type which can facilitate the expanded access to screening and diagnosis is (i) the option to use fingerstick whole blood and (ii) the stability of the specimen allowing for simple transport (even by regular mail) without the need for an intact cold chain. Once in the central laboratory, DBS samples can be stored long‐term at −20° or −80°C. Once ready for testing, the sample is eluted off the filter paper using an appropriate buffer and the eluate can then be used in the same testing systems used for serum or plasma. One other major advantage of DBS is that multiple spots can be taken at once allowing for reflex HCV RNA testing on a second (or third) spot if the initial spot is positive for HCV antibodies thus avoiding the need to bring individuals back for confirmatory HCV RNA testing, which has consistently been shown to be a major drop‐off in the cascade of care of most settings. DBS is particularly attractive for difficult‐to‐reach populations. Because phlebotomy is not required, testing can be done by peer workers with limited training and venous access, often a challenge for PWID, is less of a concern. Recent studies have confirmed the improved acceptability of DBS over standard testing approaches The performance for detection of anti‐HCV antibodies in DBS specimens compared to plasma or serum specimens has shown excellent results with sensitivity and specificity generally over 95% The main disadvantage of DBS is that the existing commercial assays have not been validated so far or received regulatory approval with this method of sample collection and transport. Some studies published detailed protocols on how to collect and analyze DBS 3.4Multianalyte testing refers to testing in the same specimen the detection of HCV along with other pathogens, for example, HIV, syphilis and/or HBV. Such an approach has several obvious advantages including the requirement for a lower specimen volume, fewer fingersticks if capillary whole blood is used, less time required than a series of tests and potentially other cost efficiencies. This approach is promising, but data on clinical sensitivity and specificity of the assays are still required as well as the evaluation of their impact on patient management 4The complexity of current algorithms for HCV diagnosis limits the ability to advance large‐scale screening programmes. Even if screening can be done, limited capacity to specialty care is often an additional barrier to care. Simplifying strategies of diagnosis/management of HCV infection will enable the potential transfer of treatment care outside of classical specialty care (i.e. infectious diseases and hepatology clinics) for most patients, likely all but those with advanced liver disease. In addition, in LMICs, lack of simple and affordable HCV diagnostic solutions represents a restraint to ensure broad access to care, as current diagnostic tools are insufficiently developed. Innovations in technologies for point‐of‐care testing  have led to the creation of chip‐sized laboratory systems that could be helpful in the future 4.1Probably the three main tools that should bring simplification to diagnosis of HCV are (i) the availability of reliable HCV cAg tests, (ii) the availability of new POC tests based on nucleic acid detection and quantification for use in decentralized settings; both of these assays could be used as one‐step procedure in diagnosing chronic HCV infection  and (iii) the potential to skip the determination of the viral genotype in the context of availability of pangenotypic all oral DAA therapy.4.1.1The HCV core antigen test (HCV cAg) targets the HCV nucleocapsid peptides 22 (p22), released from infected cells into plasma. HCV cAg is detected early and during the natural course of HCV infection as a surrogate marker of viral replication Table  23, 48. Other advantages of HCV cAg over NAAT are the following: serological marker stable at room temperature for 96 hours, short time to obtain (less than 60 minutes) In summary, HCV cAg represents a reliable HCV diagnosis tool and, being less costly than viral load tests, should facilitate HCV screening and access to care. However, this solution depends on the presence of centralized laboratory machines. In addition, caution must be paid to some limitations of the assay: most studies were conducted in high resource areas within reference laboratories where data for genotypes 4, 5 and 6 are missing 4.1.2Quantitative NAAT is widely used for measuring viral load, identifying those in need of treatment, and to assess treatment response. In the era of short‐course, highly effective therapy, there is less need for quantification of HCV RNA for HCV management. Therefore, qualitative assays, in theory less expensive, can replace quantitative NAAT, particularly in LMICs. In addition, new random access NAAT technologies offer potential for POC diagnosis 4.1.3Once diagnosis is confirmed, the current algorithm of HCV management requires HCV genotyping before therapy is initiated. However, with the pangenotypic success achieved using next‐generation DAAs  with very high rates of SVR in different clinical conditions , pre‐genotyping may no longer be required in the future. Whether genotyping can also be skipped when first‐generation DAAs (e.g. sofosbuvir/daclatasvir or generics) will be used in LMICs is questionable (at least in cirrhotic patients), as genotype 3 cirrhotic patients need longer duration of treatment and/or the addition or ribavirin when this combination is used 4.2A significant proportion of anti‐HCV antibody‐positive patients fails to have a confirmatory test in difficult‐to‐reach populations and are lost to follow‐up When DBS sampling is used, HCV RNA NAAT assays are preferred as confirmatory tests over HCV cAg; indeed HCV cAg testing has been shown to possibly have lower sensitivity on DBS compared to serum when only one spot is used DBS EIA serology + DBS NAAT ;RDT serology + DBS NAAT RDT serology + POCT NAAT EIA serology + plasma/serum‐based NAAT .The choice of whether to use DBS sampling for HCV serology or NAAT or both will depend on the healthcare setting and infrastructure, and epidemiological context. Different strategies can be suggested with varying combinations:An interesting alternative would be a biphasic strategy with first testing anti‐HCV antibody (+) patients with HCV cAg, and reserving NAAT only for those who are anti‐HCV antibody (+) but HCV cAg (−). This strategy has been evaluated in a study performed in Toronto (Canada) to determine relapse in HCV infected patients treated with DAAs 4.3To further simplify the diagnosis pathway, the ideal future algorithm would only require one test for both screening and diagnosing HCV. This test is preferably HCV cAg because of reduced costs or NAAT for HCV RNA Figure  if these5The advent of interferon‐free DAA therapies has significantly simplified monitoring treatment. In addition to the fact that pangenotypic DAA combinations could eliminate the need for genotyping before starting treatment, there is no further need to monitor viral load during treatment There remains some debate about the ability of HCV cAg to assess response to DAA treatment. There is emerging data supporting very good performances of the ARCHITECT assay as a test of cure 6Control of HCV will strongly depend on the capacity of countries to identify people who live with chronic hepatitis C and to offer them treatment. Tools for simplified screening and on and post‐treatment monitoring will be critical to make such efforts feasible. Currently costs and complexity of diagnostics algorithm are notable and important barriers to screening and treatment monitoring. With the implementation of new treatment regimens, the current pathway for HCV diagnosis can conceivably be simplified from three tests  to one or two tests if random‐access NAAT assays or reliable HCV cAg assays become affordable, particularly in high prevalence settings. A second aspect of simplification is to use the same test for diagnosis and monitoring (one single point at follow‐up visit 12 to 24 weeks). Finally, the capacity for decentralization including the use of new random‐access NAAT for HCV RNA will be crucial to further simplify diagnosis and monitoring. The future challenge will be to implement different diagnostic algorithms in different countries, based on the experience of similar countries who have already taken the first steps towards controlling HCV and explore innovative approaches to reduce the cost of these tests through large‐scale projects in different contexts .SF: scientific consulting from Abbvie, Beckman. JF: grant funding from Abbvie, Abbott, Gilead, Janssen, Merck and Scientific Consulting from Abbvie, Contravir, Gilead and Merck. SC: Grant funding from Gilead, ANRS, Ministry of Health; Scientific Consulting from Abbvie, Gilead, Merck, Abbott Diagnostics, Hologic. NL: none to declare.SF and NL wrote the draft of the outline. SF wrote the first draft of the manuscript. JF, SC and NL critically reviewed the manuscript and provided input accordingly.None.


Sphyrapicus nuchalis) and Red‐breasted (Sphyrapicus ruber) sapsuckers (Aves: Picidae) hybridize along a narrow contact zone that stretches from northern California to British Columbia. The hybrid zone between these species has been studied extensively for more than 100 years and represents an excellent system for investigations of the evolution of reproductive isolation. Shifts in the proportions of phenotypes at hybrid localities since 1910 that were inferred using specimens from museum collections were confirmed using species distribution models. We predicted the historical, current, and future distributions of parental and hybrid sapsuckers using Random Forests models to quantify how climate change is affecting hybrid zone movement in the Pacific Northwest. We found observed distribution shifts of parental sapsuckers were likely the result of climate change over the past 100 years, with these shifts predicted to continue for both sapsuckers over the next 80 years. We found Red‐breasted Sapsuckers are predicted to continue to expand, while Red‐naped Sapsuckers are predicted to contract substantially under future climate scenarios. As a result of the predicted changes, the amount of overlap in the distribution of these sapsuckers may decrease. Using hybrid phenotypes, we found the climate niche occupied by the hybrid zone is predicted to disappear under future conditions. The disappearance of this climate niche where the two parental species come into contact and hybridize may lead to a substantial reduction in genetic introgression. Understanding the impacts of global climate change on hybrid zones may help us to better understand how speciation has been shaped by climate in the past, as well as how evolution may respond to climate change in the future.Hybrid zones, where two divergent taxa meet and interbreed, offer unique opportunities to investigate how climate contributes to reproductive isolation between closely related taxa and how these taxa may respond to climatic changes. Red‐naped ( As such, climate can be a strong predictor of species distributions  the contact zone may shift with a changing climate as new climatic conditions favor one species over another  form an exemplary system to investigate climate‐driven hybrid zone shifts. Red‐naped  and Red‐breasted sapsuckers (Sphyrapicus ruber) hybridize over a narrow contact zone that stretches from British Columbia to northern California. Within this narrow contact zone where the ranges of these two species overlap, individuals with hybrid phenotypes are frequently encountered  differential aggression between parental species, with Red‐breasted Sapsuckers being aggressively dominant over Red‐naped Sapsuckers  and (2) patterns of differential mate choice, where females of both species prefer males with more red, leading to the observed eastward expansion of Red‐breasted Sapsuckers , for a total sample size of 4,824 presence localities (Tables S1–S3). We defined presence localities as any location for which a sapsucker was observed or collected (for museum specimens) at any point in time during the breeding season, which we here define as June and July . We accessed locality data for museum specimens housed at the University of Wyoming Museum of Vertebrates and the University of California at Berkeley Museum of Vertebrate Zoology using the online Arctos database (arctos.database.museum). Additionally, we accessed locality data for museum specimens housed at the University of Washington Burke Museum of Natural History and Culture directly from their online database (www.burkemuseum.org). Project eBird is a citizen science database that uses a network of over 400 regional reviewers to check the quality of the data submitted by observers . We used these localities to improve the predictive power of our species distribution models. For the current distribution models, we included species locality data from 1969 to 2013. For the historical distribution models, we pooled locality data collected between 1910 and 1940.Our study area in the Pacific Northwest included Washington, Oregon, northern California, and the western portion of Idaho and Nevada  and from museum specimens (n = 54). During the breeding season, hybrid phenotypes are only encountered within the hybrid zone where the two parental species come into contact. We assume that localities with hybrid individuals represent part of the hybrid zone  the PRISM Climate Group , which w the PRISls Table . We inclls Table  because ls Table . Though ls Table . Winter ls Table  and the ls Table , as wellls Table . We alsols Table .We chose three future climate scenarios from a general circulation model (GCM) based on the Canadian Center for Climate Modeling and Analysis (CGMC3). Three emission scenarios, representing low (B1), medium (A1B), and high (A2), were chosen for the year 2090 IPCC, .2.3m (number of independent variables permuted at each node for a tree) optimized to the out‐of‐bag error estimate  models Breiman, . RF modeWe conducted an independent validation of final RF models using an iterative 10% withhold (999 iterations) where 10% of the data were withheld  relative to current climate models for each parental species. Using the RF model of hybrid occurrence, we predicted the distribution hybrids across the landscape. To determine the effect of climate change on the future of where hybrid individuals are found in the region, and thus where the hybrid zone occurs, we also projected the distribution of hybrids into the future using the same three future climate scenarios  used to predict the distribution of Red‐naped and Red‐breasted Sapsuckers.We were unable to assess the full extent of overlap, and thus the area of the hybrid zone, between the parental species because our models of species distribution were weakest along the edges of their distribution in the contact zone. To model the distribution of the hybrid zone, we constructed distribution models based on localities where birds with hybrid phenotypes were observed. We assumed that localities where hybrid were observed represented areas within the contact zone, as hybrids typically do not occur outside of the region of the contact zone during June and July Howell, . We usedGiven the difficulty of identifying hybrids, we also ran models where we randomly added 10% and 20% simulated error into our hybrid observations to assess how robust our models were to potential identification error. We introduced error in two ways: (1) we randomly removed observations of hybrids, reducing our sample size and simulating misidentification of birds with hybrid phenotypes as parental species and (2) we randomly assigned hybrid identity to parental observations, simulating misidentification of parental species as hybrids. We weighted our selection of parental observations based on the probability they were found within the predicted distribution of the hybrid zone .We tested for significant differences between historical and current distributions for both Red‐naped and Red‐breasted sapsuckers. We directly compared these distributions to determine areas of significant range increases and decreases for each species. Significance was calculated by assessing the pairwise differences of the two distributions relative to the mean and variance of differences . We used the same climate variables from the PRISM Climate Group , the Red‐breasted Sapsucker historical model received higher support than the Red‐naped Sapsucker model, both in terms of fit  and validation . As in the models built using climate variables from the PRISM Climate Group . Model validation statistics, including PCC, were also moderate  groves for nest sites (Crockett & Hadow, While climate change may be directly impacting these distribution changes in sapsuckers through physiological intolerances or competitive exclusion, these shifts may also be the result of indirect associations that impact aspects of habitat (Thuiller et al., Shorter and warmer winters may contribute to sapsucker distribution shifts in several ways. Migration distance and timing likely both play an important role, as Red‐breasted Sapsuckers generally migrate shorter distances to wintering grounds and arrive earlier to breeding grounds than Red‐naped Sapsuckers (Trombino, Adaptation to new and changing environmental conditions in the future can alter the relationships that species currently have with their environment, greatly changing the predictability of future range change (Davis & Shaw, Climate has still not been widely demonstrated to be the cause of many moving hybrid zones (but see Carling & Zuckerberg, None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Early childhood development provides an important foundation for the development of human capital. Although there is a clear relation between stunting and child development outcomes, less information is available about the developmental and behavioural outcomes of children with severe acute malnutrition (SAM). Particularly an important research gap exists in Sub–Saharan Africa where there is a high prevalence of SAM and a high rate of co–occurring HIV (human immune deficiency virus) infection. Our first objective was to assess the prevalence and severity of developmental and behavioural disorders on a cohort of children admitted to an inpatient nutritional rehabilitation centre in Malawi. Our second objective was to compare the developmental and behavioural profiles of children with the two main phenotypes of SAM: kwashiorkor and marasmus.This was a cross–sectional observational study including all children hospitalized with complicated SAM in Blantyre, Malawi over an 8–month period from February to October 2015. At discharge, children were assessed with the well-validated Malawi Developmental Assessment Tool (MDAT) for gross motor, fine motor, language and social development. In children ≥24 months, emotional and behavioural problems were measured using the Strengths and Difficulties Questionnaire (SDQ).150 children (55% boys) with SAM were recruited; mean age of 27.2 months (standard deviation 17.9), 27 children (18%) had pre–existing neurodisabilities (ND) and 34 (23%) had a co–occurring human immune deficiency virus (HIV) infection. All children with SAM experienced profound delays in the gross and fine motor, language and social domains. Linear regression analysis demonstrated that children with kwashiorkor scored 0.75 standard deviations lower  on language MDAT domain than children with marasmus when adjusted for covariates. The prosocial behaviour score of the SDQ was low in children with SAM, indicating a lack of sensitive behaviour in social interactions.Children with SAM have severe developmental delays after a hospital admission. Our results indicate that there might be a significant difference in developmental attainment between children with kwashiorkor and with marasmus. Future studies exploring longer–term outcomes and testing possible intervention strategies are urgently needed. The Lancet, it is now estimated that children in Sub-Saharan Africa are the most “at-risk” for not reaching their developmental potential (66% in 2010) . in 2010) . During in 2010) .Previous literature has described the complex interactions between malnutrition, developmental delay and neurodisability, although clinically it is not always easy to explain a cause–effect relationship –8. It isIn contrast to the amount of evidence on stunted children, developmental and behavioural outcomes in children with severe acute malnutrition (SAM) have hardly been studied. With SAM affecting some 19 million children worldwide, this is an important evidence gap . The eveThis was a cross–sectional observational study, covering all children hospitalized for treatment of SAM in the nutrition ward of Queen Elizabeth Central Hospital (QECH), in Blantyre, Malawi, over an 8–month period from February to October 2015. QECH is a tertiary referral hospital but mainly serves as a district hospital. Children with SAM either self–present or are referred by local health category with “complicated” SAM: they have medical problems requiring inpatient care ; or have failed an “appetite test” (defined as the inability to eat Ready to Use Therapeutic Food) . The heiChildren were defined with marasmus when they presented with MUAC<11.5cm for children less than 5 years old, or a weight–for–height z–score<–3 on the WHO growth standard . ChildreOur study included children who were participating in the “F75 trial” [ClinicalTrials.gov Identifier: NCT02246296], a randomized controlled trial of a reduced carbohydrate formulation of F75 therapeutic milk vs the traditional “F75” therapeutic milk, among children aged 6 months to 8 years with SAM. Both types of milk were only used for a short duration during the stabilisation phase and therefore would likely have no effect on the developmental outcomes. Our sample size was determined by the inclusion of children in the main study. We undertook developmental and behavioural assessments on discharge from the unit when children were clinically stable, finishing all food and had a good appetite as assessed by the clinician. Children were excluded if their parents refused to give informed consent.A trained and experienced research assistant (KC) with 2 years of experience with the Malawi Developmental Assessment Tool (MDAT), as well as training on the WHO UNICEF Care for Child Development Package, administered the measures of both child development and behaviour in a quiet room next to the malnutrition unit. The caregiver was present during the complete assessment. During the study period, the development assessments were independently observed by two of the authors, both paediatricians . In addition, after 3 months KC participated in a refresher course of the development assessment tool.The MDAT is a culturally relevant developmental assessment tool that has been created for the use in African settings . It examThe MDAT has also demonstrated good sensitivity in detecting more subtle developmental problems in children with marasmus . We calcDuring the MDAT assessment, child and maternal behaviour was observed by the research assistant and reported in an observation form. This form had been adapted from the Behavior Observation Inventory from the Bayley Scales of Infant and Toddler Development . It consThe SDQ is a brief 25 item behavioural screening instrument. It is subdivided into four difficulty scales; emotional symptoms, conduct problems, inattention–hyperactivity, peer problems, and a separate fifth strength scale that enquires about the child’s behaviour in normal social interactions the “prosocial behaviour” scale ,30. All Neurodisability (ND) and HIV infection are common problems underlying SAM in our setting and are associated with poorer outcomes ,15. ChilChildren with HIV have a higher risk of developmental and behavioural problems ,39. All The following other clinically important covariates were also collected: age, sex, child disease characteristics reported during the hospital admission  and family characteristics .2 tests were used to analyse differences in child health and family characteristics between the marasmus and kwashiorkor group.We used descriptive statistics to evaluate the child’s background characteristics and the child and caregiver’s involvement in the development assessment. χFor the MDAT domains, z–scores, means and standard deviations were calculated for the entire SAM group and separately for marasmus and kwashiorkor patients. The proportion of children that had a delay in a MDAT domain  was computed for all groups. In addition, we examined the association between the type of SAM (marasmus or kwashiorkor) and MDAT domain z–scores with linear regression analysis. Second, we performed a linear regression analysis adjusted for clinically important covariates: gender, HIV, no education or some primary education of the primary caregiver and passive mother involvement during MDAT assessment. All children with pre–existing ND and children older than 6 years were excluded from the MDAT analysis.www.ibm.com, New York, NY, USA).Finally, we evaluated the SDQ scores in children with only SAM (SAM Only), in children with SAM and a concurrent HIV infection (SAM + HIV) and children with SAM and a pre–existing ND (SAM +ND). We used analyses of variance (ANOVA) to test for differences between these groups. All data was analysed with IBM SPSS version 20 software  died and 189 were discharged to community care; 39 (20.6%) children were lost to follow–up at discharge. Reasons for loss to follow–up at discharge included that some parents wanted to leave the hospital before the developmental assessment could be done and the unavailability of our research assistant. P = 0.001) and pre–existing ND  compared to those with kwashiorkor (P = 0.250).All children with SAM experienced profound delays in the gross and fine motor, language and social domains (P = 0.024) lower social MDAT z–score than children with marasmus, but this effect became non–significant when we adjusted for covariates (P = 0.056). The other MDAT domain z–scores did not differ with statistical significance between children with marasmus and kwashiorkor  –1.43 to –0.07), adjusted for covariates (P = 0.210) engagement (P = 0.670), anxiety (P = 0.600) and cooperativeness (P = 0.740) of the child and in the caregiver’s involvement (P = 0.370) between the marasmus and kwashiorkor groups  or children with SAM+HIV (P = 0.048). We found no statistically significant differences in SDQ–TDS scores between the children with SAM Only and those with an HIV infection (P = 0.130). No calculations were done to examine the different subdomains between the different groups because of the small sample size.The mean (SD) SDQ–TDS scores were 10.1 (4.2), 12.2 (5.5) and 15.5 (3.8) in the children with SAM Only, SAM+HIV and SAM + ND respectively. We report on the severe developmental delays present in a well–characterized clinical population of children admitted with SAM to a nutrition rehabilitation unit in an African setting in a first study on this topic for decades. We have confirmed previous findings showing that SAM is associated with severe developmental delay. Moreover, our results indicate that there might be a significant difference in developmental attainment between children with kwashiorkor and with marasmus. Our study is unique in adding to the very sparse literature describing child behaviour as well as child development in young children with SAM.In our study, children with kwashiorkor had a significantly worse language delay compared to children with marasmus at discharge. One explanation for this could be a difference in neurological involvement between kwashiorkor and marasmus. The striking neurological irritability of children with kwashiorkor has been identified as an important clinical feature . HoweverOur study examined the child’s social – emotional skills using direct observation (rated during the MDAT assessment) and with the use of two different questionnaires . When examining the behaviour outcomes of children with SAM, we found different SDQ scores than previously reported in a World Bank evaluation of Malawian preschool (n = 1815) children attending community based childcare centres . In the Limitations of our study include its cross–sectional design and the assessment of developmental and problems only at discharge from the ward. We did not have any previous details about the development of the child before admission and no details about the home environment. Our findings regarding a significant difference in language delay between the children in the marasmus and kwashiorkor group might also have been explained by the age difference between both groups. The kwashiorkor children were significantly older although the MDAT z–scores are adjusted for age. Unfortunately, our sample size was determined by the main trial so for an age–stratified analysis our sample size was too small.It is likely that some developmental domains will continue to improve during the final few weeks of nutritional rehabilitation in the community . This isOur study has utilized a well–validated African developmental assessment tool, the MDAT, as an outcome measure however behaviour was measured using the SDQ, which has only been translated and not validated in Malawi. There are currently no SDQ cut–off scores available for Malawi, so it is unclear how many children with complicated SAM had SDQ scores in the clinical range. This may have led to a lack of significant results from this perspective. The five questions in the observation of child and maternal behaviour were not validated in this study and could have been confounded by how well the child performed on the MDAT test. Because our sample size was lower for children with marasmus, our power estimate was probably <0.80 to correctly identify a difference between children with marasmus and kwashiorkor.Finally, we assessed only children with complicated SAM: these are by definition the most vulnerable and findings should not be directly extrapolated to the much wider group of children with uncomplicated SAM who are treated as outpatients under current protocols .Findings from this study demonstrated that children with SAM have severe developmental delays after a hospital admission. Future research should focus on the longer–term developmental and behavioural follow–up outcomes of children with SAM in a Sub–Saharan African setting. These results demonstrate the need for developmental interventions during treatment of SAM in a hospital or community setting. Both McGregor’s and Nahar’s study identified significant differences in child development outcomes between children with SAM who received an intervention and children who did not. However, both studies were not randomized and had other limitations for example a high level of selection bias and attrition bias . Therefo


Idiopathic hypercalciuria is a metabolic abnormality characterized by excessive calcium excretion in the urine with normal serum calcium levels and is a common risk factor for formation of kidney stones and/or nephrocalcinosis. These patients benefit from a normal‐calcium, reduced‐animal protein, and low‐salt diet, along with a thiazide diuretic. Nephrocalcinosis is a generalized increase in the calcium content of the kidneys that may occur at a molecular, microscopic or macroscopic level leading to progressive renal damage A 34‐year‐old otherwise healthy man was seen for back pain. He apparently had nonspecific back and leg pain for about 2 years and occasional hematuria. CT scan of the abdomen demonstrated calcinosis in the medullary region of both kidneys, suggestive of medullary nephrocalcinosis Figs.  and 2. LAll authors made substantial contribution to the preparation of this manuscript and approved the final version for submission. AK: drafted the manuscript. VB: acquired the images, revised the manuscript for critically important intellectual content and approved for final submission.None declared.Informed consent has been obtained for the publication of this clinical image.


This is a editorial focus written to highlight the findings by Yang et al in the article “The Soluble (Pro)Renin Receptor Does Not Influence Lithium‐Induced Diabetes Insipidus but Does Provoke Beiging of White Adipose Tissue in Mice.” Bipolar disorder is a common psychiatric condition characterized by periods of depression alternating with mania, affecting an estimated 1% of the population worldwide and 3% in the United States  with corresponding changes in water intake , urine osmolality  and plasma osmolality . Western blotting demonstrated decreased abundance of AQP‐2 in lithium treated animals compared to control.The downregulation of the water channel aquaporin 2 (AQP‐2) has long been thought be one of the causative factors in the pathogenesis of lithium‐induced NDI . Cells with the characteristics of BAT appearing in WAT are often referred to “beige” adipose tissue. WAT and BAT perform many of the same functions but BAT contains a larger amount of mitochondria and are classically involved in thermogenesis, especially in the young. An understanding of the role of BAT in adults is growing, and now BAT is thought to play a role in weight loss and perhaps in preventing metabolic syndrome (Poekes et al. Besides its renal side effects, lithium is well known to induce weight gain, with studies demonstrating that a 4 kg weight gain is average and a ten kilogram weight gain is typical in 20% (Vestergaard et al. In summary, this study by Yang et al. provides early mechanistic insight into two separate phenomena involving sPRR and lithium, showing that sPRR with lithium does not attenuate NDI but does result in the beiging of adipose tissue. Each of these findings evokes a number of questions that provide the basis for research in two separate fields, water channels and adipocyte biology, that will greatly further our understanding of the adverse effects of lithium.


Breast cancer is currently the most common form of cancer and the second‐leading cause of death from cancer in women. Though considerable progress has been made in the treatment of breast cancer, the heterogeneity of tumors (both inter‐ and intratumor) remains a considerable diagnostic and prognostic challenge. From clinical observation to genetic mutations, the history of understanding the heterogeneity of breast cancer is lengthy and detailed. Effectively detecting heterogeneity in breast cancer is important during treatment. Various methods of depicting this heterogeneity are now available and include genetic, pathologic, and imaging analysis. These methods allow characterization of the heterogeneity of breast cancer on a genetic level, providing greater insight during the process of establishing an effective therapeutic plan. This study reviews how the understanding of tumor heterogeneity in breast cancer evolved, and further summarizes recent advances in the detection and monitoring of this heterogeneity in patients with breast cancer. Breast cancer, an ancient disease first noted by the Egyptians more than 3500 years ago During the past two decades, the rapid development of genetic analysis technologies has enabled depiction of the heterogeneity of breast cancer on a genetic level From clinical observation to testing for genetic mutations, a lengthy and steep learning curve has been experienced in understanding the tumor heterogeneity of breast cancer Fig. . VariousIn previous studies, cumulative exposure to carcinogenic factors led to elevations in chromosomal instability and cancer‐specific driver mutations Genomic analysis is an important tool for analyzing the heterogeneity of tumors. As early as 1978, karyotyping was used for the detection of genomic abnormalities In situ hybridization (ISH), a common technique widely used to detect gene number copy alterations, localizes a specific DNA or RNA sequence in tissue by using labeled probes with known sequences Gene‐expression profiling is an important method of detecting gene‐expression heterogeneity in patients with breast cancer. Genetic microarrays and 21‐gene expression assays are the most frequently used techniques in clinical detection of RNA‐expression heterogeneity among patients.gene microarray is a collection of microscopic probes attached to a solid surface. The concept was first introduced by Schena in 1995 A The 21‐gene expression assay is based on reverse transcriptase polymerase chain reaction (RT‐PCR), which is able to qualitatively detect gene expression. In 2004, Paik and colleagues first developed a scoring system based on 21 prospectively selected genes in paraffin‐embedded tumor tissue to quantify the likelihood of distant recurrence in patients with node‐negative, ER‐positive breast cancer In 1977, Frederick Sanger first introduced his DNA sequencing technique Recently, Lee et al. developed a new technique, fluorescent in situ sequencing of RNA, for gene expression profiling Pathologic analysis plays important roles in the diagnosis and subtyping of breast cancer. Developments in pathologic technologies have promoted greater understanding of both intertumor and intratumor heterogeneity. Pathologic analysis can now combine morphology with molecular biology, providing new insights into breast cancer heterogeneity.Pathologic analysis techniques such as immunohistochemistry (IHC) and immunofluorescence (IF) have been widely used for assessing therapeutic biomarkers of breast cancer and identifying subsets of patients with different outcomes Though conventional pathologic analysis methods have great value in evaluating the heterogeneity of breast cancer, due to its limitations in quantitative analysis and multimolecular staining, these methods are not able to completely reveal the heterogeneity of breast cancer, especially intratumor heterogeneity.Recently, nanotechnology has been regarded as a promising tool to illustrate the heterogeneity of breast cancer. Optical‐based nanoparticle imaging, such as quantum dots‐based immunohistochemistry (QDs‐IHC), is an important branch of nanotechnology as applied in medicine. Most of the QDs are semiconductor nanocrystals that have properties including high fluorescence intensity, strong resistance to photobleaching and chemical degradation, size‐tunable emission wavelengths, and simultaneous multiple fluorescence under a single excitation source A variety of imaging techniques exist for the screening and diagnosis of breast cancer. Mammography, ultrasound, magnetic resonance imaging (MRI), and positron emission tomography (PET) are the most common techniques for breast cancer imaging. These imaging techniques can reveal the diverse characteristics of breast cancer Fig. . With thX‐ray‐Based imaging includes techniques such as mammography, computed tomography, and new imaging techniques such as digital breast tomosynthesis (DBT). Mammography has been widely used in breast cancer screening and allows physicians to obtain information about calcifications and breast cancer's morphologic features. The mass shape  on mammography has been associated with the Oncotype Dx (a breast cancer array incorporating the mRNA expression of 21 genes) recurrence score DBT, first introduced in 2011, improved on standard mammography by increasing breast cancer detection rates With the development of ultrasound technology, two relatively new techniques, contrast‐enhanced ultrasound (CEUS) and elastography, have added complementary information to breast cancer diagnosis. Aside from morphological features, CEUS can provide more information about tumor angiogenesis.Elastography is based on the depiction of tissue stiffness, which in breast cancer has been associated with interactions among tumor cells, stromal cells, and the extracellular matrix β3 integrin, P‐selectin, and vascular endothelial growth factor receptor 2 (VEGFR2) CEUS was reported to be a useful tool for correlating imaging features with pathological characteristics. Several studies identified heterogeneity in the expression of vascular endothelial growth factor (VEGF) using CEUS Due to its excellent soft‐tissue contrast and high sensitivity, MRI has been widely used in diagnosis of breast cancer as a complementary imaging technique to mammography and ultrasound Numerous studies have been conducted to explore the application of MRI in screening for breast cancer heterogeneity. Yun and colleagues demonstrated that MRI parameters such as standard deviation and kurtosis of apparent diffusion coefficient (ADC) values were closely related to intratumor spatial heterogeneity of necrosis patterns and vascularity in MCF‐7 and MDA‐MB‐231 xenograft models Molecular imaging is an emerging discipline with great promise in detecting intertumor and intratumor expression patterns of key molecules in breast cancer. Molecular imaging depends greatly on labeled biomarkers; these can be molecules, proteins, or antibodies. By tracing these labeled biomarkers, we can evaluate heterogeneity in biochemical changes, cellular physiology, cellular function, and metabolism within breast cancer 18F‐fluorodeoxyglucose (18F‐FDG) for metabolism, 18F‐fluorothymidine (18F‐FLT) for proliferation, and 18F‐fluoroestradiol (18F‐FES) for ER status PET is the most frequently used molecular imaging technique. PET can be used to assess various properties of tumors with an appropriate radiotracer, such as 18F‐FDG uptake was correlated with a high Ki‐67 index in triple‐negative breast cancer 18F‐FES or 89Zr‐trastuzumab could even achieve dynamic monitoring of ER and Her‐2 status during concomitant endocrine and trastuzumab therapy. In a study by van Kruchten and colleagues, 18F‐FES PET was used to determine the dose needed for ER antagonists to completely abolish ER 18F‐FES PET 18F‐FLT, intratumor proliferation heterogeneity was imaged using PET for assessing the response to neoadjuvant chemotherapy Several studies found that different 18F‐FDG PET features could be translated into the immunohistochemical characteristics of breast cancer Besides PET, QDs‐based imaging is also a promising molecular imaging technique . QDs conjugated with various biomarkers could image different targets in breast cancer QDs‐based imaging is relatively immature for clinical application. However, QDs‐based imaging will have a future role in molecular imaging for breast cancer due to its optimal applicability in imaging acquisition and ability to quantify multiple biomarkers.18F‐FES PET has been used to monitor endocrine resistance during endocrine therapy to help create more efficient treatment plans The technological advances reported in this paper have greatly increased our understanding of intertumor heterogeneity. Based on analysis of the copy number and gene‐expression profiling data from approximately 2000 breast cancer patients, Curtis et al. identified 10 novel molecular subgroups with distinct clinical outcomes The microenvironment in which cancer cells exist is another important component of breast cancer and plays an essential role in cancer progression Precision medicine has received greater attention in recent years, especially in the field of cancer treatment None declared.


EI) and to test the hypothesis that EIs positively impact on prostate contouring accuracy and consistency long term.The aim of this study was to assess variation in prostate contouring 12 months following a structured interactive educational intervention (CT) and magnetic resonance imaging (MRI) data sets were used to assess prostate contouring consistency before, immediately after and 12 months following an EI. No further EIs were provided after the initial EI. Contour variation was assessed using the volume ratio (VR), defined as the ratio of the encompassing volume to common volume.A common set of computed tomography (ROs) at baseline, four completed all assessments, and one was unavailable at 12 months follow‐up. At 12 months, mean VR deteriorated by 3.2% on CT and 1.9% on MRI compared to immediately post EI. Overall, compared to the pre‐EI baseline VR, an improvement of 11.4% and 10.8% was demonstrated on CT and MRI, respectively.Of the original five radiation oncologists (EI on prostate contouring was demonstrated. This study advocates for EIs to be included as part of continuing medical education to reduce contour variation among ROs and improve knowledge retention long term.Good retention of applied knowledge 12 months following an  Recent advances in the delivery of radiation therapy (RT) for clinically localised prostate cancer have enabled dose escalation and greater sparing of organs at risk (OAR).® extra‐cranial tracking) can monitor prostate motion during treatment to improve accuracy.Due to normal anatomical variations, physiological movement  and inherent uncertainties in patient positioning (setup error), RT to the prostate is subject to inter‐ and intra‐fraction variation. Planning target volumes (PTV) are thus generated to account for this uncertainty. Image‐guided RT using intra‐prostatic fiducial markers can reduce inter‐fractional treatment variation and PTV marginsContouring variability is a major source of error in RT delivery, having an impact on treatment accuracy similar to organ motion and setup variation.This study examines the longer term impact of an education initiative (EI) to improve TV delineation by ROs 12 months post EI. It is hypothesised that an EI would positively impact prostate contouring in the long term. This is a follow‐up study to that reported by Khoo et al.3), large prostate (66.4 cm3) and a right hip prosthesis. Four out of the five ROs from the previous study contoured the entire prostate gland for each patient on CT and MRI images. One participating RO (‘RO4’) was unable to contribute due to work relocation.This study is predominantly a quality assurance audit and thus a request for waiver of Human Research Ethics Committee review was approved by Oncology Research Australia. The same three CT and MRI data sets used in a previous reportProst‐a‐doodle softwareNo further EIs were provided. The initial EI occurred 12 months prior and consisted of three formal components. These consisted of a series of anatomy lectures, completion of contouring modules using The Philips Brilliance 16‐slice CT scanner  with 1‐mm slices and the GE Healthcare 1.5T, Excite Platform, eight‐channel coil MRI scanner  with T2‐weighted sequence with 2 mm slices were used. All data sets were imported into the Eclipse treatment planning system version 8.9 . Coregistrations of CT and MRI data sets were based on prostate fiducial markers. Neither the ROs previous contours nor their colleagues’ contours were available during any contouring session.Observer variation was measured as the ratio of encompassing volume (EV) (the volume of the union of a set of TVs) to common volume To determine intra‐observer variation, the VR was calculated for each RO, patient and imaging modality from the TVs as contoured by the RO in each of the 2 months before, immediately following and 12 months following the EI  and 1.9% (MRI) respectively. Overall, there is an improvement of 11.4% (CT) and 10.8% (MRI) from the baseline VR calculated prior to the EI. The mean intra‐observer VR averaged over all ROs and patients is shown in Figure Intra‐ and inter‐observer contouring variability is well described not only in localised prostate, but also for lung,To our knowledge, this is the first study to assess the long‐term impact of a structured EI on prostate contouring for ROs. This analysis focuses on knowledge and skill retention and has demonstrated an ongoing reduction in intra‐observer contour variation following a structured and interactive EI. While the mean VR 12 months after the EI deteriorated as a group compared to immediately after, the improvement from baseline was significant. This impact was greater on CT than MRI volumes.This study contributes to the existing literature and provides additional insights into prostate contouring variability. Fiorino et al.Retention of acquired knowledge by health care professionals is a high priority across different disciplines. After 1 year, only 60–70% of knowledge is retained while skill sets appear to deteriorate more rapidly.Given the potential adverse clinical sequelae of CTV variation on patient outcomes, there has been a significant shift in contouring teaching and assessment. The American Society for Radiation Oncology (ASTRO) and the European Society for Radiotherapy and Oncology (ESTRO) both have established contouring programmes across multiple tumour streams. ASTRO have recently incorporated online contouring programmes in its annual meeting and are now presenting an ‘eContouring’ programme for radiation oncology trainees. Common among these programmes, participants can assess their own contour variability and compare their volumes to those of the instructor and their peers. The Canadian Association of Radiation Oncology (CARO) has recently trialled a contouring ‘boot camp’ with promising results.Other means of improving contour variation include the use of published contouring atlases, institutional protocols, peer review of positron emission tomography (PET) or MRI image fusion and regular contouring quality assurance between radiation oncology departments. Intra‐ and inter‐observer variation in TV delineation has been shown to be reduced by implementing a departmental, national or international protocol.The limitations of this single institution study include small sample size, loss of follow‐up of one original participating RO and the absence of a control or gold standard group. The absence of a gold standard contour makes it impossible to make conclusions about the absolute accuracy of contours. The choice of this gold standard or reference contour varies in the literature from a mathematical average contour, an RO‐ or radiologist‐defined contour, or a consensus contour that is decided upon by a panel of experts.Possible follow‐up studies in this domain could assess the significance between RO prostate contouring consistency and the number of localised prostate cancers treated per year. Furthermore, studies randomising ROs to an EI versus no EI, assessing contouring consistency in other tumour sites and assessing the required frequency of EIs to maximise knowledge retention over time may lead to further gains in contouring quality assurance.Contour variation impacts the accuracy of RT as significantly as organ motion and setup variability. Novel methods to improve contouring consistency should be rigorously pursued to match recent technological advancements in RT delivery. This contouring audit has shown that 12 months following a structured EI consisting of anatomy tutorials, contouring modules and peer review, adequate retention of knowledge and subsequent improvement in contouring consistency was demonstrated in a small cohort of ROs treating localised prostate cancer. The improvements in contour consistency achieved herein advocate for EIs to be included as part of continuing medical education for ROs treating localised prostate cancer. Further studies are required to further define the frequency with which such EI should be incorporated into clinical practice.The authors declare no conflict of interest.


Almost the entire seafloor is covered with sediments that can be more than 10 000 m thick and represent a vast microbial ecosystem that is a major component of Earth's element and energy cycles. Notably, a significant proportion of microbial life in marine sediments can exploit energy conserved during transformations of sulfur compounds among different redox states. Sulfur cycling, which is primarily driven by sulfate reduction, is tightly interwoven with other important element cycles  and therefore has profound implications for both cellular‐ and ecosystem‐level processes. Sulfur‐transforming microorganisms have evolved diverse genetic, metabolic, and in some cases, peculiar phenotypic features to fill an array of ecological niches in marine sediments. Here, we review recent and selected findings on the microbial guilds that are involved in the transformation of different sulfur compounds in marine sediments and emphasise how these are interlinked and have a major influence on ecology and biogeochemistry in the seafloor. Extraordinary discoveries have increased our knowledge on microbial sulfur cycling, mainly in sulfate‐rich surface sediments, yet many questions remain regarding how sulfur redox processes may sustain the deep‐subsurface biosphere and the impact of organic sulfur compounds on the marine sulfur cycle. Cycling between the most oxidised  and most reduced  states of sulfur involves several inorganic sulfur compounds of intermediate oxidation states  that allow for (i) sulfur metabolite handoff to other microorganisms and (ii) shortcuts in the sulfur cycle Fig.  Jørgens. The extIn this review, we highlight recent and selected findings in marine sediment sulfur cycle microbiology and address knowledge gaps that might become emphases for future research. The review especially aims to tie‐together a perspective on various microbial ecological aspects and on biogeochemical impacts of several key aspects of the sulfur cycle in marine sediments. In some points where little is known about the associated microbial ecology, we point‐out the biogeochemical aspects that suggest their probable key influences on associated microbial ecology. The review is primarily restricted to dissimilatory metabolisms used for energy conservation.et al., et al., et al., in situ temperatures are the key determinants of SRM community structure in sediment zones with sufficient sulfate , adenylyl‐sulfate reductase (Apr), and dissimilatory (bi)sulfite reductase (Dsr) Table  [for a city Fig.   . However, Desulfobacteraceae represent just the iceberg tip of the SRM taxon diversity in marine sediments.Prototypical of SRM in general, marine Desulfobulbaceae and Syntrophobacteraceae, and a wide diversity of bacteria related to the genus Desulfatiglans, which likely represents a previously undescribed deltaproteobacterial family. More astonishing is the extraordinary, yet undescribed diversity of SRM that is only recognised to exist from molecular ecology surveys of functional marker genes for SRM, i.e. aprBA and dsrAB  are almost exclusively found in marine ecosystems  of a member of the phylum Gemmatimonadetes that was recovered from a sulfate‐rich estuarine sediment layer sulfite to sulfide, was solved by showing that the DsrC protein is a co‐substrate for DsrAB  or with organic matter after sulfurization reactions  in diverse benthic habitats  of the gammaproteobacterial family Beggiatoaceae, the motile hosts can position themselves in opposing gradients of oxygen and sulfide or shuttle themselves between oxic/suboxic and sulfidic layers. This lifestyle has developed independently and multiple times between alpha‐ and gammaproteobacterial thioautotrophs and sediment‐dwelling ciliates, oligochaetes, nematodes, flatworms and bivalves [for review see  , was discovered. In this process, multicellular filamentous bacteria bridge the oxidation of sulfide in anoxic sediment layers replete with sulfide to the reduction of oxygen or nitrate in oxic surface sediments, thereby generating and mediating electric currents in marine sediments over centimetre distances Fig. . These sBeggiatoaceae, symbiotic SOM and cable bacteria. The unambiguous identification of sulfur oxidation potential in an environmental sample is obscured by the fact that SOM have diverse and complex sulfur oxidation pathways. There is currently no clearly discernible, molecular marker that is universal among all SOM. Sulfide‐quinone oxidoreductase (Sqr) and the thioesterase subunit SoxB of the thiosulfate‐oxidising multienzyme complex (SOX‐pathway) appear to be the most widespread markers among known marine SOM  member from tidal sediments harboured the reverse DSR‐pathway   is disproportionated in oxidised or reduced sediment layers, respectively  and the deltaproteobacterial genera Desulfovibrio and Desulfomonile, the facultative anaerobic gammaproteobacterium Pantoea agglomerans, and few members of the phlya Thermodesulfobacteria and Firmicutes  appear to be specialised SCI disproportionators. This is somewhat surprising considering genome analyses of these organisms revealed genes encoding for the canonical sulfate reduction pathway, although they cannot perform sulfate reduction  and ‘sulfated’ molecules i.e. sulfate esters/‐O‐sulfonates (R‐O‐SO3–). Together, OSM represent the second most important reduced sulfur pool in marine sediment environments, accounting for up to 35–80% of the reduced sulfur  are desulfonating, polysaccharide‐degraders with a remarkable number (up to 110) sulfatase genes in their genomes, which appear to reflect a highly diverse substrate range   Fig. . OSM mayet al., et al., et al., et al., Planctomycetes and Bacteroidetes genomes from tidal flat sediments (Baker et al., Indications for the ecological significance of OSM in marine environments is reflected in the genomes of microorganisms, whereby genes encoding enzymes for desulfonation of organics to enable further catabolism of the substrates appear to be common in pelagic and benthic marine bacteria (Glöckner et al., et al., Recent research is beginning to shed light on the importance of the diagenetic sulfurization of organics by reactive sulfides and other SCI produced by sulfur‐cycling microorganisms. Although not directly related to microbial physiology, it may have a large yet overlooked influence on ecology and biogeochemistry. Evidence from recent experiments with humic acid‐like substances at lower pH has shown reactions of sulfide with organic matter may be on par with re‐oxidation with oxygen or iron oxides (Yu et al., Contemporary research has revealed that remarkably diverse microorganisms have evolved to fulfil an array of niches in marine sediments by transforming sulfur compounds among various redox states Fig. . It can


Nicrophorus vespilloides burying beetles , as well as the role of individual body size, in responsiveness to changes in social environment. The results show that body size influences the response of individuals’ signaling behavior to changes in the social environment. Moreover, there was G × E underlying the responses of males to variation in the quality of social environment experienced . This shows that individual variation in plasticity and social sensitivity of signaling behavior can evolve in response to selection on investment in mating behavior, with males selected for high mating investment having greater social sensitivity.Phenotypic plasticity is important in the evolution of traits and facilitates adaptation to rapid environmental changes. However, variation in plasticity at the individual level, and the heritable basis underlying this plasticity is rarely quantified for behavioral traits. Alternative behavioral reproductive tactics are key components of mating systems but are not often considered within a phenotypic plasticity framework . Here, using lines artificially selected for repeated mating rate, we test for genetic (G × E) sources of variation in reproductive behavior of male  Phenotypic plasticity, the environmentally sensitive production of alternative phenotypes by given genotypes  experienced by the individual  as a model. Male burying beetles actively compete with each other for access to breeding resources , and when they find a suitable carcass will emit pheromones as a signal to attract females. Emission of pheromones, as well as odors from the carcass, may also attract other males. As a consequence, intrasexual competition is common and usually leads to the establishment of a dominant male and female pair, or “resource holders,” who process the carcass and use it for breeding (Pukowski In the current study we test for genetic (G × E) sources of variation in reproductive behavior  in response to changes in the social environment experienced by individuals, using burying beetles .Relative body size would mediate this response, with larger males investing more in resource‐holding behaviors than small males.There would be underlying genetic variation for this behavioral plasticity , with males selected for high mating rates more socially sensitive to variation in the social environment than males selected for low mating rates.We predicted thatN = 160 in total), but mortality in either focal or competitor males prior to observations resulted in slightly reduced sample sizes . We did not use males from the control selection lines in our experiment because we were explicitly interested in how divergence in mating rate influences reproductive tactics. Prior to behavioral observations, all males were kept in individual containers (clear plastic container: 7 × 7 × 4 cm) filled with 2 cm of moist soil, in conditions identical to those experienced during selection. Competitor males were taken from a wild‐derived stock population, which had been maintained in the laboratory for two generations. These males were also housed individually before being used in the experiment. Prior to being added to experimental containers, we determined the mass  and size  of both focal and competitor males. Males were then placed in an experimental container, which consisted of a transparent plastic box (17 × 11 × 6 cm) containing 1 cm of moist soil and a 15–25 g freshly thawed mouse carcass. Competitor males were given a white mark on the top of their pronotum to facilitate male identification. Marking does not affect behavior  when by themselves and in the presence of a rival male (competitor). Two approaches were used to evaluate dominant males (resource holders) and subordinate males (satellite). First, we define the total amount of time that focal males spend on the carcass  as a proxy of “resource‐holding” behavior. General activity included signaling behavior, eating, self‐grooming, and walking around on the carcass  and general activity of each male was monitored by scan sampling every 10 min for 4 h prior to the onset of the scotophase, the time of maximal signaling effort in this species . This generated variation in the social environment experienced by focal males; from good  to poor . Previous work has shown that relative male size is the most important determinant of contest outcomes over carcasses  how does focal male behavior change in response to the arrival of a competitor? (2) Is this change sensitive to variation in the quality of the social environment experienced  and social environment  on the behavioral plasticity of the proportion of time spent active on the carcass (“resource defense” behavior) and signaling behavior using general linear mixed models with a Gaussian error structure and square root transformed response variables (GLMMs). Selection regime (high or low mating rate) and social environment (presence/absence of a competitor) were used as explanatory variables, with the interaction between the two included in models. Replicate nested within selection regime and male focal identity (ID) were included as random effects, and body size of focal males  was also included as a covariate (fixed effect). Response variables were (1) the proportion of time spent active  on the carcass, (2) the proportion of active time spent signaling on the carcass, and (3) the proportion of active time spent signaling off the carcass.In the second analysis, we examined the effect of the quality of the social environment experienced  and genetic background  on expression of signaling behavior. We used the relative size difference in pronotum width between focal and competitor males as the measure of competitive ability (E) . Results are presented with means and ±95% CIs obtained from the analyses. All analyses were performed using “R” version 3.0.2 .Focal males  spent a greater proportion of their time on the carcass, following the introduction of competitor males, than their rivals , competitor males: 0.72 ± 0.021 (CI); GLM with quasibinomial errors, χ22=5.21,P=0.073), indicating considerable variation among and within males in their response to a change in social environment  depended upon an interaction between selection regime (G) and social environment (E) and an interaction between the size of the focal male (P) and the social environment (E) Table . In the χ22=2.33,P=0.31), indicating high variation among and within males in their response to a change in social environment.As expected, when a competitor male was present signaling behavior both on‐ and off‐carcass changed compared to when there was no competitor present Fig. . ConsequWhen there was another male present, focal males spent slightly less of their time signaling on the carcass than when they were alone, but there was no significant main effect of selection regime on the proportion of time spent signaling on the carcass Table A, B, andFocal males also spent considerably less time signaling off the carcass (“satellite” behavior) when a competitor was present compared to when alone Fig. C, D, butDuring interactions with competitors, the proportion of time spent signaling on the carcass by focal males depended upon an interaction between selection regime (G) and the size of the focal male compared to that of his rival (R) Table : focal mA significant interaction between selection regime and relative competitive ability was also found for off‐carcass signaling behavior Table . HoweverN. vespilloides burying beetles are behaviorally plastic, primarily mediated by individual differences in body size among males, and there is genetic variation underlying this behavioral plasticity.Our experiment showed that both the proportion of time that males spent on the carcass and the proportion of time spent on signaling behaviors (on‐ and off‐carcass) was plastic in response to the introduction of a rival competitor. As predicted this behavioral plasticity in response to a change in social environment was mediated by body size, with larger males investing more time in resource‐holding behaviors  than small males. There was no G × E for plasticity of signaling behavior (either on‐ or off‐carcass) in response to the introduction of a rival male, although in both cases the effects were only marginally nonsignificant. However, there was a G × E for plasticity of time spent active on the carcass (resource‐holding behavior), with males from lines selected for high repeated mating rates increasing and males from low lines decreasing overall activity on the carcass in response to the introduction of a competitor. Moreover, there was a G × E underlying the responses of males to variation in the quality of the social environment that they experienced: males from lines selected for high mating rates did not differ from low line males in either the proportion of time spent signaling on‐ or off‐carcass, but they were more sensitive to variation in the quality of the social environment when signaling on the carcass and less sensitive when signaling off the carcass. Thus, the reproductive tactics of Population‐level plasticity depends upon individual‐level plasticity, but the latter cannot be inferred from determining plasticity at the level of the population (Nussey et al. N. vespilloides also depends upon developmental plasticity of individuals in response to variation in carcass size. This combination of current and past environments on the plasticity of behavior is thought to be common, but is rarely shown (Dingemanse and Wolf Our results confirm that relative competitive ability in burying beetles is largely determined by body size Otronen , which iAlong with variation in the size of the resource required for reproduction, the availability of carcasses in nature is stochastic, and varies temporally and spatially (Eggert and Müller N. vespilloides, and the costs involved in engaging in contests that are likely to be lost (Otronen Drosophila melanogaster mating duration of males depends on the level of competition experienced (Bretman et al. Drosophila, but rather than using multiple cues burying beetles appear to primarily rely on a single cue, relative size difference, to influence reproductive tactics.The responsiveness of males to variation in the social environment they experienced depended on genetic background, indicating that behavioral plasticity evolves as a correlated response to selection on mating behavior: We found that males showed context‐dependent social sensitivity in their signaling behavior in relation to their selection regime. This suggests that selection for increased investment in repeated mating rate, a paternity assurance trait Alcock , leads t Otronen , this ma Otronen . Such plN. vespilloides, and that this sensitivity is dependent upon variation in the genetic background of individuals. In doing so our experiments reveal social environment induced variation in the switch points and reaction norms of the expression of behavioral reproductive tactics in burying beetles: males selected for high investment in mating traits that protect paternity were more plastic in their behavioral response to variation in the quality of the social environment that they experienced when they were dominant than when subordinate. In contrast, males selected for low investment in mating traits were more plastic in behavior when subordinate. This shows that the plasticity and social sensitivity of behavioral reproductive tactics can evolve in response to selection on investment in mating traits: males that have more sex are more insecure.We provide novel evidence that alternative behavioral reproductive tactics are sensitive to social context in Supplementary MaterialClick here for additional data file.


Chronic dermal wounds are a devastating problem, which disproportionally affect individuals with conditions such as diabetes, paralysis, or simply old age. These wounds are extremely challenging to treat due to a heterogeneous combination of causative factors, creating a substantial burden on healthcare systems worldwide. Despite their large impact, there is currently a startling lack of options for effectively treating the underlying biological changes that occur within the wounds. Biomaterials possess an enticing ability to provide new comprehensive approaches to healing these devastating wounds; advanced wound dressings are now being developed that enable the ability to coordinate temporal delivery of multiple therapeutics, protect sensitive biologics from degradation, and provide supportive matrices that encourage the growth of tissue. This positions biomaterials as a potential “conductor” of wound repair, allowing them to simultaneously address numerous barriers to healing, and in turn providing a promising pathway to innovative new technologies for driving successful healing. Many times this resistance to treatment may stem from underlying biological changes that affect the ability of cells within the skin to properly carry out the process of tissue repair;[s9] cells isolated from DFUs have been shown to display differences in their response to growth factors,[s10] proliferation rates,[s11] composition of the extracellular matrix (ECM) they deposit,[s12] levels of proteases produced, and regulatory RNA molecules such as microRNAs.[s15]Despite the large impact of these wounds, there are surprisingly few options available for treating them that take advantage of recent insights from the laboratory. While conventional strategies such as debridement, negative pressure therapy, and offloading orthotics can lead to successful wound closure in many patients, Significant emphasis has been put on the development of bioengineered skin replacements, such as decellularised matrices, living skin constructs and degradable polymeric scaffolds that mimic multiple features of the native ECM and allow modulation of the matrix within the wound bed.[s25‐s28] Such scaffolds, whether natural or synthetic, have porous microstructures tagged with cell‐binding sites to support the infiltration and adhesion of cells, allow diffusion of nutrients and metabolites, have controllable degradation mechanisms, and can act as reservoirs to retain bioactive agents that stimulate innate tissue repair mechanisms , sprays (e.g. trafermin) and injections (e.g. Heberprot‐P) are unlikely to provide the control necessary to fully exploit this strategy. Using these methods does not enable the ability to easily tune the spatiotemporal delivery pattern, resulting in large initial bolus releases and high‐dosage deliveries that may lead to serious side effects, while also requiring a high level of patient compliance that many times presents a significant challenge when treating chronic wounds.[s33] process. Experiments in db/db mice demonstrated increased levels of both angiogenesis and formation of granulation tissue with the dressings. This was accomplished using a total growth factor dose of approximately 150 ng, an amount 300 times lower than previously used for the FDA‐approved becaplermin gel.[s34] Further to this, we demonstrated that the LbL technique is able to create wound dressings that facilitate localised release of siRNA targeting MMP‐9 (siMMP‐9) in db/db mice.To overcome some of these barriers, we have recently developed a self‐assembled coating for wound dressings that enables sustained release of multiple active growth factors while allowing independent modulation of their individual release kinetics.While these two examples demonstrate the flexibility of a technique such as LbL, there are many other strategies that have been developed to enable efficient delivery of biologics. For instance, recent research has demonstrated that sustained siRNA delivery to skin wounds is possible by packaging siRNA molecules within pH‐responsive polymer nanoparticles composed of 2‐dimethylaminoethyl methacrylate, which are then incorporated in biodegradable injectable polyurethane scaffolds.3[s35] In other research, it has been shown that persistent PKCδ expression inhibits insulin signalling in fibroblasts from type 1 diabetic wounds, with siRNA knockdown of PKCδ resulting in increased levels of p‐AKT, VEGF expression and rate of wound healing in vivo.[s36] Finally, sustained release of stromal cell‐derived factor‐1 (SDF‐1) from an antioxidant thermoresponsive hydrogel has been shown to significantly enhance formation of granulation tissue and epithelial maturation in non‐healing DFUs.[s37]There are many interesting biological targets that need further exploration and validation in the context of chronic wound healing, as exciting new developments are constantly being made in this area. Recent findings have elucidated an important role of Ephrin‐B1 in regulating epithelial cell‐cell junctions during re‐epithelialisation, with clinical non‐healing chronic wounds displaying strong upregulation.[s38] or potentially by using non‐invasive imaging of wounds[s39] to identify wounds that are most likely to benefit from certain treatment strategies, is crucial for maximising the success of advanced therapies.How these new insights can be integrated into a comprehensive therapeutic strategy that builds on extensive prior findings is still unknown; such a strategy will likely require a combination of proteins, nucleic acids, cells, and polymeric scaffolds that act together in a concerted manner to synergistically rewire the local wound environment to drive healing. This is where biomaterials enter the picture as an enabling technology that can simultaneously address differential delivery needs for each therapeutic , optimise dosing schedules, and provide a supportive extracellular matrix for the wound microenvironment. These strategies for treating chronic wounds will then likely need to be paired with the development of various biomarkers that can effectively stratify patient populations into treatable cohorts. For instance, patient stratification based on macrophage gene expression,[s40] ideally, biodegradable hydrogels will create no detrimental degradation products while promoting quality tissue integration and triggering minimal immunogenicity.However, despite these stimulating advances in the field of biomaterials for wound healing applications, hurdles that still need to be addressed include the manufacturability of the materials as the level of complexity increases, along with the toxicity and biocompatibility of these materials with the host tissue. Care must be taken when designing degradable hydrogels for tissue repair, as these gels can form toxic degradation products . Such products have the ability to raise the local acidity of the surrounding tissue and trigger a pronounced inflammatory response over time;[s41‐s51] and progress is being made on our ability to design materials that interact with biology over time in a bidirectional way. With help from regulatory bodies to ease advancement of promising combination therapies for a medical condition with worse survival rates than some cancers, we are optimistic that new interventions will soon be developed that will have a meaningful effect on reducing the devastating impact chronic wounds have on society.Nevertheless, we are excited by the fact that our understanding of tissue‐biomaterial interplay is growing, insightful new discoveries into the biology of wound healing and chronic wounds are being continually uncovered around the world,The authors have declared no conflicting interests.All authors contributed to the conception and writing of this article.Data S1 Supplementary References.Click here for additional data file.