Evolution of sexual dimorphism in ecologically relevant traits, for example, via resource competition between the sexes, is traditionally envisioned to stall the progress of adaptive radiation. An alternative view is that evolution of ecological sexual dimorphism could in fact play an important positive role by facilitating sex‐specific adaptation. How competition‐driven disruptive selection, ecological sexual dimorphism, and speciation interact during real adaptive radiations is thus a critical and open empirical question. Here, we examine the relationships between these three processes in a clade of salamanders that has recently radiated into divergent niches associated with an aquatic life cycle. We find that morphological divergence between the sexes has occurred in a combination of head shape traits that are under disruptive natural selection within breeding ponds, while divergence among species means has occurred independently of this disruptive selection. Further, we find that adaptation to aquatic life is associated with increased sexual dimorphism across taxa, consistent with the hypothesis of clade‐wide character displacement between the sexes. Our results suggest the evolution of ecological sexual dimorphism may play a key role in niche divergence among nascent species and demonstrate that ecological sexual dimorphism and ecological speciation can and do evolve concurrently in the early stages of adaptive radiation. This prediction can be made on the basis of biomechanical trade‐offs between foraging in aquatic  versus terrestrial (via tongue prohesion) habitat , such a comparison is mathematically equivalent and conceptually related to among‐group comparisons of standardized linear selection gradients β , as β can be defined as the vector of canonical coefficients defining the population before and after selection  and the off‐diagonals describe the among‐taxa covariance between standardized coefficients. Thus, S is a covariance matrix whose first eigenvector, smax, describes the direction through trait space where species have diverged the most in multivariate sexual dimorphism . In total, our final dataset consisted of measurements from 477 specimens from males and females of all species and subspecies in the genus, although we were unable to obtain enough data for separate analysis of the two subspecies of N. meridionalis. Summary statistics of all measurements taken, and sample sizes are given in Table p > .09) or the pooled data (p = .49). Distributions for each trait*sex*taxon combination are summarized in Figure We examined and measured specimens of 2.3S and D matrices, which were used for geometric comparisons with selection that are most consistent with theory , and all two‐ and three‐way interactions as fixed effects. We included trait as an R‐side (repeated measures) random effect with individual as the subject, modelling a separate phenotypic covariance matrix for each taxon. This residual covariance structure fit better than a common phenotypic covariance (∆AIC = 59.66). To compare trait‐specific effects, we also present univariate mixed‐model analyses. These models included sex, taxon, and their interaction as fixed effects, and the taxon‐specific residual variance as a random effect. We performed univariate tests on both the raw trait values and size‐corrected head traits  to illustrate patterns in the data that are independent of size; note that multivariate analyses can accommodate such size‐independent effects because the covariance structure of the data is explicitly modeled.To assess whether sexual dimorphism exists across the genus and differed across taxa, we fit a multivariate mixed model with trait values as the response vector, and trait type, sex, taxon .We repeated our multivariate analysis, but limited to subspecies of D variances by also modelling an R‐side random effect of trait type with individual nested within taxon as the subject. For this analysis, we standardized traits globally  to zero mean and unit variance, in order to make meaningful comparison with estimates of variance‐standardized within‐taxa nonlinear selection, where within‐population data were standardized in the same way (see below). To estimate S, we fit a multivariate mixed model with the vector of total sample standardized canonical coefficients (estimated and standardized separately for each taxon) as the response and trait type as a fixed effect to center the data. We fit trait type as a G‐side random effect with taxon as the subject to estimate S. To accommodate uncertainty in our estimates of the canonical coefficients, we analyzed bootstrapped data, where we resampled (100x) our data and each time estimated c for each taxon, and then analyzed the set of bootstrapped c estimates treating bootstrap replicate as an R‐side (repeated measures) random effect with replicate nested within taxa as the subject. Although in theory it would be possible to fit one mixed model where D is estimated as the covariance in random intercepts among taxa, while S is the covariance in the sex effect among taxa, fitting this model entails either estimating excessive (and likely meaningless) parameters  or fitting two random terms to the same set of subjects, which failed to converge.We estimated the among taxa divergence matrix, x Lande, ), by fitsmax) to describe divergence between the sexes.Our approach to data standardization aimed to be as consistent as possible across analyses; in no case did we standardize separately for males and females, as this was not performed in past work  smaxTγmax and dmaxTγmax, where T is matrix transpose and γmax is the first eigenvector of the γ matrix of nonlinear standardized selection gradients from the high density experiment in  and S (88%) was captured in one dimension.We calculated the association between divergence in sexual dimorphism, D and S in the Appendix γmax remained qualitatively equivalent and quantitatively similar.In some cases, appropriate tests of hypotheses that relate microevolutionary parameters to among‐taxa, macroevolutionary divergence require the incorporation of quantitative information on phylogenetic relatedness. For example, tests of evolution by drift, along genetic lines of least resistance, or evolution toward an optimum phenotype are most appropriately performed by parameterizing a family of Brownian motion process models of evolution Hansen, . AlthougF tests for these effects , with the exception of supplementary analysis performed in R. Mixed models were fit by REML in the glimmix procedure. All random effect covariance matrices were modeled as the Cholesky parameterization of an unstructured covariance matrix to insure positive semidefiniteness. We used the Kenwood–Roger approximation for degrees of freedom for models with fixed effects, and report type III Wald 3Notophthalmus , and sexual dimorphism differed significantly across species and subspecies . Univariate analyses reflect these results . Further, although too little is known about the natural histories of N. meridionalis and N. perstriatus to rank their life histories relative to N. viridescens, it is noteworthy that N. perstriatus, which has been reported to frequently remain aquatic for much of its life  and the small sample of fully paedomorphic N. perstriatus exhibited the strongest magnitude of sexual dimorphism , although this difference in sexual dimorphism between forms of N. perstriatus was not statistically significant .Across subspecies of dmax, occurred nearly orthogonally to the within subspecies direction of maximum disruptive selection, γmax . Current and past work in Notophthalmus thus suggests the following scenario for the evolution sexual dimorphism during probable allopatric ecological speciation. First, ecological divergence during speciation appears to reflect adaptive changes in the life history in response to geographic variation in pond hydroperiod and terrestrial habitat quality; body size divergence during speciation likely reflects divergent size optima associated with life histories characterized by complete ontogenetic niche shifts vs a more aquatic life cycle  aquatic predators that share the same feeding apparatus and inhabit standing water bodies (Petranka, We have interpreted our results predicated on the possibility that resource competition may be a perennial driver of disruptive selection for aquatic adult Our work also adds to a small but growing number of empirical studies that have related properties of the adaptive landscape, as inferred from within‐population selective surfaces, to macroevolutionary patterns of phenotypic divergence (Chenoweth, Rundle, & Blows, Despite theoretical interest, ecological causes of dimorphism and corresponding effects on adaptive radiation have received limited empirical attention. This is especially true when considered in comparison with sexual selection (Panhuis, Butlin, Zuk, & Tregenza, None declared. Click here for additional data file.


Glioblastoma multiforme is the most common and aggressive primary brain tumor, with an extremely poor prognosis. The lack of detailed knowledge about the cellular and molecular mechanisms involved in glioblastoma development restricts the design of efficient therapies. A recent study using state‐of‐art technologies explores the role of pericytes in the glioblastoma microenvironment. Glioblastoma‐activated pericytes develop an immunosuppressive phenotype, reducing T‐cell activation through the induction of an anti‐inflammatory response. Strikingly, pericytes support glioblastoma growth in vitro and in vivo. Here, we describe succinctly the results and implications of the findings reported in pericytes' and glioblastomas' biology. The emerging knowledge from this study will be essential for the treatment of brain tumors. Glioblastoma multiform is the most common brain tumor in humans with very aggressive progression Pericytes were defined, more than a century ago, as a population of contractile cells with long projections encircling the blood vessel walls Oncotarget, Valdor and colleagues show that the conditioning by brain tumor cells induces immunosuppression by pericytes β, while they do not produce proinflammatory cytokines, such as IL‐1, IL‐23, and IL12, which could be produced in other conditions by brain pericytes Interestingly, immune regulation also depends of pericytes. The reader is referred to excellent reviews that discuss these pericytes roles in detail Strikingly, pericytes promoted glioblastoma growth in vitro and in vivo. This work provides a novel possible central cellular population to be pharmacologically targeted in patients with brain tumors. Here, we discuss the findings from this study and evaluate recent advances in our understanding of the roles of pericytes in the glioblastoma microenvironment Fig. .β in brain pericytes on the glioblastoma development. Moreover, the generation of IL10‐ or TGFβ‐floxed mice to be crossed with pericyte‐specific inducible CreER driver, such as NG2‐CreERT2 The conclusions from this study claiming the immunosuppressive role of pericytes in the glioblastoma microenvironment are based on observations made in animals that received transplants of pericytes previously propagated in culture, or on experiments performed utilizing pericytes grown in vitro. Note that artificial conditions and high concentration of mitogens that characterize cell culture systems may induce some characteristics in pericytes that may not be shared by the corresponding endogenous brain pericytes in vivo under pathophysiological conditions Valdor and colleagues used a xenografted mouse model of glioblastoma, in which immunocompetent mice received human glioblastoma cancer cells α smooth muscle actin, and glutamate aspartate transporter (Glast) Valdor and colleagues examine pericytes as a homogeneous cell population in their work. Nevertheless, pericytes have been shown to be heterogeneous regarding their phenotype, distribution, origin, marker expression, and function A central nervous system pericyte subpopulation have been recently shown to take part in the formation of the scar tissue after brain lesion, which is a major obstacle to neuronal regeneration in patients with this condition β and NG2, can be expressed in other cell types in certain pathophysiological settings. For instance, PDGFRβ is a known marker of fibroblasts in the central nervous system The method of pericyte isolation used by Valdor et al. (2017) could result in the presence of other perivascular cells in these cultures. To avoid this, isolation by sorting based on several molecular membrane markers would be preferred. Although pericytes are defined by their anatomical perivascular position, not all perivascular cells are pericytes. Several cells that share molecular markers with pericytes have been described as perivascular: that is macrophages Pericytes' capacity to form several cell types is well known; the general consensus holds that pericytes behave as stem cells under certain conditions βIn addition to functioning as stem cells, pericytes can also regulate the behavior of other stem cells, being an important component of stem cell niches in several organs The study by Valdor and colleagues reveals a novel important role of pericytes in the glioblastoma microenvironment. Nevertheless, our understanding of pericytes biology in the brain still remains limited, and the complexity and interactions of different cellular components of the brain microenvironment during glioblastoma progression should be elucidated in future studies. An enormous challenge for the future will be to translate the research from experimental models into humans. Whether tumor cells during human cancer development promote the same immunosuppressive phenotype in human pericytes in the brain remains to be determined. Improving the availability of human glioblastoma samples will be essential to reach this objective.The authors indicate no potential conflict of interests.


HIV prevention interventions is a core component of the UNAIDS Fast Track strategy to end the HIV epidemic by 2030. Central to these plans is a focus on high‐prevalence geographies, in a bid to target resources to those in greatest need and maximize the reduction in new infections. Whilst this idea of geographical prioritization has the potential to improve efficiency, it is unclear how it should be implemented in practice. There are a range of prevention interventions which can be applied differentially across risk groups and locations, making allocation decisions complex. Here, we use mathematical modelling to compare the impact (infections averted) of a number of different approaches to the implementation of geographical prioritization of prevention interventions, similar to those emerging in policy and practice, across a range of prevention budgets.A strategic approach to the application of We use geographically specific mathematical models of the epidemic and response in 48 counties and major cities of Kenya to project the impact of the different geographical prioritization approaches. We compare the geographical allocation strategies with a nationally uniform approach under which the same interventions must be applied across all modelled locations.We find that the most extreme geographical prioritization strategy, which focuses resources exclusively to high‐prevalence locations, may substantially restrict impact (41% fewer infections averted) compared to a nationally uniform approach, as opportunities for highly effective interventions for high‐risk populations in lower‐prevalence areas are missed. Other geographical allocation approaches, which intensify efforts in higher‐prevalence areas whilst maintaining a minimum package of cost‐effective interventions everywhere, consistently improve impact at all budget levels. Such strategies balance the need for greater investment in locations with the largest epidemics whilst ensuring higher‐risk groups in lower‐priority locations are provided with cost‐effective interventions.HIV prevention.Our findings serve as a warning to not be too selective in the application of prevention strategies. Further research is needed to understand how decision‐makers can find the right balance between the choice of interventions, focus on high‐risk populations, and geographical targeting to ensure the greatest impact of  The most established approach for the differential allocation of intervention funds across regions is a “formula funding” strategy, whereby resources are divided between geographical areas in proportion to key indicators in each region (such as the number of PLHIV (people living with HIV), the number of people on treatment, or broader health systems, demographic or development measures) With so many examples of geographically focused policies, it is critical that their respective approaches be rigorously evaluated. Here, we use a geographically specific model of HIV transmission of Kenya 22.1This work utilizes county‐specific models of heterosexual and male‐to‐male transmission of HIV developed for Kenya with models tailored to reflect the differences in the epidemic and response across locations 2.2We sought to understand how “geographical prioritization” of HIV prevention can be applied with generalizable allocation policies and whether such strategies may improve the impact of prevention programmes. We compare different approaches to the allocation of interventions across modelled locations , behaviour change (BC), PrEP (pre‐exposure prophylaxis) and VMMC, which can be targeted by population group , low‐risk women and FSW ). In all of the modelled strategies, we assume that treatment is available to everyone  with initiation at an average CD4 cell count of 200 cells per microlitre, corresponding to those actively seeking treatment. The “accelerated access to ART” prevention intervention is in addition to this background “late ART,” and represents active outreach to those with higher CD4 counts.2.3Key features of the different approaches to the allocation of interventions across locations are outlined in Table 2.3.1Previously, we developed an optimized “geographically focused” strategy for the design of combination prevention programmes, which strategically allocates resources across populations and geographies to maximize impact for a given budget The “formula‐based” approach is also able to allocate interventions individually across locations, but utilizes a predefined order of intervention roll out , although intervention is consistently most intense in high‐prevalence areas. Whilst even low‐prevalence areas get the minimum intervention package, roll out of intervention strategies is always greatest in the higher‐prevalence locations.We compare these geographical prioritization strategies with a uniform approach where all modelled locations must receive the same set of interventions and there is no tailoring of intervention choices across locations.We assess the percentage difference in impact  between each geographical strategy and the uniform strategy at a range of budget levels .2.4As we seek to provide a simplified representation of the policy choices, for the extreme geographical prioritization and staggered implementation approaches, we limit the number of locations across which we allocate interventions through grouping modelled counties into “very high,” “high,” “medium” and “low” prevalence categories using k‐means clustering.In order to standardize the comparison between the allocation strategies, a predefined order of intervention roll out was defined for the simple geographical strategies  and uniform approach. We consider four different intervention modalities  which can be applied to high‐risk populations (FSW and MSM) or low‐risk populations  only, or to men or women only. The relative priority of the different interventions across population groups is given in Table 3We first examine the order of roll out of interventions across modelled locations under each of the strategies Figure . Each pa3.1The optimized geographically focused Figure a approacThe formula‐based Figure b approacThe extreme geographical prioritization strategy Figure c concentUnder the staggered implementation approach Figure d, even lUnder the uniform approach Figure e, all lo3.2The strategies vary substantially in how effectively they avert infections Figure , with coThe optimized geographically focused approach has complete flexibility in allocation and finds the optimal order of roll out with the greatest impact at all levels of spending (as much as 19% more of future infections averted relative to the uniform approach under the budget levels considered).The staggered implementation strategy performed consistently stronger than the uniform approach across all budget levels, with up to 10% more infections are averted for the same budget at moderate levels of prevention spending. This demonstrates that higher‐intensity implementation in the high‐priority locations is beneficial whilst maintaining a minimum package of interventions across all locations. It is unable to perform as strongly as the original optimized geographically focused approach as it is unable to be as targeted, with the order of roll out of interventions fixed for all locations.The formula‐based approach is able to generate marginal gains over the uniform approach (up to 5% under the budget levels considered). The number of PLHIV, although a useful indication of epidemic intensity, is not a direct measure of prevention need or the effect these can have on local dynamics, and so it is unable to perform as strongly.The extreme geographical prioritization strategy performs substantially less well than the uniform strategy, and may lead to less health impact than if interventions are applied uniformly across the country. The loss of impact could be much as 41% at moderate budget levels. The “jumps” in impact observed across budgets represent the change from a low‐priority intervention strategy in a higher‐prevalence category, to a higher priority intervention in a lower‐prevalence category , is found to be the least impactful at country level. Our analysis shows that limiting investment to those locations with the most intense epidemics in this way may significantly reduce impact (41% fewer infections averted at moderate budget levels) compared to a nationally uniform approach with no geographical prioritization. This strategy misses the opportunity to avert infections across the remainder of locations in the country, particularly in high‐risk populations, as it devotes resources to lower‐priority interventions in a small number of the highest priority locations. Such extreme geographical prioritization therefore restricts the number of infections it is possible to avert. Furthermore, such a strategy will have important ethical and political consequences. Often lower‐prevalence areas are rural and underserved, and require greater investment to maintain basic services. The absence of services may give the misleading signal that they are not required, even though, by international standards, HIV remains a significant public health problem.Provisions must therefore be made to ensure that essential highly cost‐effective prevention services are maintained across all settings. Country governments must ensure areas without donor funds still receive sufficient support for prevention services. Similar to that presented in the staggered implementation approach here, a “minimum package” of the most cost‐effective interventions is needed across all settings. In this way, some interventions are so favourable they are exempt from geographical prioritization, including VMMC, which as a cheap, highly effective and lifelong intervention, is one of the most cost‐effective prevention interventions available at this time. In those settings which have not yet reached VMMC targets, further expansion should be an immediate priority. Further research is needed to explore barriers to achieving target coverage for VMMC, particularly understanding factors influencing demand for VMMC services across populations. The staggered implementation strategy has many similarities to the cluster‐based approaches of India and Kenya Similarly, the formula‐based approach, through dividing funds proportional to the number of PLHIV in each location, ensures that most locations receive some essential services. Whilst this approach considers only one indicator (PLHIV), other formula‐based approaches may use multiple indicators, often not specific to HIV funding or the healthcare sector, undermining the ability to allocate funds based on the heterogeneity of the epidemic. Even with simple HIV indicators including the number of PLHIV, such approaches do not fully capture the differential opportunities for intervention.To fully maximize the impact of “geographical prioritization,” we require an understanding of the patterns of transmission between risk groups in each setting to find the optimal choice of intervention by population and location. A number of modelling tools (including Optima A number of extensions to this analysis could be explored. Examination of the impact of these strategies under different prevalence category definitions, target coverage levels or order of intervention roll out may allow for greater impact. Further work could also look at different epidemic contexts, at national and international level. A number of other factors not included in this study will influence the outcome of geographical targeting. The cost of providing services will differ between locations, as is the case for providing treatment to PLHIV 5Geographical prioritization is an important way of designing more effective and targeted prevention programmes but must be applied with caution. If funding is withdrawn from lower‐priority locations, it is critical that priority interventions are still maintained in these settings. For greatest impact, there needs to be a balance between choosing highly cost‐effective intervention modalities and strategic targeting to both priority populations and locations.SJA reports personal fees from The Bill & Melinda Gates Foundation, personal fees from Avenir Health, personal fees from Anansi Health and personal fees from the Global Fund outside the submitted work. TBH received grants and personal fees from the Bill & Melinda Gates Foundation during the conduct of the study; grants and personal fees from World Bank; grants from UNAIDS and The Rush Foundation; and personal fees from the University of Washington, New York University and Global Fund outside of the submitted work. For the remaining authors, none were declared.SJA and TBH conceived the study and developed the methods and analysis. SJA wrote the first draft of the manuscript. PDG and RO advised on the interpretation of the analysis. All authors contributed to the writing of the manuscript and reviewed and approved the final version.Funding for this study was provided by the Bill & Melinda Gates Foundation OPP1084364 through a grant for the HIV Modelling Consortium to Imperial College London.Data S1. Mathematical models.Click here for additional data file.


RNA sequencing provides a way forward, as it allows elucidation at a genome‐wide and quantitative level of the transcriptional diversity occurring within the same cell types at different anatomical positions and levels of arterio‐venous hierarchy in the organs. In this Louis‐Jeantet Prize Winner: Commentary, I give a brief overview of vascular development and how recent advances in the field pave the way for more systematic efforts to explore vascular functions in health and disease.The blood vasculature is an organ pervading all other organs . During vascular development, cell–cell signaling by extracellular ligands and cell surface receptors ensure that new vessels sprout into non‐vascularized regions and simultaneously acquire organ‐specific specializations and adaptations that match the local physiological needs. The vessels thereby specialize in their permeability, molecular transport between blood and tissue, and ability to regulate blood flow on demand. Over the past decades, we have learnt about the generic cell–cell signaling mechanisms governing angiogenic sprouting, mural cell recruitment, and vascular remodeling, and we have obtained the first insights into signals that induce and maintain vascular organotypicity. However, intra‐organ vascular diversity and arterio‐venous hierarchies complicate the molecular characterization of the vasculature's cellular building blocks. Single‐cell  A pressurized cardiovascular system is imperative for the transport of oxygen, nutrients, metabolites, and blood cells in organisms throughout the vertebrate kingdom. The vasculature is of fundamental importance for the body's physiological functions, but it is also required for embryonic development. For some organs, development and function are temporally separated, and the embryo may live and continue to grow in situations where the organ is malformed, or even missing. The vasculature, however, must develop and function simultaneously. This requires precisely coordinated cellular behaviors that shape and reshape the vessel tree while at the same time assuming new, and maintaining already established, vascular functions.The problem is amply illustrated by studies of mouse gene knockout mutants in which embryonic vascular developmental defects cause general circulatory dysfunction followed by rapid death and disintegration of the embryo. The strict dependence of organism development on vascular function makes the study of embryonic vascular development challenging; it is notoriously difficult to distinguish primary effects of the genes and proteins of interest from the secondary systemic consequences of vascular dysfunction. In this perspective, zebrafish has become a valuable complement to mammals as animal models for the study of vascular development, since part of the oxygenation of the fish larvae takes place across the body surface, allowing at least some aspects of the vasculature to develop even in the absence of a functional circulation. The transparency of zebrafish larvae has also made it possible to image vascular development in real time and at high resolution, something that is still technically very challenging in mammals.Vascular organotypicity—the adaptation and specialization of the microvasculature to match every organ's specific physiological functions—adds a further dimension of complexity to vascular development. Organotypic vascular specialization generates vast differences in functions such as permeability and molecular transport across the vessel wall. Extreme examples of this specialization include the largely impermeable vasculature of the brain—the blood–brain barrier (BBB)—and the highly permeable, yet size‐selective, capillaries of the kidney glomerulus. All organs have their unique microvasculature, reflected in differences in gene expression patterns and functions, but the details of these differences are only beginning to emerge. The development, maintenance, and derangement of vascular organotypicity are gaining increasing attention in cardiovascular medicine. The importance of these processes is underscored by the vascular contributions to disease processes in several organs, including the brain and the kidney, in metabolic disorders and in cancer.vasculogenesis—the differentiation of endothelial cells and their assembly into tubular networks, and angiogenesis, the subsequent growth, sprouting, and remodeling of the vascular tubes. It might be appropriate to dub “organotypogenesis” the third—and final—process in vascular development. All three stages require cell–cell communication, involving numerous different signals, emanating from cell‐bound (juxtacrine), as well as paracrine  or endocrine (blood‐borne) ligands, the most famous of which is vascular endothelial growth factor (VEGF‐A). Intriguingly, VEGF‐A plays a role in all three stages of vascular development. VEGF‐A's effects on endothelial cells are context‐dependent, and the molecule is a clinical drug target in cancer and eye diseases  is also the major cell surface receptor for VEGF‐A. However, the process of vascular cell differentiation from undifferentiated mesenchyme probably continues also after a functional circulatory system has formed, and hence, vasculogenic and angiogenic mechanisms are likely not temporally separate processes.et al, et al, VEGF‐A is equally the major regulator of angiogenic sprouting. VEGF‐A expression is under strong transcriptional control of environmental influences, one of which is hypoxia , which occupy small and large vessels, respectively. Pericytes and VSMC are presumed to be related and to have a common ontogeny. However, the details of mural cell development and phenotypic diversity remain largely unexplored.et al, et al, During angiogenesis, platelet‐derived growth factor‐B (PDGF‐B) is released from endothelial cells for paracrine signaling through PDGF receptor‐β (PDGF‐Rβ) on pericytes, stimulating their proliferation and recruitment along the angiogenic sprouts  and increased vascular leakage resulting in edema  vessels form in the absence of pericytes and vascular arterio‐venous hierarchies are established, but the capillaries fail to mature normally. Studies of both knockout and hypomorphic et al, The molecular basis for vascular organotypogenesis and the cellular crosstalk required for this differentiation remain unknown with a few exception  and tubular systems (re‐absorption), is an obvious example, but most organs likely display micro‐vascular heterogeneity. The brain contains small regions where the BBB is atypical or missing, such as the choroid plexus (involved in CSF production) and the circum‐ventricular organs in which hormonal exchange takes place between the brain and periphery. Arterio‐venous (A‐V) hierarchy is another level of intra‐organ vascular heterogeneity, and it is likely  that vascular functions and associated gene expression patterns vary along the A‐V axis. Thus, isolation of vascular cells in bulk from an organ for molecular profiling will inevitably provide an average, and hence erroneous, picture of the vascular cell phenotype. For example, if our aim is to reveal the nature of cell–cell signaling between capillary endothelial cells of certain specialized vessels and their associated pericytes, or between arterial endothelial cells and their neighboring SMC, it is imperative that the vascular cell subtypes can be separated before analysis. Generally, the appropriate tools (e.g. antibody markers and transgenic reporters) do not exist for such work, although there are exceptions, such as protocols for the separation of glomeruli from the rest of the kidney.et al,Rather than isolating pure populations—cohorts—of vascular cell subtypes for molecular profiling, recent developments in single‐cell RNA sequencing (scRNASeq) make it possible to isolate cells “at random”, determine their gene expression, and reveal their identity in retrospect through clustering, and their anatomical location using appropriate (combinations of) markers (Zeisel The author declares that he has no conflict of interest.


CP) in early post‐natal life are periods of plasticity during which the neuronal circuitry is most receptive to environmental stimuli. These early experiences translate to a more permanent and sophisticated neuronal connection in the adult brain systems. Multiple studies have pointed to the development of inhibitory circuitry as one of the central factors for the onset of critical periods. We discuss several molecular mechanisms regulating inhibitory circuit maturation and CP, from gene transcription level to protein signaling level. Also, beyond the level of gene sequences, we briefly consider recent information on dynamic epigenetic regulation of gene expression through histone methylation and acetylation and their implication on timed development of the inhibitory circuitry for the onset of CP.Critical periods ( Neuronal circuits are exceptionally sensitive to being shaped by patterns of cellular activity during early brain development called critical periods Hensch . The criThe aim of studies investigating mechanisms underlying activation and regulation of CPs in the central nervous system (CNS) are to identify mechanisms that may allow for reactivation of neural circuit plasticity in adulthood when these circuits are no longer plastic. An important application would be to aid rewiring of neural circuits after damage to the CNS such as in the case of stroke to regain normal function. In children with neurodevelopmental disorders involving synaptic plasticity, understanding mechanisms to reactivate critical period can also potentially help to correct alterations in neural circuits via pharmacological intervention.et al. et al. et al. et al. et al. The visual cortex is the best‐studied experimental model used to study critical period as it is easy to manipulate visual experience independently in each eye and measure changes in plasticity. Direct electrophysiological measurements of neuronal function measure ocular dominance, a phenomenon whereby visual cortical neurons are activated to different degrees by the presentation of visual stimuli to one eye versus the other. During the CP, cortical responses can be manipulated by deprivation of sensory inputs via the closure of one eye, known as monocular deprivation (MD), leading to irreversible synaptic reorganization of neural circuits. This in turn results in the decrease in ability of the deprived eye to elicit cortical neuronal responses and in the increase in number of neurons responsive to the open eye. This phenomenon is known as the ocular dominance plasticity, first described in kittens by Nobel award winners Hubel and Wiesel , neuropeptide somatostatin, and the ionotropic serotonin receptor 5HT3a (5HT3aR)‐expressing interneurons have been shown to account for almost 100% of GABAergic interneurons in the neocortex  is a growth factor required for the formation of GABAergic synapses in hippocampal and cortical cultures  in the endogenous promoter IV blocking the its activity‐dependent expression. These mice have decreased spontaneous inhibitory postsynaptic currents (sIPSCs) in cortical culture and lesser GABAergic synapses in the cortex  exhibited decreased inhibition  influences growth cone motility and bouton stability. Furthermore, GABA signaling through post‐synaptic or glia receptors could trigger retrograde factors, promoting axon branching and synapse formation. Fiorentino et al., demonstrated that one of the possible mechanisms utilized by GABA to regulate synapse formation is via the metabotropic GABAb receptors on pyramidal neurons that trigger secretion of BDNF and promotes the development of perisomatic GABAergic synapses in hippocampal neurons  – p75 signaling pathway. The presence of proBDNF results in degradation of GABAa receptors and repression of its synthesis resulting in decreased inhibitory transmission. The cleavage of proBDNF to mature BDNF is mediated by tissue‐type plasminogen activator and the expression of tissue‐type plasminogen activator is found to be implicated in experience‐dependent plasticity in the visual system  is a key player in cell–cell adhesion of neuronal circuits  form around inhibitory neurons, specifically PV+ neurons  that have opposing actions. An experience‐dependent epigenetic mark, acetylated histone, is abundant at loose chromatin sites of active gene transcription and these marks also exhibit a correlative decrease as critical period plasticity ends  genes by HDAC1. Whisker‐deprived animals showed an increase in HDAC1 expression and activity with a corresponding decrease in inhibitory synapses of PV interneurons. Chromatin Immunoprecipitation analysis revealed direct associations between HDAC1 and promoter regions of Bdnf and Pvalb which were down‐regulated because of the increased histone deacetylation , has been associated with many cellular processes, mainly in cell cycle progression. Prmts can catalyze the formation of mono‐ or di‐methylated arginine residues. Depending on the type of arginine dimethylation, PRMTs can be classified as a Type I or Type II enzyme (Bedford and Clarke et al. et al. PRMTs are strongly implicated in stem cell and cancer biology. Also, many groups are beginning to look to PRMTs as potential therapeutic targets (Cha and Jho et al. Prmt8 knockout mice, Tnr transcription levels were heightened by 1.5‐fold resulting in the increased PNN formation (Fig. et al. moreover, showed that the visual acuity of Prmt8 knockout mice were lower than wildtype mice suggesting a premature closure of critical period because of the hastened PNNs formation. These findings suggest that PRMT8 methylation activity is critical for the complete maturation of inhibitory circuitry by regulating the formation of PNNs that essentially act as structural brakes to critical period plasticity (Lee et al. Because of its unique tissue localization, PRMT8 is suspected to play an important role in neurodevelopment and synaptogenesis. There are evidence to suggest that it plays a role in early neuronal development (Lin ion Fig. . Also, tet al. et al. et al. With more models to study critical period plasticity emerging in different modalities, GABAergic PV expression has been shown to be a key regulator of critical period plasticity. However, experiences can regulate the maturation of PV+ interneurons via different pathways and studying these processes in different modalities may help us to better understand the differences in regulation between modalities. Further work has to be done to find out if similar pathways of regulation are common in different regions of the brain. Neuroepigenetics is an emerging field crucial for the downstream regulation of gene expression required for PV+ interneuron maturation and can perhaps be used as targets of pharmaceutical intervention as lines of evidence show that using HDAC inhibitors have reactivated critical period plasticity in the visual cortex (Putignano


Several presentations focused on the role of calreticulin and other ER chaperones in a variety of disease states, including haemophilia, obesity, diabetes, Sjogren's syndrome, Chagas diseases, multiple sclerosis, amyotrophic lateral sclerosis, neurological malignancies , haematological malignancies , lung adenocarcinoma, renal pathology with emphasis in fibrosis and drug toxicity. In addition, the role of calreticulin and calnexin in growth and wound healing was discussed, as well as the possible use of extracellular calreticulin as a marker for certain diseases. It was agreed that the 13th International Calreticulin Workshop will be organized in 2019 in Montreal, Quebec, Canada.Starting from 1994, every 2 years, an international workshop is organized focused on calreticulin and other endoplasmic reticulum chaperones. In 2017, the workshop took place at Delphi Greece. Participants from North and South America, Europe, Asia and Australia presented their recent data and discussed them extensively with their colleagues. Presentations dealt with structural aspects of calreticulin and calnexin, the role of Ca Importantly, the ER is a multifunctional organelle able to detect and integrate incoming signals, modulate its own luminal dynamics and generate output signals in response to environmental changes. Disruption of ER homoeostasis (proteostasis and lipidostasis) therefore results not only in cellular disease but also has detrimental effects at organ and systemic levels as well. To support its diverse cellular functions, the ER contains many luminal and integral membrane proteins that have dynamic interactions with membrane lipids as well as lipid metabolites. Calr is a major Ca2+ buffering chaperone and ER‐resident protein involved in a variety of biological processes including quality control of protein folding, regulation of Ca2+ homoeostasis and MHC class I antigen processing. Calr plays an important role in both the innate and adaptive immune response. Cell surface Calr impacts the phagocytosis of apoptotic cells and has potential application as a therapeutic for diabetic wound healing and cancer therapy. The ER membrane protein Canx, which has a high degree of structural similarity to Calr, works in conjunction with Calr as molecular chaperones in the ER protein quality control system. Several aspects of Calr and Canx structure and function, and their impact on ER homoeostasis, were discussed at the Delphi meeting.Many vital cellular processes occur in the ER, notably Ca2+. Under cellular stress conditions, Calr easily multimerizes, and through XLMS, a model of the protein dimer was determined. The dimer forms in parallel fashion, and the P‐domain of Calr is involved in intermolecular bonds. This model fits with the presence of higher orders of multimerization observed and supports the ability of Calr to interact with many partners. While most of these interactions are short‐lived, and thus difficult to detect, Peter's group has been able to obtain preliminary interaction data for a number of proteins, including protein disulphide isomerase 3 (PDIA3) and complement component 1 (C1q).Using chemical cross‐linking combined with mass spectrometry (XLMS), the group of Peter Hojrup  has probed the structure of the flexible arms of Calr and the influence of CaDrosophila melanogaster. Three‐dimensional reconstruction revealed a cage‐like structure with a large central cavity and flexibility that precluded determination of a high‐resolution structure. It was suggested that the central cavity contains the catalytic site and hydrophobic surfaces to enhance the binding of misfolded substrates and exclude hydrophilic, folded proteins, possibly the mechanism behind UGGT function Using NMR spectroscopy, Kalle Gehring's group  presented elegant studies on direct binding of oxidoreductase ERp29 (PDIA9) to the P‐domain of Canx and the role of D348 in Canx, which when mutated abrogated interactions between Canx and PDIA9, PDIA3 and cyclophilin B (CypB). Using X‐ray crystallography, the structures of the D‐domain of PDIA9 bound the P‐domains of Calr and calmegin were presented. These structures reveal that the tip of the P‐domain functions as a plurivalent adapter to bind different folding factors. The structural diversity of the bound chaperones suggests that they became specialized for glycoprotein folding through convergent evolution of their P‐domain binding sites. In addition, this group has used negative‐stain electron microscopy to determine the structure at 18 Å resolution of UDP‐glucose:glycoprotein (UGGT) from 2+ homoeostasis and ER architecture. Data were presented on the regulation of DHHC6 function, some of its targets and consequences of palmitoylation. Approximately 10% of the human proteome undergoes palmitoylation and targets include major channels and receptors, enzymes and signalling molecules Gisou van der Goot's group  discussed the role of palmitoylation in the ER. The ER hosts many Asp‐His‐His‐Cys (DHHC) domain‐containing palmitoyltransferase enzymes that mediate protein palmitoylation. Palmitoylation occurs on cysteine residues and allows soluble proteins to become peripheral membrane proteins. DHHC6 is of particular interest as it modifies key ER proteins involved in protein folding and quality control, Ca2+‐sensing protein stromal interacting molecule 1 (STIM1), acting as an intracellular ligand to control the gating of Ca2+‐permeable channels of the Ca2+ release‐activated calcium modulator (ORAI) and transient receptor potential cation channel (TRPC) families. STIM/ORAI coupling occurs at membrane contact sites, which are evolutionarily conserved structures that sustain non‐vesicular lipid transfer at sites of close apposition (~10–20 nm) between the ER and other cellular membranes. In neutrophils, STIM/ORAI interactions occur on phagocytic vacuoles and enhance the efficiency of phagocytosis by promoting actin dynamics and by enabling phagocytic vacuoles to gain oxidative and lytic properties via Ca2+‐dependent fusion with granules and lysosomes. Using ultrastructural, genetic and functional approaches, the Demaurex group showed that STIM1 increases the formation of ER‐phagosome contact sites to generate local Ca2+ elevations that promote the phagocytic process. At ER‐phagosome contact sites, STIM1 triggers the opening of phagosomal Ca2+ channels and synergizes with its adapter protein junctate to promote inositol trisphosphate (InsP3)‐mediated Ca2+ release from the recruited ER stores. In dendritic cells, Stim1 ablation reduced local and global Ca2+ signals as well as cross‐presentation and chemotaxis without altering cell differentiation, maturation or phagocytic capacity. Phagosomal proteolysis and insulin‐regulated aminopeptidase (IRAP) activity, as well as fusion of phagosomes with endosomes and lysosomes were impaired by STIM1 deficiency. Local Ca2+ signals occurring at membrane contact sites between the ER and phagosomes may regulate the efficiency of bacterial killing by neutrophils and the ability of dendritic cells to induce antiviral and anticancer immunity.Nicolas Demaurex  focused on the ER Ca2+ handling by the ER and especially via the InsP3 receptor/Ca2+ release channels plays a crucial role in the autophagy process. Autophagy is a conserved, catabolic pathway that under basal conditions assures cell homoeostasis by degrading protein aggregates, long‐lived proteins and dysfunctional organelles and is up‐regulated during stress conditions. In follow‐up of previous work, Jan Parys presented the latest findings concerning the Ca2+ dependence of resveratrol‐induced autophagy and of autophagy triggered by the ER stress inducer azetidine carboxylic acid (AZC). Interestingly, resveratrol‐induced autophagy is even strictly dependent on the InsP3 receptor, as a mammalian cell line deficient in InsP3 receptors lost its sensitivity for resveratrol‐induced autophagy 2+ should be further explored, as although both compounds affect ER Ca2+‐store handling, they do so in a different way.Jan Parys  emphasized that Cavia direct ER to lysosomes transport defined as ER‐ to lysosome‐associated degradation (ERLAD).Maurizio Molinari  reported on mechanisms operating in mammalian cells to clear aggregated polypeptides generated in the lumen of the ER. Eukaryotic cells host two major catabolic pathways, the ubiquitin proteasome system (UPS) and autophagy. The majority of misfolded proteins produced in the ER are retrotranslocated across the ER membrane and degraded by the UPS in a series of processes defined as ER‐associated degradation. It is postulated that the UPS‐resistant aggregates accumulating in the ER lumen are removed by macroautophagy. About 40 autophagy (ATG) gene products regulate formation of double membrane structures, the phagophores, that capture cytosolic material, mature to autophagosomes that eventually fuse with lysosomes to eliminate their content (damaged organelles or aggregated proteins). Molinari's group showed that generation of double membrane phagophores and autophagosomes is dispensable for clearance of misfolded ordered and proteasome‐resistant polymers generated in the ER. These are cleared from cells 2+‐binding characteristics of ER chaperones and how Ca2+ binding affects substrate interactions with ER chaperones. Calr is known to have multiple low‐affinity Ca2+‐binding sites within an acidic C‐terminal region that is mutated in myeloproliferative neoplasms (MPN) 2+‐binding acidic region of Calr are shown to reduce stability, induce self‐association and alter cellular chaperone function.Much remains to be understood about the Ca2+ binding by BiP and the formation of BiP oligomers was unknown. Using isothermal titration calorimetric studies, the Raghavan laboratory showed that BiP contains low‐affinity Ca2+ binding sites in the ER range of Ca2+ concentrations. The role of the C‐terminal acidic region in low‐affinity Ca2+ binding by BiP is being investigated. BiP oligomers, previously described, are destabilized by truncations of the C‐terminal acidic region of BiP. Thus, the C‐terminal regions of BiP and Calr regulate their self‐associations, but in opposite directions.The ER chaperone BiP also contains acidic C‐terminal region, but the relevance of this region to Cavia the ATPase domain of BiP, which was independent of nucleotides. Then using misfolded protein substrate, CH1, they demonstrated that it bound solely to the canonical substrate‐binding domain of BiP, and not to the luminal domain of Ire1 via a conformational change. This observation forms the basis of the proposed allosteric model for UPR induction that provides a rationale for how UPR detects misfolded proteins3.The UPR is an essential cell signalling system that detects the presence of misfolded proteins within the ER, and then initiates a number of cellular responses that aims restore ER protein homoeostasis Characterization of the IRE1α interactome followed by functional validation by the group of Claudio Hetz  and his collaborators unveiled several novel regulators of the UPR, highlighting the ER chaperone heat‐shock protein 47 (Hsp47) as the major hit. Cellular and biochemical analyses revealed that Hsp47 instigates IRE1α signalling through a physical interaction. At the molecular level, Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate its oligomerization. Regulation of IRE1α signaling by Hsp47 is evolutionary conserved as validated using genetic manipulation in fly and mouse models of ER stress. Therefore, it was concluded that Hsp47 adjusts IRE1α signalling by fine‐tuning the threshold to engage an adaptive UPR.Robert Binder  presented recent findings from his group regarding the regulation of the signalling cascades within antigen‐presenting cells, triggered by ER heat‐shock proteins (HSP) via the GFFKR motif, a possible implication of cytosolic Calr as a mediator of drug resistance 2+. His group then assessed whether integrin function can modulate surface Calr levels in immunogenic cell death (ICD) C. James Lim  discussed how cell adhesion is a physiologically relevant stimulus shown to increase the interaction of Calr with α‐integrins Renilla luciferase that is virtually inactive until the glycan is enzymatically removed by protein N‐glycanase (PNGase), which converts the asparagine to which the glycan is attached into an aspartic acid residue. Cytosolic entry after phagocytosis or endocytosis followed by deglycosylation by endogenous cytosolic PNGase liberates the activity of the enzyme, providing a probe to analyse the translocation function as well as downstream processes that affect the reconstitution of cytosolic activity. Characterization of the luciferase probe and its potential for unravelling the cellular processes involved in liberating its cytosolic activity were discussed.Peter Cresswell  presented on protein entry into the cytosol during antigen cross‐presentation. The precise mechanism(s) for this event remain obscure, but previous work from his group argues that the property is not restricted to dendritic cells (DCs) or even to classical antigen‐presenting cells. To further enhance our understanding of the pathway, they have developed a glycosylated derivative of 2+.Leslie Gold's group  in collaboration with Paul Eggleton's group  presented data suggesting that extracellular Calr  specifically sequesters and binds to lipopolysaccharide (LPS), enhancing both cellular and innate immune processes. This interaction may represent a combined damage  and pathogen (LPS)‐associated molecular pattern danger signalling molecule to the immune system. It appears that LPS binds multimeric eCalr and may even induce aggregation of this protein. Notably, eCalr dose dependently bound to immobilized LPS by ELISA, which was increased twofold by Cain vitro by incubation with hydrolysable ATP. FVIII aggregation in the cell causes mitochondrial oxidative stress, and a mitochondrial‐targeted antioxidant reduces oxidative stress and promotes solubility and secretion of FVIII. FVIII gene transduction into hepatocytes in mice also causes FVIII aggregation, which when combined with a high‐fat diet treatment leads to hepatocellular carcinoma. The aggregation of FVIII in the ER presents a unique example of a natural protein that is prone to amyloid formation in the ER, indicating that cellular biochemical machinery exists to dissociate these aggregates for productive folding and secretion of functional protein.Haemophilia A (HA) results from deficiency in coagulation factor VIII (FVIII). Preferred therapy requires prophylactic protein replacement with recombinant‐derived FVIII (rFVIII). Although several proteins that interact with FVIII within the ER have been identified, including the ER chaperones BiP, Canx, Calr and the ER to Golgi intermediate compartment members LMAN1/ERGIC‐53 and multiple coagulation factor deficiency 2 (MCFD2), both the mechanism and the other factors governing proper folding and efficient secretion of FVIII are still poorly understood. Randal Kaufman  provided evidence that FVIII forms aggregates within the ER that have a structure characteristic of amyloid. Significantly, the aggregation is reversible in a process that requires cellular energy in which the aggregates can be refolded properly to secrete functional FVIII. These aggregates can also be dissociated Giannis Spyrou  presented data on ERdj5 (PDIA19), an ER chaperone protein belonging to the thioredoxin superfamily with a highly potent reductase activity and an integral part of the ER‐associated protein degradation system (ERAD), which binds to BiP to facilitate the cleavage of S‐S bonds in terminally misfolded proteins before they can be translocated to the cytosol for proteasomal degradation. Presented data revealed that ERdj5 function affects metabolic pathways, immune responses and protein aggregate formation in various afflicted tissues. The ERdj5 gene knockout mice develop an obese and diabetic phenotype with elevated blood glucose, insulin resistance, altered lipid and carbohydrate metabolism rates compared to the wild‐type controls. These mice also develop inflammatory lesions in the salivary glands resembling the pathological manifestations of Sjögren's syndrome and are positive for antinuclear autoantibodies in the serum. The presented studies provided evidence for the crucial connection between the ER protein quality control system and diverse classes of human disease, such as NAFLD, diabetes and autoimmunity.Trypanosoma cruzi, the causative agent for Chagas diseases. T. cruzi trypomastigotes up‐regulate the expression of thrombospondin‐1 (TSP‐1) which is important in the process of cellular infection. They have shown that the N‐terminal domain of TSP‐1 (NTSP) interacts with T. cruzi Calr  in a complex of purified parasite membrane surface proteins, thus predicting one of the domains of TSP‐1 molecule that is important in mediating interaction with TcCalr. Using fluorescence activated cell sorting (FACS) and membrane protein expression analysis, they showed that transgenic parasites had more TcCalr on their surface compared to control parasites. Invasive trypomastigotes overexpressing TcCalr showed a higher level of infectivity compared to control parasites. Therefore, TcCalr is a virulent factor that enhances T. cruzi infection.Pius Nde  presented his latest work on Evidence suggests that changes in expression of ER chaperones can influence cell surface protein stabilization and function in the nervous system. Marek Michalak  presented the latest findings on a potential role of Canx in the pathology of multiple sclerosis.in vivo in zebrafish models of ALS. Therefore, therapeutics based on PDI may be attractive candidates for ALS. However, in addition to its protective functions, aberrant, toxic roles for PDI have recently been described. These functions need to be fully characterized before effective therapeutic strategies can be designed.Sonam Parakh  presented the latest findings from Julie Atkin's laboratory, regarding amyotrophic lateral sclerosis (ALS). There is increasing evidence that ER chaperones PDIA1 (PDI) and PDIA3 (ERp57) are protective against neurodegenerative diseases related to protein misfolding, including ALS, which is a rapidly progressing disorder affecting motor neurons in the brain, brainstem and spinal cord, resulting in paralysis and death usually 2–5 years post‐diagnosis. Work from the group has demonstrated that overexpression of PDI is protective against mutant superoxide dismutase (SOD1) misfolding and aggregation, ER stress and apoptosis, in neuronal cell lines. PDI family members are protective against other ALS‐associated proteins and against multiple features characteristic of cellular pathology in neuronal cells and primary neurons, including misfolded protein aggregation, mislocalization to the cytoplasm, ER stress, inhibition of ER–Golgi transport and induction of apoptosis. PDI family members are protective Eric Chevet  talked about proteostasis imbalance as an emerging major hallmark of cancer, driving tumour aggressiveness as genetic and pharmacological evidence suggests that the ER plays a critical role in cancer development. This concept was evaluated in the case of glioblastoma multiform (GBM), the most lethal primary brain cancer with an overall survival of 15 months and no effective treatment. Previous work from the group has demonstrated that the ER stress sensor IRE1α contributes to GBM progression, impacting tissue invasion and tumour vascularization. IRE1α also regulates the stability of certain miRNAs and mRNAs through a process known as RIDD. Somatic mutations in the IRE1α gene have been identified in GBM and other forms of cancer. Data were presented regarding the contribution of IRE1α signalling to GBM based on the systematic comparison of mutant forms identified in cancer, and demonstrated its significance to the disease. A novel mutation associated with GBM was uncovered, with functional consequences to tumour formation. Taking advantage of the specific signalling outputs of the RNase domain of IRE1α engaged by distinct GBM‐related mutations, specific expression signatures were defined that were confronted to human GBM transcriptomes. This approach allowed them to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumour outcome. These data led to the demonstration of a dual role of IRE1α downstream signalling in cancer and open a new therapeutic window to abrogate tumour progression Calr, functionally a chaperone involved in the correct folding of N‐linked glycoproteins Calr mutations develop a maturation defect in myeloperoxidase (MPO), a glycoprotein folded by CalrCalr mutations can affect the chaperone function of Calr. The mechanism(s) of MPO deficiency in MPN and the possible implications for future basic and clinical research in the field were discussed.Alexandre Theocharides  presented data on Philadelphia‐negative MPN, chronic haematopoietic stem cell disorders characterized by increased proliferation of one or more haematopoietic cell lineages. MPN are characterized by frequent somatic mutations in the Janus kinase 2 (JAK2) and Ilyas Chachoua  and Anita Roy  from Stefan Constantinescu laboratory presented data on how mutants of Calr are associated with MPNs, essential thrombocythemia and myelofibrosis. The phenotype induced by Calr mutants is due to pathologic activation of the thrombopoietin receptor  in the secretory pathway and at the cell surface. Localization of mutant Calr in ERGIC, Golgi and surface was demonstrated by confocal immunofluorescence microscopy. Using FRAP (Fluorescence Recovery after Photobleaching), a change in mobility of TpoR upon expression of Calr mutants at the plasma membrane was shown. Using structure‐guided mutagenesis as well as serial truncations from the C‐terminus of Calr mutants, the sequences required by TpoR and Calr mutants for interaction and eventual activation of TpoR‐JAK‐signal transducer and activator of transcription 5 (STAT5) signalling were defined. Using CRISPR/Cas9 technology, knockin heterozygous cell line and mouse models of murine Calr del52 and del61 were generated. Although the frameshift in the mouse Calr leads to a slightly different sequence, the biophysical features remain similar to those of human Calr mutants and effects on TpoR appear similar. Early data indicate the appearance of a thrombocytosis phenotype and histological features reminiscent of MPNs in the mouse models. These data further support the notion that the major driver event induced by mutant Calr is activation of TpoR Activation of ER stress coping responses has been associated with nephrotoxicity and kidney fibrosis. Cyclosporine (CsA) is an immunosuppressant widely used in organ transplantation and in treatment of various autoimmune diseases. CsA binds to cyclophilin A and the complex associates with and inhibits calcineurin. CsA‐dependent inhibition prevents activation of promoters of T cell activation and overall immune response. Prolonged intake of CsA induces secondary side effects including fibrosis and chronic nephrotoxicity. The group of Jody Groenendyk and Marek Michalak  discovered that CsA interacts with prostaglandin endoperoxidase and cyclooxygenase 2 (COX‐2), an inducible, ER‐associated enzyme. The CsA‐COX‐2 complex binds to IRE1α to enhance its activity and contributes, at least in part, to development of fibrosis. Increased COX‐2 activity is associated with renal tissue damage and poor outcome for kidney transplant patients. COX‐2 enzyme is also up‐regulated during cardiac allograft rejection and correlates with a poor outcome. CsA treatment causes an increase in tumour necrosis factor‐α (TNFα) secretion, induction of the fibrotic cascade and a disruption in the redox balance. Tauroursodeoxycholic acid (TUDCA) is a bile acid and proteostasis promoter that is shown to reduce the UPR and prevent cardiac fibrosis. Data were presented showing that application of TUDCA prevents fibrosis in a nephrotoxic mouse model system.Transforming growth factor‐β (TGF‐β) is a key factor in the pathogenesis of diabetic nephropathy, a leading cause of end‐stage renal disease. Joanne Murphy‐Ullrich's group  previously showed that Calr is important for collagen transcription, secretion and assembly into the extracellular matrix and that Calr is critical for TGF‐β stimulation of type I collagen transcription through Calr's action on ER calcium release and NF‐AT activation. In recent work, her group used Calr fl/fl mice to knockdown Calr expression in a tissue‐specific manner in adult mice through targeted ultrasound delivery of cre‐recombinase plasmid and showed that Calr knockdown reduced neointimal hyperplasia and collagen in response to acute vascular injury in the carotid artery in vitro studies were confirmed in vivo, in the animal fibrotic model biochemically and morphologically. Among them, the σ‐isoform exhibited the highest expression. In addition, transgenic animals that were heterozygous for Calr were expressing reduced amounts of 14‐3‐3 proteins. Furthermore, studies using human biopsy material from patients suffering from IgA nephropathy, membranous nephropathy, lupus nephropathy and antineutrophil cytoplasm‐associated (ANCA) nephropathy revealed a pronounced up‐regulation of the expression of 14‐3‐3 proteins compared to control biopsies, almost exclusively in distal tubule epithelial cells. Further studies were proposed to establish the 14‐3‐3 family as an important diagnostic marker.Previous work in Aris Charonis group  has established an increase in Calr expression in a mouse model of fibrosis localized exclusively in tubular epithelial cells. In a human tubular epithelial cell line, proteomic analysis was performed comparing control and overexpressing Calr cells. Among several alterations in expression, the up‐regulated family of 14‐3‐3 proteins was selected for further study. These proteins are considered docking proteins, mediators of several signalling pathways and their roles in nephropathies remain largely unknown. Proteomic analysis data were confirmed by PCR and Western blotting. In addition, cell lines where Calr expression was down‐regulated by sh‐RNA exhibited in PCR and Western blotting down‐regulation of all members of the 14‐3‐3 family. These in vitro exogenous Calr  can ameliorate most cellular defects that impair the healing of diabetic wounds. eCalr promotes cellular migration and proliferation of keratinocytes to resurface the wound, recruits macrophages to phagocytose cellular debris, attracts fibroblasts into the wound and induces extracellular matrix (ECM) production to reconstruct the wound defect. It was shown that eCRT could not induce ECM proteins or migration of Calr null MEFs. A novel paradigm evolved showing that intracellular ER Calr  and eCalr are both required for the induction of collagen, fibronectin and elastin synthesis for tissue regeneration in the following manner: eCalr binds to the surface receptor LRP1 (CD91), LRP1 signalling induces TGF‐β1 and TGF‐β3 proteins that are released from the cell and subsequently in an autocrine manner, induce canonical TGF‐β signalling via Smad 2 and 3 transcription factors for iCalr‐dependent ECM induction. The data presented showed the importance of the convergence of intracellular and extracellular Calr in ECM induction essential for wound healing and tissue regeneration.Chronic wound healing, which occurs as a consequence of diabetes, in pressure ulcers, and venous stasis ulcers, is characterized by a paucity of granulation tissue due to the lack of recruitment and proliferation of cells necessary for healing. Leslie Gold's group (New York) has shown In vitro studies using human dermal fibroblasts transfected with a β‐catenin (TOPFLASH) promoter provided evidence that hair follicle induction by eCRT is via the Wnt/β‐catenin pathway with the up‐regulation of specific Wnts. Furthermore, a chemical Wnt agonist dose dependently increased Calr transcription and protein synthesis. Further studies revealing the interaction between Calr and Wnt/β‐catenin may be important for potential therapies for deep wound treatment and degenerative skin conditions, and hair loss.Following excisional wound repair, epidermal appendages, such as hair follicles and sebaceous glands are not recovered in these deep tissue wounds. In a separate presentation, Leslie Gold (New York) reported that topical application of Calr to experimental wounds in diabetic mice induced melanin‐containing hair follicle neogenesis, whereas buffer‐treated wounds showed normal scar tissue formation. This finding implies that Calr promotes a regenerative healing process rather than classic adult wound repair. Luis Agellon  presented newly characterized features of Canx deficiency in mice. Adult mice lacking Canx are substantially smaller as compared to their wild‐type littermates (PMID 20400506), which is likely associated with growth arrest occurring between days 15 and 25 after birth, despite the resumption of normal growth rate after day 25. Nutrient absorption in the digestive tract is facilitated by membrane‐bound transporters, and these proteins may require the chaperone function of Canx for proper synthesis. The digestive tract of adult Canx‐deficient mice exhibit changes that appear to be adaptive strategies to ensure nutrient assimilation and survival.There is a need for new biomarkers that will aid in disease diagnosis, activity and response to therapy. Biomarkers can also be of support in the understanding of disease aetiology. With the expansion of functions ascribed to Calr, there has also been an increase in studies monitoring extracellular Calr and anti‐Calr antibodies in various diseases especially cancers and autoimmune disease. The recent work of Holoshitz and colleagues have demonstrated that the binding of Calr and especially citrullinated Calr  to the signal transduction share epitope peptide encoded by a number of major histocompatibility complex class II (HLA‐DRB1) alleles present in a high proportion of rheumatoid arthritis (RA) patients may correlate with erosive bone damage. This hypothesis has been shown to be the case in mouse models of arthritis. Paul Eggleton  presented data on the detection of citCalr prior to developing RA and evaluated whether citCalr is a target for autoantibodies in RA cohorts with and without lung disease/bronchiectasis (BR). The detection of citCalr in BR and development of anti‐citCalr in BR patients suggests citCalr antigens are early targets of antigenicity in these patients, prior to the onset of RA.Nasrin Mesaeli  presented data on a genetically modified mouse model overexpressing Calr under the tunica interna endothelial cell kinase (Tie2) promoter which showed development of metastatic lung adenocarcinoma. Microarray analysis of lung tissue from Tie2‐Calr mice as compared to the wild‐type mice showed a significant increase in a number of long non‐coding RNAs. One of these long non‐coding RNA (LncRNA) known as metastasis‐associated lung adenocarcinoma transcript‐1  has been implicated in the development of metastatic lung cancer. Malat‐1 is a novel LncRNA that is localized to nucleus. Furthermore, its 3’ end can be spliced to form tRNA‐like structure called mascRNA that translocate to cytoplasm. Her data illustrate the involvement of this LncRNA in the increased rate of tumour cell migration and altered cell cycle. The exact targets of this LncRNA are not known. Using RNA sequencing analysis of tumour cells after knockdown of Malat‐1, changes were presented in cluster of genes involved in cell proliferation and migration.2+ homoeostasis dictates the sensitivity of the cellular sterol sensing mechanism During the meeting, a special session was organized for young researchers to short 10 min talks. The session was extremely well received by the participants. Abdullah Treffa  discussed mechanisms of Calr binding and release from cancer cells and the implications of her results for immunotherapy; Najla Arshad  talked about characterization of tumour‐associated Calr mutants and their effect on antigen presentation by MHC class I; Kristofer Marjon  presented data on the secretion of Calr from macrophages to label target cells for phagocytosis; Danilo Medinas  discussed the role of PDIA3 in the pathogenesis of ALS; Sonam Parakh  gave a presentation on ER dysfunction is a common pathological mechanism induced by mutant proteins in ALS; Hector Vega  presented data on a frameshift mutation in the Calr gene contributing to the pathophysiology of sudden unexplained death; Qian Wang  presented data on new, a muscle‐specific IRE1α interacting proteins; Wen‐An Wang  provided evidence how the ER CaThe current knowledge and future challenges for the involvement of Calr in physiology and pathology are briefly presented in Table 2+ homoeostasis and stress responses with major emphasis on the ER and other intracellular organelles. In addition, as the functions of molecular chaperones expand into the fields of myeloproliferative diseases, neurodegenerative diseases, autoimmune diseases, cancer and metabolism, this workshop provides the optimal venue to bring scientists from diverse disciplines together with a common goal to advance the understanding of the functions of Calr and other ER proteins as relevant therapeutic targets. The 13th International Calreticulin Workshop will be organized in 2019 in the beautiful city of Montreal, Quebec, Canada.The 12th International Calreticulin workshop offered an excellent exchange of information on the latest findings in the area of protein structure, protein folding and CaThe authors declare that there is no conflict of interest.


Current guidelines recommend orchidopexy for cryptorchidism by 12 months of age, yet this is not universally adhered to. The aim of this systematic review and meta‐analysis was to compare outcomes between orchidopexies performed before and after 1 year of age.MEDLINE and Embase were searched (September 2015) using terms relating to cryptorchidism, orchidopexy and the outcomes of interest. Studies were eligible for inclusion if they compared orchidopexy at less than 1 year of age (early) with orchidopexy at 1 year or more of age (delayed) and reported the primary outcome (testicular atrophy) or one of the secondary outcomes . Studies were excluded when more than 50 per cent of infants had intra‐abdominal testes, or the population included infants with disorders of sexual differentiation. Additional studies were identified through reference list searching. Unpublished data were sought from the ORCHESTRA study investigators.Fifteen eligible studies were identified from 1387 titles. There was no difference in atrophy rate between early orchidopexy and delayed orchidopexy . Testicular volume was greater (mean difference 0·06 (95 per cent c.i. 0·01 to 0·10) ml; 346 testes) and there were more spermatogonia per tubule (mean difference 0·47 (0·31 to 0·64); 382 testes) in infants undergoing early orchidopexy, with no difference in complication rate . No study reported malignancy rate.Atrophy and complication rates do not appear different between early and delayed orchidopexy, and fertility potential may be better with early orchidopexy. Imprecision of the available data limits the robustness of these conclusions. Cryptorchidism affects between 2·4 and 5 per cent of live‐born boysThe current theory is that there is a short time frame between 3 and 6 months of age when the normal maturation of gonocytes into type A spermatogonia should occur, and this process is temperature sensitiveThis guidance is not adhered to universally. A recent international surveyet al.A recent systematic review by Chan and colleaguesThe review was conducted according to a prespecified protocol registered on the Prospero International Prospective Register of Systematic Reviews (CRD42016025930).The population of interest for this review were boys without disorders of sexual differentiation who were identified as having unilateral or bilateral cryptorchidism, and for whom an orchidopexy was performed. The intervention of interest was orchidopexy performed before 1 year of age, with orchidopexy performed at 1 year of age or later as a comparator. The primary outcome was testicular atrophy, regardless of method of measurement, with secondary outcomes including fertility potential, testicular malignancy, neurodevelopmental outcomes, and surgical or anaesthetic complication . Studies were therefore eligible for inclusion if they compared orchidopexy performed before 1 year of age with orchidopexy performed at or after 1 year of age in boys with either unilateral or bilateral cryptorchidism, and reported at least one outcome of interest. It was considered likely that the outcomes of interest would be reported using multiple different methods of measurement, at multiple different time points. As the intention of this review was to understand the current state of evidence, as opposed to drawing robust conclusions for management, the decision was made not to restrict eligibility of studies based on their use of a specific outcome measure. Meta‐analysis was, however, performed only for studies reporting comparable outcome measures.As it is believed that infants with intra‐abdominal testes have worse outcomes than those with testes located in the inguinal canal or suprascrotal pouch, studies that assessed outcomes purely for infants undergoing treatment for intra‐abdominal cryptorchidism were excluded from the review. Studies where there was a mixed population of infants were, however, included, if the majority of testes were in the inguinal canal or suprascrotal pouch. This decision was made, as it was considered that exclusion of all studies with a heterogeneous population would prevent the review from addressing its primary objective. To aid transparency of the comparability of the intervention and control groups, the authors sought to describe the testicular positions reported in each included study. Studies including infants with disorders of sexual differentiation were also excluded, as these children are likely to represent a different population to those without disorders of sexual differentiation.Appendix S1, supporting information). All study designs, except expert opinion, were eligible for inclusion, and no limits were placed based on year of publication, language or geographical location. Hand‐searching of reference lists from included manuscripts was undertaken to identify additional eligible studies.Multiple search strategies were used to identify relevant articles from MEDLINE and Embase. The initial search was performed on 3 September 2015, and rerun on 15 December 2016 to confirm that no additional eligible articles had been published between the initial search and submission for publication. Search terms were identified from database thesauri and free text relating to cryptorchidism, orchidopexy and the outcomes of interest. Terms were combined using Boolean operators . A summary for each outcome, which could range from very low to high quality, was produced.Mean differences were calculated for continuous variables using the inverse variance method. Risk ratios (RRs) were calculated for dichotomous variables using the Mantel–Haenszel method. Meta‐analyses were performed using Review Manager (RevMan) version 5.3 .Where studies reported continuous outcomes using mean(s.d.) value for subgroups of the intervention and control groups used in the present review, data from these subgroups were combined into one summary measure using Microsoft® Excel® for Mac™ 2011 version 14.7.4 (170508) .Table S1, supporting information). After hand‐searching of the reference lists of the 12 eligible studies identified from database searching, a further three eligible studies were identified. In total, 15 published studiesFig. Table et al.et al.After removal of duplicates, 1387 titles were reviewed, with 1006 excluded at title review stage, 335 at abstract review and 34 on review of full papers There were no studies reporting outcomes directly relating to malignancy or neurodevelopment.24,30,32,36 including 912 testes contributed data to analysis of the primary outcome, testicular atrophy, with no difference in rates seen between boys who underwent orchidopexy before 1 year of age and those who had orchidopexy at or after 1 year of age.This systematic review and meta‐analysis comparing outcomes following orchidopexy before 1 year of age with outcomes following orchidopexy at or after the age of 1 year for cryptorchidism included 15 published studies and data from one unpublished study. Seven of the published studies were drawn from the same two populations of infants, and the quality of evidence provided for all reported outcomes was graded as very low. Four studiesThere was also no evidence of difference in overall rates of postoperative complications between the two groups. Meta‐analysis of secondary outcomes relating to fertility suggested that boys who underwent orchidopexy before 1 year of age had a statistically significantly larger testicle at the time of orchidopexy, significantly larger‐diameter seminiferous tubules, and a significantly higher number of spermatogonia per seminiferous tubule than those who had orchidopexy at or after 1 year of age. Although not suitable for meta‐analysis, other markers of fertility potential reported in eligible studies also appeared to suggest greater fertility potential in infants who underwent orchidopexy before 1 year of age.These results therefore suggest a potential benefit of performing orchidopexy before 1 year of age, with no current evidence of additional harm. These results must, however, take into consideration the robustness of the primary data included in the review.Although robust methodology was used in the design of this study, there are multiple limitations that affect interpretation of the findings. First, no limits were placed on the age at which orchidopexy at or after 1 year of age could be performed, making the control group particularly heterogeneous. Further heterogeneity was introduced as boys with intra‐abdominal cryptorchidism and those who had undergone hormonal therapy before orchidopexy were included in the review. Distribution of these children amongst the control and intervention groups was not always clearly described, making it difficult to interpret how these factors may have confounded the review's results. Patient flow and study methodology were also unclear in many of the included studies, making it difficult to assess the impact of loss to follow‐up and potential chance, confounding and bias on the results of the review.Given the heterogeneity in choice of outcome measures relating to measures of fertility potential, it was impossible to produce one summary measure of fertility potential. It is therefore possible that the results of the meta‐analyses do not reflect the true picture. Each of the fertility measures assessed is also only a surrogate for true fertility, and although some, such as sperm countet al.A previous systematic review by Chan and colleaguesThe results of this systematic review and meta‐analysis suggest that there is currently insufficient evidence to advocate a wholesale move to performing all orchidopexies before 1 year of age, but there is some evidence that this may improve fertility potential. Given the equivocal state of the available evidence, in order to support the BAPU consensus statement, high‐quality RCTs must be conducted. These trials must assess harms as well as benefits, and also document the rates of spontaneous testicular descent seen in the delayed intervention group. Significant preliminary work must, however, be completed before attempting such RCTs. This will include identification of an appropriate, reliable, non‐invasive surrogate marker of fertility, and establishment of mechanisms for linking study participants to cancer registries or routinely collected data in order to identify long‐term risk of malignancy. Results from such an RCT could be combined with the final results of the GAS study to provide the robust evidence required either to support or to refute the BAPU consensus statement.Appendix S1 Search strategyTable S1 Characteristics of studies excluded at full paper reviewClick here for additional data file.


Batrachochytrium dendrobatidis (Bd). While this fungus is widespread, not all Bd‐positive populations have been associated with declines, which could be attributed to differences in pathogen virulence or host susceptibility. In a laboratory experiment, we examined the effects of Bd isolate origin, two from areas with Bd‐associated amphibian population declines  and two from areas without Bd‐related population declines , on the terrestrial growth and survival of American toad (Anaxyrus americanus) metamorphs reared in larval environments with low or high intraspecific density. We predicted that (1) Bd isolates from areas experiencing declines would have greater negative effects than Bd isolates from areas without declines, and (2) across all isolates, growth and survival of smaller toads from high‐density larval conditions would be reduced by Bd exposure compared to larger toads from low‐density larval conditions. Our results showed that terrestrial survival was reduced for smaller toads exposed to Bd with variation in the response to different isolates, suggesting that smaller size increased susceptibility to Bd. Toads exposed to Bd gained less mass, which varied by isolate. Bd isolates from areas with population declines, however, did not have more negative effects than isolates from areas without recorded declines. Most strikingly, our study supports that host condition, measured by size, can be indicative of the negative effects of Bd exposure. Further, Bd isolates’ impact may vary in ways not predictable from place of origin or occurrence of disease‐related population declines. This research suggests that amphibian populations outside of areas experiencing Bd‐associated declines could be impacted by this pathogen and that the size of individuals could influence the magnitude of Bd's impact.Understanding factors that influence host–pathogen interactions is key to predicting outbreaks in natural systems experiencing environmental change. Many amphibian population declines have been attributed to an amphibian chytrid fungus,  We chose American toad metamorphs in this study because they are sensitive to Bd in the laboratory  egg masses were collected on 12 and 18 April 2014 from Rush Run Wildlife Area . Eggs were hatched and held in the laboratory at 17 C with a 12‐hr:12‐hr light:dark cycle until tadpoles reached free‐swimming stage . We fed2.2Bd exposure of varying isolates  with five replicates of each treatment, for a total of 50 experimental units in the terrestrial phase. An experimental unit was a group of eight toads housed in a single terrarium. Two isolates were from areas with records of Bd‐associated amphibian population declines , and two were from areas without noted Bd‐associated declines , which were acquired from Joyce Longcore . Treatments were randomly assigned to terraria (34 cm × 22 cm × 27 cm) containing groups of eight toads, a water dish, an upturned dish for refugia, and 1.5 cm of pea gravel covered by 2.5 cm of topsoil. Terraria were held at 22 °C on a 12‐hr:12‐hr light:dark cycle.We examined the effects of larval density  and On 22 June 2014 , we weighed individuals and gave each a toe clip for identification within the terrarium; two toes or less were cut from each toad. To prevent infection and relieve pain, cut toes were treated with an antiseptic and analgesic ointment, Bactine . We sprayed terraria everyday with dechlorinated water to keep soil moist and changed water in water dishes each week. We fed groups of toads calcium‐dusted crickets three times per week. Crickets increased in size (from 3.8 to 6.4 mm) and number (from three to 12 crickets/toad/feeding) throughout the time of the experiment.Bd treatments for 12 hr. Each toad was exposed in a ventilated plastic Petri dish containing 8 ml dechlorinated water and 1 ml of a Bd treatment solution (see below). After the exposure, toads were returned to the terraria. We cultured Bd on 1% tryptone agar plates using standard protocols , individuals were exposed to Bd exposure and larval density on hosts. We monitored survival each day and weighed individual toads weekly to measure growth. To confirm infection by Bd from toads, we made wet‐mount slides of epidermal skin sloughs and examined skin samples using a compound light microscope (100×–400× magnification) for Bd zoosporangia for toads that died from experimental day 1 until experimental day 40 , and their interaction on American toad metamorph survival over 73 days using a generalized linear mixed model with a binomial distribution and a logit link function. Terrarium was designated as a random factor. We tested for the effect of Bd exposure, larval density, and their interaction on American toad initial mass on experimental day 0, the number of days that toads survived, change in mass , and growth over 66 days using mixed linear models with Gaussian distributions and terraria designated as a random factor. The last day all animals were weighed was day 66. To improve the model fit, we log‐transformed initial mass on experimental day 0, days survived, and masses when analyzing growth over 66 days. To test for treatment differences, we performed multiple‐comparisons tests of the least‐squared means of 73 days survival and log‐transformed number of days toads survived and change in mass. All analyses were completed with SAS 9.4 using the PROC GLIMMIX  procedure, or the PROC MIXED procedure  (SAS Institute). Type three tests of fixed effects were evaluated for generalized linear mixed models, and type III sums of squares were evaluated for linear mixed models.We tested for the effect of 3Bd exposed metamorphs died without observable Bd zoosporangia: three on experimental day 1 during Bd exposures, one on experimental day 2, and one on experimental day 7. All other exposed toads that died during the experiment that were examined had Bd zoosporangia visible with light microscopy. Deceased toads in control treatments that were not exposed to Bd did not show signs of infection.We measured infection in toads that died using light microscopy of skin samples. Five Bd and the interaction of Bd and larval density . However, toad size did not differ significantly by Bd treatments  or the interaction of density and Bd . Growth of American toad metamorphs over time was reduced with exposure to the Bd isolate from Panama and Maine compared to exposure with Bd isolates from Ohio, California, and the Bd control (Figure Change in mass of American toads was impacted by ty Table , but nott Figure c. When es Figure c, but ths Figure c. Examinhs Table d. At the4Bd is widespread in its occurrence, but the presence of Bd is not always associated with amphibian declines (Daszak et al., Bd isolates from locations associated with the presence and absence of amphibian population declines. We predicted that exposure to Bd isolates from areas where amphibian population declines have occurred, California and Panama, would result in greater negative effects than isolates from areas where population declines related to disease have not been observed, Ohio and Maine, and that these negative effects of Bd would be stronger for hosts reared in high larval density conditions compared to low larval density. Our results provide a partial explanation for the pattern that exists between Bd occurrence and amphibian declines by demonstrating that host responses to Bd can vary with isolate and larval environment, which influences host body condition.The world is evermore connected thanks to globalization, which has lead to increased translocation of hosts and their pathogens. This movement results in novel pairings of pathogen strains and naïve hosts. Evaluating variation in host responses to pathogen strains across varying environmental conditions is essential to understanding how the translocation of organisms influences the outcomes of novel pathogen and host pairings in natural environments. In natural populations, an amphibian chytrid fungus Bd isolates exists, but does not vary based on whether or not isolates are associated with locations of amphibian population declines, pointing to the need for caution against introductions of novel strains. Local selection pressures may drive these observed variations in the effects of isolates on host growth and survival, even though isolates from areas of decline did not impact American toads more strongly than isolates from areas where declines are absent. For instance, the effects of the local Ohio isolate did not impact hosts less than any of the other isolates, including those from areas associated with declines. The geographic range of Bd is large, encompassing locations of diverse environments (Olson et al., Bd in parts of the world may not be solely caused by variation in isolate virulence. While genetic variation in isolates could contribute to declines in some areas under some conditions, likely variation in host responses to other factors within the environment that influences host susceptibility is important for determining the outcome of pathogen exposures on hosts.Our results indicate that variation in host responses to We found no clear pattern with isolate origin and virulence, suggesting that effects of pathogens based on their locations of origin are unpredictable in this system. Movement of a pathogen to a new location may pose risks to hosts at the new location regardless of its place of origination and effects on hosts native to the place of origination. Characterizing isolate virulence by origin has limited usefulness and isolates of chytrid fungi need not be novel to potentially threaten amphibian populations, even in localities without previously noted declines. We found that the native Ohio isolate caused negative effects on hosts similar to isolates from areas associated with amphibian population declines. We reiterate the call to limit human‐aided host and pathogen movement through trade and translocation. Limitation of the movement of hosts will prevent novel pairings of pathogens and hosts, which could result in unanticipated consequences.Bd. Across isolates, survival and growth of smaller hosts reared as larvae at high densities were generally reduced in response to Bd exposure, indicating that host condition can determine the outcome of pathogen exposures on hosts. The effect of density can vary with the identity of the isolate, indicating that under certain conditions exposure to high‐density larval environments can increase the effect of some isolates. The conditions that hosts experience may induce or increase susceptibility; and the magnitude of this effect varies according to which isolate hosts are exposed. These results support that host condition is an important factor that in part determines susceptibility to disease (Lochmiller & Deerenberg, Bd (Lochmiller & Deerenberg, Bd, while conditions that increase competition for food resources, such as drying ponds or increased density of hosts during breeding may increase risk.Perhaps most interestingly, our results also show that increased competition in the larval stage, which affects size at metamorphosis, may increase the impact of Bd on populations of amphibians in the Midwestern United States for species like the American toad are not well understood because research in this field generally focuses on areas in which widespread mortality events have occurred, such as Central and South America and Australia. While no reported mortality events from Bd have been reported in the Midwestern United States, our research suggests that amphibian hosts may still be at risk to Bd, especially if conditions impact host condition and increase susceptibility. Our results support the limitation of movement of hosts and pathogens to new environments because of the unpredictability of outcomes of novel pairings, and conservation of high‐quality aquatic and terrestrial habitat for amphibian hosts in populations that may be at risk of outbreaks of chytridiomycosis to decrease the strength of effects on hosts. Consideration must be given to variation in host responses across strains and environmental conditions if our goal is to limit effects of pathogens on host populations.The effects of The authors declare no conflict of interests.SLR and MDB conceived the study design. AKB conducted the experiment under the guidance of SLR and MDB. MDB and SLR analyzed the data. AKB, SLR, and MDB interpreted the results. AKB and SLR wrote the manuscript, and MDB provided editorial advice.The dataset will be made available in Dryad upon publication.