High fitness costs of climate change‐induced camouflage mismatch

Anthropogenic climate change has created myriad stressors that threaten to cause local extinctions if wild populations fail to adapt to novel conditions. We studied individual and population‐level fitness costs of a climate change‐induced stressor: camouflage mismatch in seasonally colour molting species confronting decreasing snow cover duration. Based on field measurements of radiocollared snowshoe hares, we found strong selection on coat colour molt phenology, such that animals mismatched with the colour of their background experienced weekly survival decreases up to 7%. In the absence of adaptive response, we show that these mortality costs would result in strong population‐level declines by the end of the century. However, natural selection acting on wide individual variation in molt phenology might enable evolutionary adaptation to camouflage mismatch. We conclude that evolutionary rescue will be critical for hares and other colour molting species to keep up with climate change.     

Characterization of V. cholerae T3SS‐dependent cytotoxicity in cultured intestinal epithelial cells

AM‐19226 is a pathogenic, non‐O1/non‐O139 serogroup strain of Vibrio cholerae that uses a Type 3 Secretion System (T3SS) mediated mechanism to colonize host tissues and disrupt homeostasis, causing cholera. Co‐culturing the Caco2‐BBE human intestinal epithelial cell line with AM‐19226 in the presence of bile results in rapid mammalian cell death that requires a functional T3SS. We examined the role of bile, sought to identify the mechanism, and evaluated the contributions of T3SS translocated effectors in in vitro cell death. Our results suggest that Caco2‐BBE cytotoxicity does not proceed by apoptotic or necrotic mechanisms, but rather displays characteristics consistent with osmotic lysis. Cell death was preceded by disassembly of epithelial junctions and reorganization of the cortical membrane skeleton, although neither cell death nor cell‐cell disruption required VopM or VopF, two effectors known to alter actin dynamics. Using deletion strains, we identified a subset of AM‐19226 Vops that are required for host cell death, which were previously assigned roles in protein translocation and colonization, suggesting that they function other than to promote cytotoxicity. The collective results therefore suggest that cooperative Vop activities are required to achieve cytotoxicity in vitro, or alternatively, that translocon pores destabilize the membrane in a bile dependent manner.     

Olfactory navigation during spawning migrations: a review and introduction of the Hierarchical Navigation Hypothesis

Migrations are characterized by periods of movement that typically rely on orientation towards directional cues. Anadromous fish undergo several different forms of oriented movement during their spawning migration and provide some of the most well‐studied examples of migratory behaviour. During the freshwater phase of the migration, fish locate their spawning grounds via olfactory cues. In this review, we synthesize research that explores the role of olfaction during the spawning migration of anadromous fish, most of which focuses on two families: Salmonidae (salmonids) and Petromyzontidae (lampreys). We draw attention to limitations in this research, and highlight potential areas of investigation that will help fill in current knowledge gaps. We also use the information assembled from our review to formulate a new hypothesis for natal homing in salmonids. Our hypothesis posits that migrating adults rely on three types of cues in a hierarchical fashion: imprinted cues (primary), conspecific cues (secondary), and non‐olfactory environmental cues (tertiary). We provide evidence from previous studies that support this hypothesis. We also discuss future directions of research that can test the hypothesis and further our understanding of the spawning migration.     

Limitations of a morphological criterion of adaptive inference in the fossil record

Experimental analyses directly inform how an anatomical feature or complex functions during an organism's lifetime, which serves to increase the efficacy of comparative studies of living and fossil taxa. In the mammalian skull, food material properties and feeding behaviour have a pronounced influence on the development of the masticatory apparatus. Diet‐related variation in loading magnitude and frequency induce a cascade of changes at the gross, tissue, cellular, protein and genetic levels, with such modelling and remodelling maintaining the integrity of oral structures vis‐à‐vis routine masticatory stresses. Ongoing integrative research using rabbit and rat models of long‐term masticatory plasticity offers unique insight into the limitations of functional interpretations of fossilised remains. Given the general restriction of the palaeontological record to bony elements, we argue that failure to account for the disparity in the hierarchical network of responses of hard versus soft tissues may overestimate the magnitude of the adaptive divergence that is inferred from phenotypic differences. Second, we note that the developmental onset and duration of a loading stimulus associated with a given feeding behaviour can impart large effects on patterns of intraspecific variation that can mirror differences observed among taxa. Indeed, plasticity data are relevant to understanding evolutionary transformations because rabbits raised on different diets exhibit levels of morphological disparity comparable to those found between closely related primate species that vary in diet. Lastly, pronounced variation in joint form, and even joint function, can also characterise adult conspecifics that differ solely in age. In sum, our analyses emphasise the importance of a multi‐site and hierarchical approach to understanding determinants of morphological variation, one which incorporates critical data on performance.     

Circulating ceramides are inversely associated with cardiorespiratory fitness in participants aged 54–96 years from the Baltimore Longitudinal Study of Aging

Cardiorespiratory fitness (VO₂ peak) declines with age and is an independent risk factor for morbidity and mortality in older adults. Identifying biomarkers of low fitness may provide insight for why some individuals experience an accelerated decline of aerobic capacity and may serve as clinically valuable prognostic indicators of cardiovascular health. We investigated the relationship between circulating ceramides and VO₂ peak in 443 men and women (mean age of 69) enrolled in the Baltimore Longitudinal Study of Aging (BLSA). Individual species of ceramide were quantified by HPLC–tandem mass spectrometry. VO₂ peak was measured by a graded treadmill test. We applied multiple regression models to test the associations between ceramide species and VO₂ peak, while adjusting for age, sex, blood pressure, serum LDL, HDL, triglycerides, and other covariates. We found that higher levels of circulating C18:0, C20:0, C24:1 ceramides and C20:0 dihydroceramides were strongly associated with lower aerobic capacity (P < 0.001, P < 0.001, P = 0.018, and P < 0.001, respectively). The associations held true for both sexes (with men having a stronger association than women, P value for sex interaction <0.05) and were unchanged after adjusting for confounders and multiple comparison correction. Interestingly, no significant association was found for C16:0, C22:0, C24:0, C26:0, and C22:1 ceramide species, C24:0 dihydroceramide, or total ceramides. Our analysis reveals that specific long‐chain ceramides strongly associate with low cardiovascular fitness in older adults and may be implicated in the pathogenesis of low fitness with aging. Longitudinal studies are needed to further validate these associations and investigate the relationship between ceramides and health outcomes.     

Case report of whole genome sequencing in the XY female: identification of a novel <Emphasis Type="Italic">SRY</Emphasis> mutation and revision of a misdiagnosis of androgen insensitivity syndrome

Background

The 46,XY female is characterised by a male karyotype and female phenotype arising due to any interruption in the sexual development pathways in utero. The cause is usually genetic and various genes are implicated.

Case presentation

Herein we describe a 46,XY woman who was first diagnosed with androgen insensitivity syndrome (testicular feminisation) at 18 years; however, this was later questioned due to the presence of intact Müllerian structures. The clinical phenotype suggested several susceptibility genes including SRY, DHH, NR5A1, NR0B1, AR, AMH, and AMHR2. To study candidate genes simultaneously, we performed whole genome sequencing. This revealed a novel and likely pathogenic missense variant (p.Arg130Pro, c.389G>C) in SRY, one of the major genes implicated in complete gonadal dysgenesis, hence securing this condition over androgen insensitivity syndrome as the cause of the patient’s disorder of sexual development.

Conclusion

This case highlights the emerging clinical utility of whole genome sequencing as a tool in differentiating disorders of sexual development.

     

Proteome-wide Identification of Novel Ceramide-binding Proteins by Yeast Surface cDNA Display and Deep Sequencing

Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D₂ synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands.The bioactive sphingolipid ceramide is involved in the regulation of a wide variety of cellular processes, including apoptosis, autophagy, and cell cycle progression in cancer (1–3). Ceramide has also been implicated in a number of disease states, including inflammation and inflammatory disorders (4) and neurodegenerative diseases (5).Despite the wide range of processes regulated by ceramide, the precise molecular mechanisms by which ceramide acts as a signaling molecule are not clear. It has been suggested that plasma membrane ceramide acts to stabilize lipid rafts, which act as platforms for the concentration of signaling molecules (6, 7). Another possible mechanism of ceramide signaling is through direct interaction with target proteins. However, relatively few direct protein interactions with ceramide have been described. Examples of proteins that are regulated by direct ceramide binding include KSR (8), Raf-1 (9), protein kinase C-ζ (10), PP2A inhibitor SET (11), and cathepsin D (12). Thus, the identification of additional ceramide-binding proteins could lead to a better mechanistic understanding of how ceramide functions as a signaling molecule.Although various techniques have been used previously, in general, efforts to systematically screen for protein-lipid interactions have proved challenging (13–15). The commonly used yeast two-hybrid system is ineffective when the bait cannot be expressed inside the yeast cell and phage and bacterial display is limited due to prokaryotic expression of eukaryotic proteins. Column-based affinity purification (16, 17) and protein chip methods (18, 19) have been utilized, but they also have drawbacks, including the difficulty in recovering low abundance proteins and cost of setup and quality control (14).We have previously described the generation and application of yeast surface cDNA display libraries to novel protein-ligand discovery (13, 15, 20–24). Here, we describe their application for proteome-wide identification of human ceramide-binding proteins. Utilizing deep sequencing to comprehensively interrogate enriched selection outputs, we have identified a large number of ceramide-binding proteins, many of which represent novel interactions. For example, we have identified and validated EF-hand and STI1 domain-containing proteins as ceramide-specific binding proteins, suggesting that ceramide may regulate cellular pathways by interacting directly with those proteins.     

Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment.