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Fish papillomaviruses form a newly discovered group broadly recognized as the Secondpapillomavirinae subfamily. This study expands the documented genomes of the fish papillomaviruses from six to 16, including one from the Antarctic emerald notothen, seven from commercial market fishes, one from data mining of sea bream sequence data, and one from a western gull cloacal swab that is likely diet derived. The genomes of secondpapillomaviruses are ∼6 kilobasepairs (kb), which is substantially smaller than the ∼8 kb of terrestrial vertebrate papillomaviruses. Each genome encodes a clear homolog of the four canonical papillomavirus genes, E1, E2, L1, and L2. In addition, we identified open reading frames (ORFs) with short linear peptide motifs reminiscent of E6/E7 oncoproteins. Fish papillomaviruses are extremely diverse and phylogenetically distant from other papillomaviruses suggesting a model in which terrestrial vertebrate-infecting papillomaviruses arose after an evolutionary bottleneck event, possibly during the water-to-land transition.

MicroRNAs (miRNAs) are important gene expression regulators implicated in many biological processes, but we lack a global understanding of how miRNA genes evolve and contribute to developmental canalization and phenotypic diversification. Whole-genome duplication events likely provide a substrate for species divergence and phenotypic change by increasing gene numbers and relaxing evolutionary pressures. To understand the consequences of genome duplication on miRNA evolution, we studied miRNA genes following the teleost genome duplication (TGD). Analysis of miRNA genes in four teleosts and in spotted gar, whose lineage diverged before the TGD, revealed that miRNA genes were retained in ohnologous pairs more frequently than protein-coding genes, and that gene losses occurred rapidly after the TGD. Genomic context influenced retention rates, with clustered miRNA genes retained more often than nonclustered miRNA genes and intergenic miRNA genes retained more frequently than intragenic miRNA genes, which often shared the evolutionary fate of their protein-coding host. Expression analyses revealed both conserved and divergent expression patterns across species in line with miRNA functions in phenotypic canalization and diversification, respectively. Finally, major strands of miRNA genes experienced stronger purifying selection, especially in their seeds and 3′-complementary regions, compared with minor strands, which nonetheless also displayed evolutionary features compatible with constrained function. This study provides the first genome-wide, multispecies analysis of the mechanisms influencing metazoan miRNA evolution after whole-genome duplication.

Efforts to study the effects of environmental change on early development of Antarctic fishes would benefit from the ability to control reproduction by in vitro fertilization. Here, we established a spectrophotometric method to determine spermatozoa concentration in the semen of the blackfin icefish Chaenocephalus aceratus and we evaluated the potential for short (refrigeration) and long-term (cryopreservation) storage. The former will maximize the use of semen over several hours and the latter alleviate the risk that semen is unavailable when a spawning female is available. Seminal plasma osmolality and pH of GnRH-injected male semen were 423.65 ± 35.6 mOsm kg−1 and 8.05 ± 0.11, respectively. The predictive power of regression models between spermatozoa concentration and absorbance was determined from 200 to 600 nm and revealed a best-fitted correlation at 505 nm. The standard curve was tested against measured concentrations and the average difference between measured and estimated concentrations was 0.27 ± 10.23%. Semen could be kept on ice for up to 6 h with minimal loss ( 90% post-thawing motility using a salt-based extender (pH 7.5–7.85 and ~ 280 mOsm kg−1) and 10% DMSO after 72 h immersed in liquid nitrogen. Spermatozoa morphometry revealed significant inter-individual variability. Average head length, head width, and flagella length were 3.78 ± 1.03, 2.87 ± 0.78, and 17.75 ± 7.15 µm, respectively. Spermatids composed 9.7 ± 0.8% of total spermatogenic cells, probably indicating active spermatogenesis, but perhaps resulting to some degree from the semen extraction technique used. Variations on these baseline methods may facilitate semen preservation and ultimately in vitro fertilization in this and other notothenioid species, thereby enhancing our understanding of the responses of early-life stages of Antarctic fishes to future warming of the Southern Ocean.

Antarctic dragonfishes (Bathydraconidae) of the suborder Notothenioidei are found only in the Southern Ocean where they diversified in habitats from the surface to the bathypelagic zone thousands of meters deep. Among dragonfishes, the pelagic Gymnodraconinae sister species Acanthodraco dewitti and Psilodraco breviceps remain poorly known. Although A. dewitti is thought to be restricted to Antarctic waters and P. breviceps to be endemic to South Georgia Island, several P. breviceps specimens have occasionally been reported in coastal Antarctica. Here we investigated the molecular genetic identity of the two species and their geographic distribution. Three mitochondrial genetic markers (mt-cyb, mt-co1, and mt-nd2) identified two dragonfish larvae collected on the West Antarctic Peninsula as A. dewitti and showed that all six specimens with available genetic data and reported to be P. breviceps collected in Antarctic waters were also A. dewitti. These results support the allopatric distribution of the two species, with P. breviceps being endemic to South Georgia Island and A. dewitti being endemic to Antarctic waters, potentially with a circumpolar distribution. The biogeography of the sister species A. dewitti and P. breviceps is likely similar to the allopatric distribution of the congeneric sister dragonfish species Parachaenichthys charcoti and P. georgianus. These considerations suggest that the Antarctic Circumpolar Current may geographically isolate the sub-Antarctic and Antarctic species of both sister species pairs, limiting gene flow and promoting speciation. Furthermore, we provide a detailed description of the A. dewitti larvae to supply characteristic morphological features differentiating A. dewitti and P. breviceps larvae.

Background Circulating miRNAs (c-miRNAs) are found in most, if not all, biological fluids and are becoming well-established non-invasive biomarkers of many human pathologies. However, their features in non-pathological contexts and whether their expression profiles reflect normal life history events have received little attention, especially in non-mammalian species. The aim of the present study was to investigate the potential of c-miRNAs to serve as biomarkers of reproductive and metabolic states in fish. Results The blood plasma was sampled throughout the reproductive cycle of female rainbow trout subjected to two different feeding regimes that triggered contrasting metabolic states. In addition, ovarian fluid was sampled at ovulation, and all samples were subjected to small RNA-seq analysis, leading to the establishment of a comprehensive miRNA repertoire (i.e., miRNAome) and enabling subsequent comparative analyses to a panel of RNA-seq libraries from a wide variety of tissues and organs. We showed that biological fluid miRNAomes are complex and encompass a high proportion of the overall rainbow trout miRNAome. While sharing a high proportion of common miRNAs, the blood plasma and ovarian fluid miRNAomes exhibited strong fluid-specific signatures. We further revealed that the blood plasma miRNAome significantly changed depending on metabolic and reproductive states. We subsequently identified three evolutionarily conserved muscle-specific miRNAs or myomiRs (miR-1-1/2-3p, miR-133a-1/2-3p, and miR-206-3p) that accumulated in the blood plasma in response to high feeding rates, making these myomiRs strong candidate biomarkers of active myogenesis. We also identified miR-202-5p as a candidate biomarker for reproductive success that could be used to predict ovulation and/or egg quality. Conclusions Together, these promising results reveal the high potential of c-miRNAs, including evolutionarily conserved myomiRs, as physiologically relevant biomarker candidates and pave the way for the use of c-miRNAs for non-invasive phenotyping in various fish species.

International audience; BACKGROUND AND OBJECTIVES: Rituximab is an anti-CD20 monoclonal antibody approved in several diseases, including chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), rheumatoid arthritis (RA), and anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). The influence of underlying disease on rituximab pharmacokinetics has never been investigated for several cancer and non-cancer diseases simultaneously. This study aimed at assessing this influence using an integrated semi-mechanistic model accounting for target-mediated elimination of rituximab. METHODS: Rituximab concentration-time data from five studies previously published in patients with CLL, DLBCL, FL, RA, and AAV were described using a two-compartment model with irreversible binding of rituximab to its target antigen. Both underlying disease and target antigen measurements were assessed as covariates. RESULTS: Central volume of distribution was [95% confidence interval] 1.7-fold [1.6-1.9] higher in DLBCL than in RA, FL, and CLL, and it was 1.8-fold [1.6-2.1] higher in RA, FL, and CLL than in AAV. First-order elimination rate constants were 1.8-fold [1.7-2.0] and 1.3-fold [1.2-1.5] higher in RA, DLBCL, and FL than in CLL and AAV, respectively. Baseline latent antigen level (L(0)) was 54-fold [30-94], 20-fold [11-36], and 29-fold [14-64] higher in CLL, DLBCL, and FL, respectively, than in RA and AAV. In lymphoma, L(0) increased with baseline total metabolic tumor volume (p = 6.10(-7)). In CLL, the second-order target-mediated elimination rate constant (k(deg)) increased with baseline CD20 count on circulating B cells (CD20(cir), p = 0.0081). CONCLUSIONS: Our results show for the first time that rituximab pharmacokinetics is strongly influenced by underlying disease and disease activity. Notably, neoplasms are associated with higher antigen amounts that result in decreased exposure to rituximab compared to inflammatory diseases. Our model might be used to estimate unbound target amounts in upcoming studies.

Long-read sequencing is driving a new reality for genome science where highly contiguous assemblies can be produced efficiently with modest resources. Genome assemblies from long-read sequences are particularly exciting for understanding the evolution of complex genomic regions that are often difficult to assemble. In this study, we leveraged long-read sequencing data to generate a high-quality genome assembly for an Antarctic eelpout, Opthalmolycus amberensis, the first for the globally distributed family Zoarcidae. We used this assembly to understand how O. amberensis has adapted to the harsh Southern Ocean and compared it to another group of Antarctic fishes: the notothenioids. We showed that selection has largely acted on different targets in eelpouts relative to notothenioids. However, we did find some overlap; in both groups, genes involved in membrane structure, thermal tolerance, and vision have evidence of selection. We found evidence for historical shifts of transposable element activity in O. amberensis and other polar fishes, perhaps reflecting a response to environmental change. We were specifically interested in the evolution of two complex genomic loci known to underlie key adaptations to polar seas: hemoglobin and antifreeze proteins (AFPs). We observed unique evolution of the hemoglobin MN cluster in eelpouts and related fishes in the suborder Zoarcoidei relative to other Perciformes. For AFPs, we identified the first species in the suborder with no evidence of afpIII sequences (Cebidichthys violaceus) in the genomic region where they are found in all other Zoarcoidei, potentially reflecting a lineage-specific loss of this cluster. Beyond polar fishes, our results highlight the power of long-read sequencing to understand genome evolution.

We report on 2 cousins, a girl and a boy, born to first-cousin Lebanese parents with Hamamy syndrome, exhibiting developmental delay, intellectual disability, severe telecanthus, abnormal ears, dentinogenesis imperfecta, and bone fragility. Whole-exome sequencing studies performed on the 2 affected individuals and one obligate carrier revealed the presence of a homozygous c.503G>A (p.Arg168His) missense mutation in IRX5 in both sibs, not reported in any other family. Review of the literature and differential diagnoses are discussed.

The role of osteoblast placement in skeletal morphological variation is relatively well understood, but alternative developmental mechanisms affecting bone shape remain largely unknown. Specifically, very little attention has been paid to variation in later mineralization stages of intramembranous ossification as a driver of morphological diversity. We discover the occurrence of specific, sometimes large, regions of nonmineralized osteoid within bones that also contain mineralized tissue. We show through a variety of histological, molecular, and tomographic tests that this “extended” osteoid material is most likely nonmineralized bone matrix. This tissue type is a significant determinant of gill cover bone shape in the teleostean suborder Cottoidei. We demonstrate repeated evolution of extended osteoid in Cottoidei through ancestral state reconstruction and test for an association between extended osteoid variation and habitat differences among species. Through measurement of extended osteoid at various stages of gill cover development in species across the phylogeny, we gain insight into possible evolutionary developmental origins of the trait. We conclude that this fine-tuned developmental regulation of bone matrix mineralization reflects heterochrony at multiple biological levels and is a novel mechanism for the evolution of diversity in skeletal morphology. This research lays the groundwork for a new model in which to study bone mineralization and evolutionary developmental processes, particularly as they may relate to adaptation during a prominent evolutionary radiation of fishes.

MicroRNAs (miRNAs) can have tissue-specific expression and functions; they can originate from dedicated miRNA genes, from non-canonical miRNA genes, or from mirror-miRNA genes and can also experience post-transcriptional variations. It remains unclear, however, which mechanisms of miRNA production or modification are tissue-specific and the extent of their evolutionary conservation. To address these issues, we developed the software Prost! (PRocessing Of Short Transcripts), which, among other features, allows accurate quantification of mature miRNAs, takes into account post-transcriptional processing, such as nucleotide editing, and helps identify mirror-miRNAs. Here, we applied Prost! to annotate and analyze miRNAs in three-spined stickleback (Gasterosteus aculeatus), a model fish for evolutionary biology reported to have a miRNome larger than most teleost fish. Zebrafish (Danio rerio), a distantly related teleost with a well-known miRNome, served as comparator. Despite reports suggesting that stickleback had a large miRNome, results showed that stickleback has 277 evolutionary-conserved mir genes and 366 unique mature miRNAs (excluding mir430 gene replicates and the vaultRNA-derived mir733), similar to zebrafish. In addition, small RNA sequencing data from brain, heart, testis, and ovary in both stickleback and zebrafish identified suites of mature miRNAs that display organ-specific enrichment, which is, for many miRNAs, evolutionarily-conserved. These data also supported the hypothesis that evolutionarily-conserved, organ-specific mechanisms regulate miRNA post-transcriptional variations. In both stickleback and zebrafish, miR2188-5p was edited frequently with similar nucleotide editing patterns in the seed sequence in various tissues, and the editing rate was organ-specific with higher editing in the brain. In summary, Prost! is a critical new tool to identify and understand small RNAs and can help clarify a species’ miRNA biology, as shown here for an important fish model for the evolution of developmental mechanisms, and can provide insight into organ-specific expression and evolutionary-conserved miRNA post-transcriptional mechanisms.

Ancestors of the Antarctic icefishes (family Channichthyidae) were benthic and had no swim bladder, making it energetically expensive to rise from the ocean floor. To exploit the water column, benthopelagic icefishes were hypothesized to have evolved a skeleton with “reduced bone,” which gross anatomical data supported. Here, we tested the hypothesis that changes to icefish bones also occurred below the level of gross anatomy. Histology and micro‐CT imaging of representative craniofacial bones (i.e., ceratohyal, frontal, dentary, and articular) of extant Antarctic fish species specifically evaluated two features that might cause the appearance of “reduced bone”: bone microstructure (e.g., bone volume fraction and structure linear density) and bone mineral density (BMD, or mass of mineral per volume of bone). Measures of bone microstructure were not consistently different in bones from the icefishes Chaenocephalus aceratus and Champsocephalus gunnari, compared to the related benthic notothenioids Notothenia coriiceps and Gobionotothen gibberifrons. Some quantitative measures, such as bone volume fraction and structure linear density, were significantly increased in some icefish bones compared to homologous bones of non‐icefish. However, such differences were rare, and no microstructural measures were consistently different in icefishes across all bones and species analyzed. Furthermore, BMD was similar among homologous bones of icefish and non‐icefish Antarctic notothenioids. In summary, “reduced bone” in icefishes was not due to systemic changes in bone microstructure or BMD, raising the prospect that “reduced bone” in icefish occurs only at the gross anatomic level (i.e., smaller or fewer bones). Given that icefishes exhibit delayed skeletal development compared to non‐icefish Antarctic fishes, combining these phenotypic data with genomic data might clarify genetic changes driving skeletal heterochrony., Antarctic icefishes are amazing examples of evolutionary adaptation. Here, we show quantitatively that icefish skeletons did not change bone microstructure or bone mineral density, relative to closely related fish species.

International audience; Structured abstract Background & aim: Resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer (CRC) is frequent and prognostic biomarkers are lacking. MicroRNAs (miR) are good candidates in this context. We aimed to characterize cetuximab and panitumumab exposure influence on miR expression in colorectal cancer cells to identify those regulating the EGFR pathway and implicated in resistance to treatment. Finally, we aimed to identify miR expression in serum of patients with advanced CRC treated with cetuximab or panitumumab. Results: Cetuximab and panitumumab exposure induced significant expression variations of 17 miR out of a miRnome panel of 752. Six of those miR interacted with at least one downstream element of the EGFR pathway. Conclusion: After the bioinformatics two-phase process, 5 miR rarely described before could be potential actors of anti-EGFR monoclonal antibody resistance: miR-95-3p, miR-139-5p, miR-145-5p, miR-429 and miR-1247-5p. In vivo, we detected the expression of miR-139-5p and miR-145-5p in serum of patients with metastatic CRC.

International audience; Southern Ocean ecosystems are globally important and vulnerable to global drivers of change, yet they remain challenging to study. Fish and squid make up a significant portion of the biomass within the Southern Ocean, filling key roles in food webs from forage to mid-trophic species and top predators. They comprise a diverse array of species uniquely adapted to the extreme habitats of the region. Adaptations such as antifreeze glycoproteins, lipid-retention, extended larval phases, delayed senescence, and energy-conserving life strategies equip Antarctic fish and squid to withstand the dark winters and yearlong subzero temperatures experienced in much of the Southern Ocean. In addition to krill exploitation, the comparatively high commercial value of Antarctic fish, particularly the lucrative toothfish, drives fisheries interests, which has included illegal fishing. Uncertainty about the population dynamics of target species and ecosystem structure and function more broadly has necessitated a precautionary, ecosystem approach to managing these stocks and enabling the recovery of depleted species. Fisheries currently remain the major local driver of change in Southern Ocean fish productivity, but global climate change presents an even greater challenge to assessing future changes. Parts of the Southern Ocean are experiencing ocean-warming, such as the West Antarctic Peninsula, while other areas, such as the Ross Sea shelf, have undergone cooling in recent years. These trends are expected to result in a redistribution of species based on their tolerances to different temperature regimes. Climate variability may impair the migratory response of these species to environmental change, while imposing increased pressures on recruitment. Fisheries and climate change, coupled with related local and global drivers such as pollution and sea ice change, have the potential to produce synergistic impacts that compound the risks to Antarctic fish and squid species. The uncertainty surrounding how different species will respond to these challenges, given their varying life histories, environmental dependencies, and resiliencies, necessitates regular assessment to inform conservation and management decisions. Urgent attention is needed to determine whether the current management strategies are suitably precautionary to achieve conservation objectives in light of the impending changes to the ecosystem.

Vibratory behaviours are widespread in social insects, but the produced vibrations remain poorly explored. Communication using vibrations is an efficient way to transmit information in subterranean environments where visual and odorant signals are less efficient. In termites, different vibratory behaviours are performed in different contexts like reproductive regulation and alarm signalling, but only few studies explored the structure of the produced vibrations (i.e., duration, number of pulses, amplitude). Here, we described several types of vibrations produced by a vibratory behaviour widespread in termites (body-shaking), which can be transmitted through the substrate and detected by other colony members. We analysed the structures of the emitted vibrations and the occurrence of the body-shaking events in presence/absence of reproductives and/or in presence/absence of a stress stimuli (flashlight) in the subterranean termite Reticulitermes flavipes. Interestingly, only the presence of the reproductives did influence the number of pulses and the duration of the emitted vibrations. Moreover, the first part of the emitted vibrations seems to be enough to encode reproductive information, but other parts might hold other type of information. Body-shaking occurrence did increase in presence of reproductives but only briefly under a flashlight. These results show that vibratory cues are complex in termites and their diversity might encode a plurality of social cues.

Circulating miRNAs (c-miRNAs) are found in most, if not all, biological fluids and are becoming well established biomarkers of many human pathologies. The aim of the present study was to investigate the potential of c-miRNAs as biomarkers of reproductive and metabolic states in fish, a question that has received little attention. Plasma was collected throughout the reproductive cycle from rainbow trout females subjected to two different feeding levels to trigger contrasting metabolic states; ovarian fluid was sample at ovulation. Fluid samples were subjected to small RNA-seq analysis followed by quantitative PCR validation for a subset of promising c-miRNA biomarkers. A comprehensive miRNA repertoire, which was lacking in trout, was first established to allow subsequent analysis. We first showed that biological fluids miRNAomes are complex and encompass a high proportion of the overall species miRNAome. While sharing a high proportion of common miRNAs, plasma and ovarian fluid miRNAomes exhibited strong fluid-specific signatures. We further showed that the plasma miRNAome exhibited major significant changes depending on metabolic and reproductive state. We subsequently identified three (miR-1-1/2-3p, miR-133-a-1/2-3p and miR-206-3p) evolutionarily conserved muscle-specific miRNA that accumulate in the plasma in response to high feeding rates, making these myomiRs strong candidate biomarkers of active myogenesis. We also identified miR-202-5p as a candidate biomarker for reproductive success that could be used to predict ovulation and/or egg quality. These highly promising results reveal the high potential of c-miRNAs as physiologically relevant biomarkers and pave the way for the use of c-miRNAs for non-invasive phenotyping in various fish species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231's early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231's efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models.Importance:The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

Both SARS-CoV-2 infection and vaccination elicit potent immune responses. A number of studies have described immune responses to SARS-CoV-2 infection. However, beyond antibody production, immune responses to COVID-19 vaccines remain largely uncharacterized. Here, we performed multimodal single-cell sequencing on peripheral blood of patients with acute COVID-19 and healthy volunteers before and after receiving the SARS-CoV-2 BNT162b2 mRNA vaccine to compare the immune responses elicited by the virus and by this vaccine. Phenotypic and transcriptional profiling of immune cells, coupled with reconstruction of the B and T cell antigen receptor rearrangement of individual lymphocytes, enabled us to characterize and compare the host responses to the virus and to defined viral antigens. While both infection and vaccination induced robust innate and adaptive immune responses, our analysis revealed significant qualitative differences between the two types of immune challenges. In COVID-19 patients, immune responses were characterized by a highly augmented interferon response which was largely absent in vaccine recipients. Increased interferon signaling likely contributed to the observed dramatic upregulation of cytotoxic genes in the peripheral T cells and innate-like lymphocytes in patients but not in immunized subjects. Analysis of B and T cell receptor repertoires revealed that while the majority of clonal B and T cells in COVID-19 patients were effector cells, in vaccine recipients clonally expanded cells were primarily circulating memory cells. Importantly, the divergence in immune subsets engaged, the transcriptional differences in key immune populations, and the differences in maturation of adaptive immune cells revealed by our analysis have far-ranging implications for immunity to this novel pathogen.

Model organism research is essential to understand disease mechanisms. However, laboratory-induced genetic models can lack genetic variation and often fail to mimic the spectrum of disease severity. Evolutionary mutant models (EMMs) are species with evolved phenotypes that mimic human disease. EMMs complement traditional laboratory models by providing unique avenues to study gene-by-environment interactions, modular mutations in noncoding regions, and their evolved compensations. EMMs have improved our understanding of complex diseases, including cancer, diabetes, and aging, and illuminated mechanisms in many organs. Rapid advancements of sequencing and genome-editing technologies have catapulted the utility of EMMs, particularly in fish. Fish are the most diverse group of vertebrates, exhibiting a kaleidoscope of specialized phenotypes, many that would be pathogenic in humans but are adaptive in the species' specialized habitat. Importantly, evolved compensations can suggest avenues for novel disease therapies. This review summarizes current research using fish EMMs to advance our understanding of human disease.

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization.

Made available in DSpace on 2021-06-25T11:54:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-09 National Institute of Health (USA) Deutsche Forschungsgemeinschaft National Science Foundation (USA) Office of Polar Programs Agence Nationale de la Recherche Schweizerischer Nationalfonds zur Forderung der Wissenschaftlichen Forschung The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish. IINRAE, LPGP, Rennes, France Univ Lausanne, Dept Ecol & Evolut, Lausanne, Switzerland Swiss Inst Bioinformat, Lausanne, Switzerland Hunan Normal Univ, Coll Life Sci, State Key Lab Dev Biol Freshwater Fish, Changsha, Peoples R China Uppsala Univ, Dept Organismal Biol, Systemat Biol, Uppsala, Sweden Univ Montpellier, Inst Genom Fonct, INSERM, IGF,CNRS, Montpellier, France INRAE, Math & Informat Appl Toulouse, SIGENAE, Castanet Tolosan, France Univ Wurzburg, Physiol Chem, Bioctr, Wurzburg, Germany Texas State Univ, Xiphophorus Genet Stock Ctr, Dept Chem & Biochem, San Marcos, TX USA Univ Wurzburg, Dev Biochem, Bioctr, Wurzburg, Germany Univ Oregon, Inst Neurosci, Eugene, OR USA NOAA, Environm & Fisheries Sci Div, Northwest Fisheries Sci Ctr, Natl Marine Fisheries Serv, Seattle, WA USA Sophia Univ, Fac Sci & Technol, Dept Mat & Life Sci, Tokyo, Japan Northeastern Univ, Ctr Marine Sci, Dept Marine & Environm Sci, Nahant, MA 01908 USA Sao Paulo State Univ, Inst Biosci, Dept Struct & Funct Biol, Reprod & Mol Biol Grp, Botucatu, SP, Brazil Tokyo Univ Marine Sci & Technol, Dept Aquat Marine Biosci, Tokyo, Japan Inst Marine Res, Bergen, Norway Chinese Acad Sci, Inst Hydrobiol, Beijing, Peoples R China IGB, Leibniz Inst Freshwater Ecol & Inland Fishe, Berlin, Germany Michigan State Univ, Dept Integrat Biol, Ecol Evolut & Behav Program, E Lansing, MI 48824 USA Sao Paulo State Univ, Inst Biosci, Dept Struct & Funct Biol, Reprod & Mol Biol Grp, Botucatu, SP, Brazil National Institute of Health (USA): R01GM085318 National Institute of Health (USA): R35GM139635 Deutsche Forschungsgemeinschaft: SCHA 408/10-1 National Science Foundation (USA) Office of Polar Programs: OPP-1543383 National Science Foundation (USA) Office of Polar Programs: PLR-1247510 National Science Foundation (USA) Office of Polar Programs: PLR-1444167 Agence Nationale de la Recherche: SCHA 408/10-1 Agence Nationale de la Recherche: ANR-10-INBS-09 Schweizerischer Nationalfonds zur Forderung der Wissenschaftlichen Forschung: PP00P3_170664

International audience; In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.

Modern genetic data sets present unprecedented opportunities to understand the evolutionary origins of taxonomic groups comprising hundreds to thousands of species. When the timing of key events are known, it is also possible to investigate biogeographic history in the context of major phenomena (e.g., continental drift). In this study, we investigated the biogeographic history of the suborder Zoarcoidei, a globally distributed fish group that includes species inhabiting both poles and multiple taxa that produce antifreeze proteins to survive chronic subfreezing temperatures. We first generated a multi-locus, time-calibrated phylogeny for the group. We then used biogeographic modeling to reconstruct ancestral ranges across the tree and quantify the type and frequency of biogeographic events (e.g., founder, dispersal). With these results, we considered how the cooling of the Southern and Arctic Oceans, which reached their present-day subfreezing temperatures 10-15 million years ago (Mya) and 2-3 Mya, respectively, may have shaped the evolutionary history of Zoarcoidei, with an emphasis on the most speciose and widely distributed family, eelpouts (family Zoarcidae). Our phylogenetic results clarified standing issues in the Zoarcoidei taxonomy and showed that the group began to diversify in the Oligocene ∼31-32 Mya, with the center of origin for all families in north temperate waters. Within-area speciation was the most common biogeographic event in the group’s history (80% of all events) followed by dispersal (20%). Finally, we found mixed evidence for polar ocean cooling underpinning Zoarcoidei diversification, with support limited to eelpout speciation in the Southern Ocean over the last 10 million years.

Both SARS-CoV-2 infection and vaccination elicit potent immune responses. A number of studies have described immune responses to SARS-CoV-2 infection. However, beyond antibody production, immune responses to COVID-19 vaccines remain largely uncharacterized. Here, we performed multimodal single-cell sequencing on peripheral blood of patients with acute COVID-19 and healthy volunteers before and after receiving the SARS-CoV-2 BNT162b2 mRNA vaccine to compare the immune responses elicited by the virus and by this vaccine. Phenotypic and transcriptional profiling of immune cells, coupled with reconstruction of the B and T cell antigen receptor rearrangement of individual lymphocytes, enabled us to characterize and compare the host responses to the virus and to defined viral antigens. While both infection and vaccination induced robust innate and adaptive immune responses, our analysis revealed significant qualitative differences between the two types of immune challenges. In COVID-19 patients, immune responses were characterized by a highly augmented interferon response which was largely absent in vaccine recipients. Increased interferon signaling likely contributed to the observed dramatic upregulation of cytotoxic genes in the peripheral T cells and innate-like lymphocytes in patients but not in immunized subjects. Analysis of B and T cell receptor repertoires revealed that while the majority of clonal B and T cells in COVID-19 patients were effector cells, in vaccine recipients clonally expanded cells were primarily circulating memory cells. Importantly, the divergence in immune subsets engaged, the transcriptional differences in key immune populations, and the differences in maturation of adaptive immune cells revealed by our analysis have far-ranging implications for immunity to this novel pathogen.

People with NR5A1 mutations experience testicular dysgenesis, ovotestes, or adrenal insufficiency, but we do not completely understand the origin of this phenotypic diversity. NR5A1 is expressed in gonadal soma precursor cells before expression of the sex-determining gene SRY. Many fish have two co-orthologs of NR5A1 that likely partitioned ancestral gene subfunctions between them. To explore ancestral roles of NR5A1, we knocked out nr5a1a and nr5a1b in zebrafish. Single-cell RNA-seq identified nr5a1a-expressing cells that co-expressed genes for steroid biosynthesis and the chemokine receptor Cxcl12a in 1-day postfertilization (dpf) embryos, as does the mammalian adrenal–gonadal (interrenal-gonadal) primordium. In 2dpf embryos, nr5a1a was expressed stronger in the interrenal-gonadal primordium than in the early hypothalamus but nr5a1b showed the reverse. Adult Leydig cells expressed both ohnologs and granulosa cells expressed nr5a1a stronger than nr5a1b. Mutants for nr5a1a lacked the interrenal, formed incompletely differentiated testes, had no Leydig cells, and grew far larger than normal fish. Mutants for nr5a1b formed a disorganized interrenal and their gonads completely disappeared. All homozygous mutant genotypes lacked secondary sex characteristics, including male breeding tubercles and female sex papillae, and had exceedingly low levels of estradiol, 11-ketotestosterone, and cortisol. RNA-seq showed that at 21dpf, some animals were developing as females and others were not, independent of nr5a1 genotype. By 35dpf, all mutant genotypes greatly under-expressed ovary-biased genes. Because adult nr5a1a mutants form gonads but lack an interrenal and conversely, adult nr5a1b mutants lack a gonad but have an interrenal, the adrenal, and gonadal functions of the ancestral nr5a1 gene partitioned between ohnologs after the teleost genome duplication, likely owing to reciprocal loss of ancestral tissue-specific regulatory elements. Identifying such elements could provide hints to otherwise unexplained cases of Differences in Sex Development.

Calcific aortic valve disease (CAVD) is a significant cause of illness and death worldwide. Identification of early predictive markers could help optimize patient management. RNA-sequencing was carried out on human fetal aortic valves at gestational weeks 9, 13, and 22 and on a case-control study with adult noncalcified and calcified bicuspid and tricuspid aortic valves. In dimension reduction and clustering analyses, diseased valves tended to cluster with fetal valves at week 9 rather than normal adult valves, suggesting that part of the disease program might be due to reiterated developmental processes. The analysis of groups of coregulated genes revealed predominant immune-metabolic signatures, including innate and adaptive immune responses involving lymphocyte T-cell metabolic adaptation. Cytokine and chemokine signaling, cell migration, and proliferation were all increased in CAVD, whereas oxidative phosphorylation and protein translation were decreased. Discrete immune-metabolic gene signatures were present at fetal stages and increased in adult controls, suggesting that these processes intensify throughout life and heighten in disease. Cellular stress response and neurodegeneration gene signatures were aberrantly expressed in CAVD, pointing to a mechanistic link between chronic inflammation and biological aging. Comparison of the valve RNA-sequencing data set with a case-control study of whole blood transcriptomes from asymptomatic individuals with early aortic valve calcification identified a highly predictive gene signature of CAVD and of moderate aortic valve calcification in overtly healthy individuals. These data deepen and broaden our understanding of the molecular basis of CAVD and identify a peripheral blood gene signature for the early detection of aortic valve calcification. This work was supported by the Ministerio de Ciencia, Innovacion y Universidades from Spain (MCIU) Grants SAF2016-78370-R, CB16/11/00399, CIBER CV, and RD16/0011/0021, TERCEL, Fundacion BBVA Grant BIO14_298, and Fundacion La Marato TV3 Grant 20153431 (to J.L.d.l.P.); and the Fondation Pour la Recherche Medicale Grant DPC20111123002 from l'Institut National de la Sante et de la Recherche Medicale and l'Association Francaise Contre les Myopathies Grant TRIM-RD) (to S.Z). The cost of this publication was supported in part with funds from the European Regional Development Fund. The CNIC is supported by the Ministerio de Ciencia, Innovacion y Universidades (MCIU), the Instituto de Salud Carlos III (ISCIII), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (Grant SEV-2015-0505). Sí

microRNAs (miRNAs) are post-transcriptional regulators of gene expression and can play an important role in modulating organismal development and physiology in response to environmental stress. However, the role of miRNAs in mediating adaptation to diverse environments in natural study systems remains largely unexplored. Here, we characterized miRNAs and their expression in Poecilia mexicana, a species of small fish that inhabits both normal streams and extreme environments in the form of springs rich in toxic hydrogen sulphide (H2 S). We found that P. mexicana has a similar number of miRNA genes as other teleosts. In addition, we identified a large population of mature miRNAs that were differentially expressed between locally adapted populations in contrasting habitats, indicating that miRNAs may contribute to P. mexicana adaptation to sulphidic environments. In silico identification of differentially expressed miRNA-mRNA pairs revealed, in the sulphidic environment, the downregulation of miRNAs predicted to target mRNAs involved in sulphide detoxification and cellular homeostasis, which are pathways essential for life in H2 S-rich springs. In addition, we found that predicted targets of upregulated miRNAs act in the mitochondria (16.6% of predicted annotated targets), which is the main site of H2 S toxicity and detoxification, possibly modulating mitochondrial function. Together, the differential regulation of miRNAs between these natural populations suggests that miRNAs may be involved in H2 S adaptation by promoting functions needed for survival and reducing functions affected by H2 S. This study lays the groundwork for further research to directly demonstrate the role of miRNAs in adaptation to H2 S. Overall, this study provides a critical stepping-stone towards a comprehensive understanding of the regulatory mechanisms underlying the adaptive variation in gene expression in a natural system.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro(Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLproregimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLproinhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2cells and validates PF-00835231’s early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231’s efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2cells or human polarized airway epithelial cultures. Thus, our study providesin vitroevidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-humanin vitromodels.ImportanceThe arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro(Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3Din vitromodels of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Here, we determine the ability of sera from 101 recovered healthcare workers to neutralize both authentic SARS-CoV-2 and SARS-CoV-2 pseudotyped virus and address their antibody titers against SARS-CoV-2 nucleoprotein and spike receptor-binding domain. Interestingly, the majority of individuals have low neutralization capacity and only 6% of the healthcare workers showed high neutralizing titers against both authentic SARS-CoV-2 virus and the pseudotyped virus. We found the antibody response to SARS-CoV-2 infection generates antigen-specific isotypes as well as a diverse combination of antibody isotypes, with high titers of IgG, IgM and IgA against both antigens correlating with neutralization capacity. Importantly, we found that neutralization correlated with antibody titers as quantified by ELISA. This suggests that an ELISA assay can be used to determine seroneutralization potential. Altogether, our work provides a snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides evidence that possessing multiple antibody isotypes may play an important role in SARS-CoV-2 neutralization.

We present a genome assembly for Cottoperca gobio (channel bull blenny, (Günther, 1861)); Chordata; Actinopterygii (ray-finned fishes), a temperate water outgroup for Antarctic Notothenioids. The size of the genome assembly is 609 megabases, with the majority of the assembly scaffolded into 24 chromosomal pseudomolecules. Gene annotation on Ensembl of this assembly has identified 21,662 coding genes.

The evolution of antifreeze glycoproteins has enabled notothenioid fish to flourish in the freezing waters of the Southern Ocean. Whilst successful at the biodiversity level to life in the cold, paradoxically at the cellular level these stenothermal animals have problems producing, folding and degrading proteins at their ambient temperatures of down to −1.86°C. In this first multi-species transcriptome comparison of the amino acid composition of notothenioid proteins with temperate teleost proteins, we show that, unlike psychrophilic bacteria, Antarctic fish provide little evidence for the mass alteration of protein amino acid composition to enhance protein folding and reduce protein denaturation in the cold. The exception was the significant over-representation of positions where leucine in temperate fish proteins was replaced by methionine in the notothenioid orthologues. Although methionine may increase stability in critical proteins, we hypothesise that a more likely explanation for the extra methionines is that they have been preferentially assimilated into the genome because they act as redox sensors. This redox hypothesis is supported by the enrichment of duplicated genes within the notothenioid transcriptomes which centre around Mapk signalling, a major pathway in the cellular cascades associated with responses to environmental stress. Whilst notothenioid fish show cold-associated problems with protein homeostasis, they may have modified only a selected number of biochemical pathways to work efficiently below 0°C. Even a slight warming of the Southern Ocean might disrupt the critical functions of this handful of key pathways with considerable impacts for the functioning of this ecosystem in the future.

International audience; With the rapidly developing high-throughput sequencing technologies known as next generation sequencing or NGS, our approach to gene hunting and diagnosis has drastically changed. In

Background The caudal fin of actinopterygians transitioned from a heterocercal dorsoventrally asymmetrical fin to a homocercal externally symmetrical fin in teleosts through poorly understood evolutionary developmental mechanisms. We studied the caudal skeleton of major living actinopterygian lineages, including polypteriformes, acipenseriformes, Holostei (gars and bowfin), and teleosts, compared with reports of extinct neopterygians and basal teleosteans. We focused on the hypural diastema complex, which includes (1) a gap between hypurals 2 and 3, that (2) separates two plates of connective tissue at (3) the branching of caudal vasculature; these features had been considered as a shared, derived trait of teleosts, a synapomorphy. Results These studies revealed that gars and teleosts share all three features of the hypural diastema complex. Absence of a complex with these features from bowfin, fossil Holostei, and stem Teleostei argues in favor of repetitive, independent emergence in several neopterygian and basal Teleostei lineages, or less likely, many independent losses. We further observed that, in gars and teleosts, the earliest developing lepidotrichia align with the horizontal adult body axis, thus participating in external symmetry. Conclusions These results suggest that the hypural diastema complex in teleosts and gars represents a homoplasy among neopterygians and that it emerged repeatedly by parallel evolution due to shared inherited underlying genetic and developmental programs (latent homology). Because the hypural diastema complex exists in gars with heterocercal tails, this complex is independent of homocercality. Developmental Dynamics 247:832-853, 2018. © 2018 Wiley Periodicals, Inc.

BACKGROUND: Infection with Mycobacterium tuberculosis is associated with inconsistent and incomplete elimination of the bacteria, despite development of antigen-specific T-cell responses. One mechanism used by M tuberculosis is to limit availability of antigen for activation of CD4 T cells. METHODS: We examined the utility of systemic administration of epitope peptides to activate pre-existing T cells in mice infected with M tuberculosis. RESULTS: We found that systemic peptide administration (1) selectively activates T cells specific for the epitope peptide, (2) loads major histocompatibility complex class II on lung macrophages and dendritic cells, (3) activates CD4 T cells in the lung parenchyma, (4) and has little antimycobacterial activity. CONCLUSIONS: Further studies revealed that CD4 T cells in lung lesions are distant from the infected cells, suggesting that, even if they can be activated, the positioning of CD4 T cells and their direct interactions with infected cells may be limiting determinants of immunity in tuberculosis.

Background The caudal fin of actinopterygians experienced substantial morphological changes during evolution. In basal actinopterygians, the caudal fin skeleton supports an asymmetrical heterocercal caudal fin, while most teleosts have a symmetrical homocercal caudal fin. The transition from the ancestral heterocercal form to the derived homocercal caudal fin remains poorly understood. Few developmental studies provide an understanding of derived and ancestral characters among basal actinopterygians. To fill this gap, we examined the development of the caudal fin of spotted gar Lepisosteus oculatus, one of only eight living species of Holostei, the sister group to the teleosts. Results Our observations of animals from fertilization to more than a year old provide the most detailed description of the development of caudal fin skeletal elements in any Holostean species. We observed two different types of distal caudal radials replacing two transient plates of connective tissue, identifying two hypaxial ensembles separated by a space between hypurals 2 and 3. These features have not been described in any gar species, but can be observed in other gar species, and thus represent anatomical structures common to lepisosteiformes. Conclusions The present work highlights the power and importance of ontogenic studies and provides bases for future evolutionary and morphological investigations on actinopterygians fins. Developmental Dynamics 247:724-740, 2018. © 2018 Wiley Periodicals, Inc.

The green birdmouth wrasse Gomphosus caeruleus is present all year round on the coral reefs of Reunion Island (Indian Ocean). A group of individuals was followed on one of these reefs with the objective of studying the reproduction mode of the species, the influence of environmental factors, and social behaviors on the control of reproduction. Our observations revealed that G. caeruleus is, like many Labridae, a protogynous hermaphrodite species, probably diandric, that the reproduction of G. caeruleus is, like in other reef fish species, influenced by the lunar cycle with a peak of reproductive activity during waxing gibbous phase, and that G. caeruleus displays social behavior leading to alternating haremic mating system on a single territory and lek-like mating systems without aggressions between males. These observations enhanced our knowledge of the reproduction of Labridae and reef species.

Modern genetic data sets present unprecedented opportunities to understand the evolutionary origins of diverse taxonomic groups. When the timing of key events is known, it is possible to investigate biogeographic history in the context of major phenomena (e.g., cooling of a major ocean). In this study, we investigated the biogeographic history of the suborder Zoarcoidei, a globally distributed fish group that includes species inhabiting both poles that produce antifreeze proteins to survive chronic subfreezing temperatures. We first generated a multi-locus, time-calibrated phylogeny for the group. We then used biogeographic modeling to reconstruct ancestral ranges across the tree and to quantify the type and frequency of biogeographic events (e.g., founder, dispersal). With these results, we considered how the cooling of the Southern and Arctic Oceans, which reached their present-day subfreezing temperatures 10–15 million years ago (Mya) and 2–3 Mya, respectively, may have shaped the group’s evolutionary history, with an emphasis on the most speciose and widely distributed family, eelpouts (family Zoarcidae). Our phylogenetic results clarified the Zoarcoidei taxonomy and showed that the group began to diversify in the Oligocene ~31–32 Mya, with the center of origin for all families in north temperate waters. Within-area speciation was the most common biogeographic event in the group’s history (80% of all events) followed by dispersal (20%). Finally, we only found evidence, albeit limited, for ocean cooling underpinning diversification of eelpouts living in the high Antarctic over the last 10 million years.

Background: Aberrant signaling between germ cells and somatic cells can lead to reproductive disease and depends on diffusible signals, including TGFB-family proteins. The TGFB-family protein Gsdf (gonadal soma derived factor) controls sex determination in some fish and is a candidate for mediating germ cell/soma signaling. Results: Zebrafish expressed gsdf in somatic cells of bipotential gonads and expression continued in ovarian granulosa cells and testicular Sertoli cells. Homozygous gsdf knockout mutants delayed leaving the bipotential gonad state, but then became a male or a female. Mutant females ovulated a few oocytes, then became sterile, accumulating immature follicles. Female mutants stored excess lipid and down-regulated aromatase, gata4, insulin receptor, estrogen receptor, and genes for lipid metabolism, vitellogenin, and steroid biosynthesis. Mutant females contained less estrogen and more androgen than wild types. Mutant males were fertile. Genomic analysis suggests that Gsdf, Bmp15, and Gdf9, originated as paralogs in vertebrate genome duplication events. Conclusions: In zebrafish, gsdf regulates ovarian follicle maturation and expression of genes for steroid biosynthesis, obesity, diabetes, and female fertility, leading to ovarian and extra-ovarian phenotypes that mimic human polycystic ovarian syndrome (PCOS), suggesting a role for a related TGFB signaling molecule in the etiology of PCOS. This article is protected by copyright. All rights reserved.

Splenic diffuse red pulp lymphoma is an indolent small B-cell lymphoma recognized as a provisional entity in the World Health Organization 2008 classification. Its precise relationship to other related splenic B-cell lymphomas with frequent leukemic involvement or other lymphoproliferative disorders remains undetermined. We performed whole-exome sequencing to explore the genetic landscape of ten cases of splenic diffuse red pulp lymphoma using paired tumor and normal samples. A selection of 109 somatic mutations was then evaluated in a cohort including 42 samples of splenic diffuse red pulp lymphoma and compared to those identified in 46 samples of splenic marginal zone lymphoma and eight samples of hairy-cell leukemia. Recurrent mutations or losses in BCOR (the gene encoding the BCL6 corepressor) - frameshift (n=3), nonsense (n=2), splicing site (n=1), and copy number loss (n=4) - were identified in 10/42 samples of splenic diffuse red pulp lymphoma (24%), whereas only one frameshift mutation was identified in 46 cases of splenic marginal zone lymphoma (2%). Inversely, KLF2, TNFAIP3 and MYD88, common mutations in splenic marginal zone lymphoma, were rare (one KLF2 mutant in 42 samples; 2%) or absent (TNFAIP3 and MYD88) in splenic diffuse red pulp lymphoma. These findings define an original genetic profile of splenic diffuse red pulp lymphoma and suggest that the mechanisms of pathogenesis of this lymphoma are distinct from those of splenic marginal zone lymphoma and hairy-cell leukemia.

The blackfin icefish Chaenocephalus aceratus represents a key component of the fish fauna surrounding the Antarctic Peninsula that may be especially vulnerable to the effects of climate change. Although the sensitivity of adults to elevated temperature has been evaluated, little is known about the potential impact of a warmer temperature on other life history stages including embryos and larvae. To investigate thermal effects on embryogenesis and larval development, one must establish optimal zootechnical parameters for maintaining reproductively competent broodstock in captivity. During two consecutive years, we conducted an evaluation of body morphometrics and reproductive traits and investigated appropriate husbandry practices. Absolute and relative fecundities of females were estimated as 14,367 ± 5733 and 8.87 ± 2.35 (Mean ± SD), respectively. Mean wet and dry masses (±SD) of eggs were 24.5 ± 11.0 and 15.3 ± 4.8 mg, respectively and the mean egg diameter (±SD) was 0.31 ± 0.03 cm. Post-capture mortality was high and no spawning state females survived for a sufficiently long time to allow release or extraction of mature eggs. Injecting males with gonadotropin-releasing hormone (GnRH) increased their gonadosomatic index by 2-fold and stimulated spermiation. Mean (±SD) sperm cell count per mL post-GnRH treatment was 9.9 × 109 ± 2.5 × 109, and the spermatocrit was 16.9 ± 3.8%. Our findings indicate that extending the duration of female survival post-capture will be necessary for the production of fertile eggs. Suggestions for future research are discussed.

Aims Rituximab is a monoclonal antibody directed against CD20, which is approved in rheumatoid arthritis (RA). This study aimed at assessing the influence of CD19+ cell counts as target-antigen amount, and of immunoglobulin G (IgG) serum concentrations on rituximab pharmacokinetics in RA patients. Methods In a cohort of 64 RA patients who had received repetitive courses of rituximab, the influence of CD19+ cell count, IgG serum concentration, body surface area (BSA), sex, and disease activity score in 28 joints (DAS28) on rituximab pharmacokinetic parameters was assessed using a population pharmacokinetic analysis. Results A two-compartment model, with first-order distribution and elimination best described the data. The volume of distribution of central compartment (Vc) and clearance (CL) of rituximab were estimated at 4.7 L and 0.56 L/day, respectively. Distribution (T½-α) and elimination (T½-β) half-lives were 0.9 days and 17.3 days, respectively. As expected, the central volume of distribution increased with BSA (p = 0.012) and was higher in male than in female (p = 0.004). We found that the elimination rate constant (k10) increased with CD19+ count (p = 0.00022) and IgG concentration (p = 7.4 10-8), and that k10 decreased with time (p = 0.00015), partly explained by a change in target-antigen amount. Conclusions The association between CD19+ count and k10 may be explained by target-mediated drug disposition, while the association between IgG serum concentration and k10 may be explained by a saturation of the neonatal Fc receptor (FcRn) at high IgG concentrations, resulting in decreased recycling of rituximab.

Aims-During embryogenesis, the onset of circulatory blood flow generates a variety of hemodynamic forces which reciprocally induce changes in cardiovascular development and performance. It has been known for some time that these forces can be detected by as yet unknown mechanosensory systems which in turn promote cardiogenic events such as outflow tract and aortic valve development. PIEZO1 is a mechanosensitive ion channel present in endothelial cells where it serves to detect hemodynamic forces making it an ideal candidate to play a role during cardiac development. We sought to determine whether PIEZO1 is required for outflow tract and aortic valve development.Methods and results-By analysing heart development in zebrafish we have determined thatpiezo1is expressed in the developing outflow tract where it serves to detect hemodynamic forces. In particular, we have found that mechanical forces generated during the cardiac cycle activate Piezo1 which triggers nitric oxide to be released in the outflow tract. Consequently, disrupting Piezo1 signalling leads to defective outflow tract and aortic valve development and indicates this gene may be involved in the etiology of congenital heart diseases. Based on these findings, we analysed genomic data generated from a cohort of bicuspid aortic valve patients and identified 3 probands who each harboured a novel variant inPIEZO1. Subsequentin vitroandin vivoassays indicates that these variants behave as dominant negatives leading to an inhibition of normal PIEZO1 mechanosensory activity and defective aortic valve development.Conclusion-These data indicate that the mechanosensitive ion channelpiezo1is required for OFT and aortic valve development and, furthermore, dominant negative variants ofPIEZO1appear to be associated with BAV in humans.

The retina has a very high energy demand but lacks an internal blood supply in most vertebrates. Here we explore the hypothesis that oxygen diffusion limited the evolution of retinal morphology by reconstructing the evolution of retinal thickness and the various mechanisms for retinal oxygen supply, including capillarization and acid-induced haemoglobin oxygen unloading. We show that a common ancestor of bony fishes likely had a thin retina without additional retinal oxygen supply mechanisms and that three different types of retinal capillaries were gained and lost independently multiple times during the radiation of vertebrates, and that these were invariably associated with parallel changes in retinal thickness. Since retinal thickness confers multiple advantages to vision, we propose that insufficient retinal oxygen supply constrained the functional evolution of the eye in early vertebrates, and that recurrent origins of additional retinal oxygen supply mechanisms facilitated the phenotypic evolution of improved functional eye morphology.

Hawthorn (Crataegus) is used for its cardiotonic, hypotensive, vasodilative, sedative, antiatherosclerotic, and antihyperlipidemic properties. One of the main goals of this work was to find a well-defined optimized extraction protocol usable by each of us that would lead to repeatable, controlled, and quantified daily uptake of active components from hawthorn at a drinkable temperature (below 60 &deg, C). A thorough investigation of the extraction mode in water (infusion, maceration, percolation, ultrasounds, microwaves) on the yield of extraction and the amount of phenolic compounds, flavonoids, and proanthocyanidin oligomers as well as on the Ultra High Performance Liquid Chromatography (UHPLC) profiles of the extracted compounds was carried out. High-resolution Fourier transform ion cyclotron resonance mass spectrometry was also implemented to discriminate the different samples and conditions of extraction. The quantitative and qualitative aspects of the extraction as well as the kinetics of extraction were studied, not only according to the part (flowers or leaves), the state (fresh or dried), and the granulometry of the dry plant, but also the stirring speed, the temperature, the extraction time, the volume of the container (cup, mug or bowl) and the use of infusion bags.

International audience; Background:The past few decades have witnessed a tremendous development in the field of genetics. Theimplementation of next generation sequencing (NGS) technologies revolutionized the field of molecular biologyand made the genetic information accessible at a large scale. However, connecting a rare genetic variation to acomplex phenotype remains challenging. Indeed, identifying the cause of a genetic disease requires amultidisciplinary approach, starting with the establishment of a clear phenotype with a detailed family history andending, in some cases, with functional assays that are crucial for the validation of the pathogenicity of a mutation.Methods:Two hundred Lebanese patients, presenting a wide spectrum of genetic disorders (neurodevelopmental,neuromuscular or metabolic disorders, etc.), sporadic or inherited, dominant or recessive, were referred, over thelast three and a half years, to the Medical Genetics Unit (UGM) of Saint Joseph University (USJ). In order to identify the genetic basis of these diseases, Whole Exome Sequencing (WES), followed by a targeted analysis, was performed for each case. In order to improve the genetic diagnostic yield, WES data, generated during the first 2 years of this study, were reanalyzed for all patients who were left undiagnosed at the genetic level. Reanalysis was based on updated bioinformatics tools and novel gene discoveries.Results:Our initial analysis allowed us to identify the specific genetic mutation causing the disease in 49.5% of the cases, in line with other international studies. Repeated WES analysis enabled us to increase the diagnostics yield to 56%.Conclusion:The present article reports the detailed results of both analysis and pinpoints the contribution of WES data reanalysis to an efficient genetic diagnosis. Lessons learned from WES reanalysis and interpretation are also shared.

Anti-Mullerian hormone (Amh) inhibits female reproductive duct development, signals oocyte reserve, and marks polycystic ovarian syndrome. Zebrafish lacks Mullerian ducts and the typical Amh receptor, questioning evolving roles of Amh. Yan et al. made knockout mutations in zebrafish... Fetal mammalian testes secrete Anti-Müllerian hormone (Amh), which inhibits female reproductive tract (Müllerian duct) development. Amh also derives from mature mammalian ovarian follicles, which marks oocyte reserve and characterizes polycystic ovarian syndrome. Zebrafish (Danio rerio) lacks Müllerian ducts and the Amh receptor gene amhr2 but, curiously, retains amh. To discover the roles of Amh in the absence of Müllerian ducts and the ancestral receptor gene, we made amh null alleles in zebrafish. Results showed that normal amh prevents female-biased sex ratios. Adult male amh mutants had enormous testes, half of which contained immature oocytes, demonstrating that Amh regulates male germ cell accumulation and inhibits oocyte development or survival. Mutant males formed sperm ducts and some produced a few offspring. Young female mutants laid a few fertile eggs, so they also had functional sex ducts. Older amh mutants accumulated nonvitellogenic follicles in exceedingly large but sterile ovaries, showing that Amh helps control ovarian follicle maturation and proliferation. RNA-sequencing data partitioned juveniles at 21 days postfertilization (dpf) into two groups that each contained mutant and wild-type fish. Group21-1 upregulated ovary genes compared to Group21-2, which were likely developing as males. By 35 dpf, transcriptomes distinguished males from females and, within each sex, mutants from wild types. In adult mutants, ovaries greatly underexpressed granulosa and theca genes, and testes underexpressed Leydig cell genes. These results show that ancestral Amh functions included development of the gonadal soma in ovaries and testes and regulation of gamete proliferation and maturation. A major gap in our understanding is the identity of the gene encoding a zebrafish Amh receptor; we show here that the loss of amhr2 is associated with the breakpoint of a chromosome rearrangement shared among cyprinid fishes.

Charcot-Marie-Tooth disease (CMT) is a hereditary sensory-motor neuropathy characterized by a strong clinical and genetic heterogeneity. Over the past few years, with the occurrence of whole-exome sequencing (WES) or whole-genome sequencing (WGS), the molecular diagnosis rate has been improved by allowing the screening of more than 80 genes at one time. In CMT, except the recurrent PMP22 duplication accounting for about 60% of pathogenic variations, pathogenic copy number variations (CNVs) are rarely reported and only a few studies screening specifically CNVs have been performed. The aim of the present study was to screen for CNVs in the most prevalent genes associated with CMT in a cohort of 200 patients negative for the PMP22 duplication. CNVs were screened using the Exome Depth software on next generation sequencing (NGS) data obtained by targeted capture and sequencing of a panel of 81 CMT associated genes. Deleterious CNVs were identified in four patients (2%), in four genes: GDAP1, LRSAM1, GAN, and FGD4. All CNVs were confirmed by high-resolution oligonucleotide array Comparative Genomic Hybridization (aCGH) and/or quantitative PCR. By identifying four new CNVs in four different genes, we demonstrate that, although they are rare mutational events in CMT, CNVs might contribute significantly to mutational spectrum of Charcot-Marie-Tooth disease and should be searched in routine NGS diagnosis. This strategy increases the molecular diagnosis rate of patients with neuropathy.

International audience; Aging is often considered to affect both the peripheral (i.e. the cochlea) and central (brainstem and thalamus-cortex) auditory systems. We investigated the effects of aging on the cochlea, brainstem and cortex of female Sprague-Dawley rats. The auditory nerve threshold remained stable between the ages of nine and 21 months, as did distortion product otoa-coustic emissions and the number of ribbon synapses between inner hair cells and nerve fibers. The first clear signs of aging appeared in the brainstem, in which response amplitude decreased, with thresholds remaining stable until the age of 15 months, and increasing slightly thereafter. The responses of primary auditory cortex neurons revealed specific effects of aging: at 21 months, receptive fields were spectrally narrower and the temporal reliability of responses to communication sounds was lower. However, aging had a null or even positive effect on neuronal responses in the presence of background noise, responses to amplitude-modulated sounds, and responses in gap-detection protocols. Overall, inter-animal variability remained high relative to the variability across groups of different ages, for all parameters tested. Beha-vioral performance for the modulation depth of amplitude modulation noise was worse in 21-month old animals than in other animals. Age-related alterations of cortical and behavioral responses were thus observed in animals displaying no signs of aging at the peripheral level. These results suggest that intrinsic, central aging effects can affect the perception of acoustic stimuli independently of the effects of aging on peripheral receptors.