Proteomic Analysis of the Reproductive Organs of the Hermaphroditic Gastropod Lymnaea stagnalis Exposed to Different Endocrine Disrupting Chemicals

Many studies have reported perturbations of mollusc reproduction following exposure to low concentrations (ng/L range) of endocrine disrupting chemicals (EDCs). However, the mechanisms of action of these molecules on molluscs are still poorly understood. Investigation of the modifications of protein expression in organisms exposed to chemicals using proteomic methods can provide a broader and more comprehensive understanding of adverse impacts of pollution on organisms than conventional biochemical biomarkers (e.g., heat-shock proteins, metallothioneins, GST, EROD). In this study we have investigated the impacts of four chemicals, which exhibit different endocrine disrupting properties in vertebrates, on the proteome of the hermaphroditic freshwater pulmonate gastropod Lymnaea stagnalis after 21 days of exposure. Testosterone, tributyltin, chlordecone and cyproterone acetate were chosen as tested compounds as they can induce adverse effects on the reproduction of this snail. The 2D-DIGE method was used to identify proteins whose expression was affected by these compounds. In addition to modifying the expression of proteins involved in the structure and function of the cytoskeleton, chemicals had impacts on the expression of proteins involved in the reproduction of L. stagnalis. Exposure to 19.2 µg/L of chlordecone increased the abundance of ovipostatin, a peptide transmitted during mating through seminal fluid, which reduces oviposition in this species. The expression of yolk ferritin, the vitellogenin equivalent in L. stagnalis, was reduced after exposure to 94.2 ng Sn/L of tributyltin. The identification of yolk ferritin and the modification of its expression in snails exposed to chemicals were refined using western blot analysis. Our results showed that the tested compounds influenced the abundance of yolk ferritin in the reproductive organs. Alteration in proteins involved in reproductive pathways (e.g., ovipostatin and yolk ferritin) could constitute relevant evidence of interaction of EDCs with reproductive pathways that are under the control of the endocrine system of L. stagnalis.     

Monocytes Loaded with Indocyanine Green as Active Homing Contrast Agents?Permit Optical Differentiation of Infectious and Non-Infectious Inflammation

Distinguishing cutaneous infection from sterile inflammation is a diagnostic challenge and currently relies upon subjective interpretation of clinical parameters, microbiological data, and nonspecific imaging. Assessing characteristic variations in leukocytic infiltration may provide more specific information. In this study, we demonstrate that homing of systemically administered monocytes tagged using indocyanine green (ICG), an FDA-approved near infrared dye, may be assessed non-invasively using clinically-applicable laser angiography systems to investigate cutaneous inflammatory processes. RAW 264.7 mouse monocytes co-incubated with ICG fluoresce brightly in the near infrared range. In vitro, the loaded cells retained the ability to chemotax toward monocyte chemotactic protein-1. Following intravascular injection of loaded cells into BALB/c mice with induced sterile inflammation (Complete Freund’s Adjuvant inoculation) or infection (Group A Streptococcus inoculation) of the hind limb, non-invasive whole animal imaging revealed local fluorescence at the inoculation site. There was significantly higher fluorescence of the inoculation site in the infection model than in the inflammation model as early as 2 hours after injection (p<0.05). Microscopic examination of bacterial inoculation site tissue revealed points of near infrared fluorescence, suggesting the presence of ICG-loaded cells. Development of a non-invasive technique to rapidly image inflammatory states without radiation may lead to new tools to distinguish infectious conditions from sterile inflammatory conditions at the bedside.     

Alcohol Impairs Predation Risk Response and Communication in Zebrafish

The effects of ethanol exposure on Danio rerio have been studied from the perspectives of developmental biology and behavior. However, little is known about the effects of ethanol on the prey-predator relationship and chemical communication of predation risk. Here, we showed that visual contact with a predator triggers stress axis activation in zebrafish. We also observed a typical stress response in zebrafish receiving water from these conspecifics, indicating that these fish chemically communicate predation risk. Our work is the first to demonstrate how alcohol effects this prey-predator interaction. We showed for the first time that alcohol exposure completely blocks stress axis activation in both fish seeing the predator and in fish that come in indirect contact with a predator by receiving water from these conspecifics. Together with other research results and with the translational relevance of this fish species, our data points to zebrafish as a promising animal model to study human alcoholism.     

Production of a Dominant-Negative Fragment Due to G3BP1 Cleavage Contributes to the Disruption of Mitochondria-Associated Protective Stress Granules during CVB3 Infection

Stress granules (SGs) are dynamic cytosolic aggregates containing messenger ribonucleoproteins and target poly-adenylated (A)-mRNA. A key component of SGs is Ras-GAP SH3 domain binding protein-1 (G3BP1), which in part mediates protein-protein and protein-RNA interactions. SGs are modulated during infection by several viruses, however, the function and significance of this process remains poorly understood. In this study, we investigated the interplay between SGs and Coxsackievirus type B3 (CVB3), a member of the Picornaviridae family. Our studies demonstrated that SGs were formed early during CVB3 infection; however, G3BP1-positive SGs were actively disassembled at 5 hrs post-infection, while poly(A)-positive RNA granules persisted. Furthermore, we confirmed G3BP1 cleavage by 3C^pro at Q325. We also demonstrated that overexpression of G3BP1-SGs negatively impacted viral replication at the RNA, protein, and viral progeny levels. Using electron microscopy techniques, we showed that G3BP1-positive SGs localized near mitochondrial surfaces. Finally, we provided evidence that the C-terminal cleavage product of G3BP1 inhibited SG formation and promoted CVB3 replication. Taken together, we conclude that CVB3 infection selectively targets G3BP1-SGs by cleaving G3BP1 to produce a dominant-negative fragment that further inhibits G3BP1-SG formation and facilitates viral replication.     

PUMA: A Unified Framework for Penalized Multiple Regression Analysis of GWAS Data

Penalized Multiple Regression (PMR) can be used to discover novel disease associations in GWAS datasets. In practice, proposed PMR methods have not been able to identify well-supported associations in GWAS that are undetectable by standard association tests and thus these methods are not widely applied. Here, we present a combined algorithmic and heuristic framework for PUMA (Penalized Unified Multiple-locus Association) analysis that solves the problems of previously proposed methods including computational speed, poor performance on genome-scale simulated data, and identification of too many associations for real data to be biologically plausible. The framework includes a new minorize-maximization (MM) algorithm for generalized linear models (GLM) combined with heuristic model selection and testing methods for identification of robust associations. The PUMA framework implements the penalized maximum likelihood penalties previously proposed for GWAS analysis (i.e. Lasso, Adaptive Lasso, NEG, MCP), as well as a penalty that has not been previously applied to GWAS (i.e. LOG). Using simulations that closely mirror real GWAS data, we show that our framework has high performance and reliably increases power to detect weak associations, while existing PMR methods can perform worse than single marker testing in overall performance. To demonstrate the empirical value of PUMA, we analyzed GWAS data for type 1 diabetes, Crohns''s disease, and rheumatoid arthritis, three autoimmune diseases from the original Wellcome Trust Case Control Consortium. Our analysis replicates known associations for these diseases and we discover novel etiologically relevant susceptibility loci that are invisible to standard single marker tests, including six novel associations implicating genes involved in pancreatic function, insulin pathways and immune-cell function in type 1 diabetes; three novel associations implicating genes in pro- and anti-inflammatory pathways in Crohn''s disease; and one novel association implicating a gene involved in apoptosis pathways in rheumatoid arthritis. We provide software for applying our PUMA analysis framework.     

Novel Methods for Analysing Bacterial Tracks Reveal Persistence in Rhodobacter sphaeroides

Tracking bacteria using video microscopy is a powerful experimental approach to probe their motile behaviour. The trajectories obtained contain much information relating to the complex patterns of bacterial motility. However, methods for the quantitative analysis of such data are limited. Most swimming bacteria move in approximately straight lines, interspersed with random reorientation phases. It is therefore necessary to segment observed tracks into swimming and reorientation phases to extract useful statistics. We present novel robust analysis tools to discern these two phases in tracks. Our methods comprise a simple and effective protocol for removing spurious tracks from tracking datasets, followed by analysis based on a two-state hidden Markov model, taking advantage of the availability of mutant strains that exhibit swimming-only or reorientating-only motion to generate an empirical prior distribution. Using simulated tracks with varying levels of added noise, we validate our methods and compare them with an existing heuristic method. To our knowledge this is the first example of a systematic assessment of analysis methods in this field. The new methods are substantially more robust to noise and introduce less systematic bias than the heuristic method. We apply our methods to tracks obtained from the bacterial species Rhodobacter sphaeroides and Escherichia coli. Our results demonstrate that R. sphaeroides exhibits persistence over the course of a tumbling event, which is a novel result with important implications in the study of this and similar species.     

Reconstructing Native American Migrations from Whole-Genome and Whole-Exome Data

There is great scientific and popular interest in understanding the genetic history of populations in the Americas. We wish to understand when different regions of the continent were inhabited, where settlers came from, and how current inhabitants relate genetically to earlier populations. Recent studies unraveled parts of the genetic history of the continent using genotyping arrays and uniparental markers. The 1000 Genomes Project provides a unique opportunity for improving our understanding of population genetic history by providing over a hundred sequenced low coverage genomes and exomes from Colombian (CLM), Mexican-American (MXL), and Puerto Rican (PUR) populations. Here, we explore the genomic contributions of African, European, and especially Native American ancestry to these populations. Estimated Native American ancestry is in MXL, in CLM, and in PUR. Native American ancestry in PUR is most closely related to populations surrounding the Orinoco River basin, confirming the Southern America ancestry of the Taíno people of the Caribbean. We present new methods to estimate the allele frequencies in the Native American fraction of the populations, and model their distribution using a demographic model for three ancestral Native American populations. These ancestral populations likely split in close succession: the most likely scenario, based on a peopling of the Americas thousand years ago (kya), supports that the MXL Ancestors split kya, with a subsequent split of the ancestors to CLM and PUR kya. The model also features effective populations of in Mexico, in Colombia, and in Puerto Rico. Modeling Identity-by-descent (IBD) and ancestry tract length, we show that post-contact populations also differ markedly in their effective sizes and migration patterns, with Puerto Rico showing the smallest effective size and the earlier migration from Europe. Finally, we compare IBD and ancestry assignments to find evidence for relatedness among European founders to the three populations.