A broad-taxa approach as an important concept in ecotoxicological studies and pollution monitoring.

Aquatic invertebrates play a pivotal role in (eco)toxicological assessments because they offer ethical, cost-effective and repeatable testing options. Additionally, their significance in the food chain and their ability to represent diverse aquatic ecosystems make them valuable subjects for (eco)toxicological studies. To ensure consistency and comparability across studies, international (eco)toxicology guidelines have been used to establish standardised methods and protocols for data collection, analysis and interpretation. However, the current standardised protocols primarily focus on a limited number of aquatic invertebrate species, mainly from Arthropoda, Mollusca and Annelida. These protocols are suitable for basic toxicity screening, effectively assessing the immediate and severe effects of toxic substances on organisms. For more comprehensive and ecologically relevant assessments, particularly those addressing long-term effects and ecosystem-wide impacts, we recommended the use of a broader diversity of species, since the present choice of taxa exacerbates the limited scope of basic ecotoxicological studies. This review provides a comprehensive overview of (eco)toxicological studies, focusing on major aquatic invertebrate taxa and how they are used to assess the impact of chemicals in diverse aquatic environments. The present work supports the use of a broad-taxa approach in basic environmental assessments, as it better represents the natural populations inhabiting various ecosystems. Advances in omics and other biochemical and computational techniques make the broad-taxa approach more feasible, enabling mechanistic studies on non-model organisms. By combining these approaches with in vitro techniques together with the broad-taxa approach, researchers can gain insights into less-explored impacts of pollution, such as changes in population diversity, the development of tolerance and transgenerational inheritance of pollution responses, the impact on organism phenotypic plasticity, biological invasion outcomes, social behaviour changes, metabolome changes, regeneration phenomena, disease susceptibility and tissue pathologies. This review also emphasises the need for harmonised data-reporting standards and minimum annotation checklists to ensure that research results are findable, accessible, interoperable and reusable (FAIR), maximising the use and reusability of data. The ultimate goal is to encourage integrated and holistic problem-focused collaboration between diverse scientific disciplines, international standardisation organisations and decision-making bodies, with a focus on transdisciplinary knowledge co-production for the One-Health approach.

Intrinsically High Capacity of Animal Cells From a Symbiotic Cnidarian to Deal With Pro-Oxidative Conditions.

The cnidarian-dinoflagellate symbiosis is a mutualistic intracellular association based on the photosynthetic activity of the endosymbiont. This relationship involves significant constraints and requires co-evolution processes, such as an extensive capacity of the holobiont to counteract pro-oxidative conditions induced by hyperoxia generated during photosynthesis. In this study, we analyzed the capacity of Anemonia viridis cells to deal with pro-oxidative conditions by in vivo and in vitro approaches. Whole specimens and animal primary cell cultures were submitted to 200 and 500 µM of H2O2 during 7 days. Then, we monitored global health parameters (symbiotic state, viability, and cell growth) and stress biomarkers (global antioxidant capacity, oxidative protein damages, and protein ubiquitination). In animal primary cell cultures, the intracellular reactive oxygen species (ROS) levels were also evaluated under H2O2 treatments. At the whole organism scale, both H2O2 concentrations didn't affect the survival and animal tissues exhibited a high resistance to H2O2 treatments. Moreover, no bleaching has been observed, even at high H2O2 concentration and after long exposure (7 days). Although, the community has suggested the role of ROS as the cause of bleaching, our results indicating the absence of bleaching under high H2O2 concentration may exculpate this specific ROS from being involved in the molecular processes inducing bleaching. However, counterintuitively, the symbiont compartment appeared sensitive to an H2O2 burst as it displayed oxidative protein damages, despite an enhancement of antioxidant capacity. The in vitro assays allowed highlighting an intrinsic high capacity of isolated animal cells to deal with pro-oxidative conditions, although we observed differences on tolerance between H2O2 treatments. The 200 µM H2O2 concentration appeared to correspond to the tolerance threshold of animal cells. Indeed, no disequilibrium on redox state was observed and only a cell growth decrease was measured. Contrarily, the 500 µM H2O2 concentration induced a stress state, characterized by a cell viability decrease from 1 day and a drastic cell growth arrest after 7 days leading to an uncomplete recovery after treatment. In conclusion, this study highlights the overall high capacity of cnidarian cells to cope with H2O2 and opens new perspective to investigate the molecular mechanisms involved in this peculiar resistance.

Stem cells of aquatic invertebrates as an advanced tool for assessing ecotoxicological impacts.

Environmental stressors are assessed through methods that quantify their impacts on a wide range of metrics including species density, growth rates, reproduction, behaviour and physiology, as on host-pathogen interactions and immunocompetence. Environmental stress may induce additional sublethal effects, like mutations and epigenetic signatures affecting offspring via germline mediated transgenerational inheritance, shaping phenotypic plasticity, increasing disease susceptibility, tissue pathologies, changes in social behaviour and biological invasions. The growing diversity of pollutants released into aquatic environments requires the development of a reliable, standardised and 3R (replacement, reduction and refinement of animals in research) compliant in vitro toolbox. The tools have to be in line with REACH regulation 1907/2006/EC, aiming to improve strategies for potential ecotoxicological risks assessment and monitoring of chemicals threatening human health and aquatic environments. Aquatic invertebrates' adult stem cells (ASCs) are numerous and can be pluripotent, as illustrated by high regeneration ability documented in many of these taxa. This is of further importance as in many aquatic invertebrate taxa, ASCs are able to differentiate into germ cells. Here we propose that ASCs from key aquatic invertebrates may be harnessed for applicable and standardised new tests in ecotoxicology. As part of this approach, a battery of modern techniques and endpoints are proposed to be tested for their ability to correctly identify environmental stresses posed by emerging contaminants in aquatic environments. Consequently, we briefly describe the current status of the available toxicity testing and biota-based monitoring strategies in aquatic environmental ecotoxicology and highlight some of the associated open issues such as replicability, consistency and reliability in the outcomes, for understanding and assessing the impacts of various chemicals on organisms and on the entire aquatic environment. Following this, we describe the benefits of aquatic invertebrate ASC-based tools for better addressing ecotoxicological questions, along with the current obstacles and possible overhaul approaches.

Cnidarian Cell Cryopreservation: A Powerful Tool for Cultivation and Functional Assays.

Cnidarian primary cell cultures have a strong potential to become a universal tool to assess stress-response mechanisms at the cellular level. However, primary cell cultures are time-consuming regarding their establishment and maintenance. Cryopreservation is a commonly used approach to provide stable cell stocks for experiments, but it is yet to be established for Cnidarian cell cultures. The aim of this study was therefore to design a cryopreservation protocol for primary cell cultures of the Cnidarian Anemonia viridis, using dimethyl sulfoxide (DMSO) as a cryoprotectant, enriched or not with fetal bovine serum (FBS). We determined that DMSO 5% with 25% FBS was an efficient cryosolution, resulting in 70% of post-thaw cell survival. The success of this protocol was first confirmed by a constant post-thaw survival independently of the cell culture age (up to 45 days old) and the storage period (up to 87 days). Finally, cryopreserved cells displayed a long-term recovery with a maintenance of the primary cell culture parameters and cellular functions: formation of cell aggregates, high viability and constant cell growth, and unchanged intrinsic resistance to hyperthermal stress. These results will further bring new opportunities for the scientific community interested in molecular, cellular, and biochemical aspects of cnidarian biology.

Emergence of antibodies endowed with proteolytic activity against High-mobility group box 1 protein (HMGB1) in patients surviving septic shock.

High-mobility group box 1 (HMGB1) concentration in serum or plasma has been proposed as an important biological marker in various inflammation-related pathologies. We previously showed that low titer autoantibodies against HMGB1 could emerge during the course of sepsis. Importantly their presence was positively related with patients' survival. In this study, we focused on plasma samples from 2 patients who survived sepsis and exhibited high titer antibodies to HMGB1. These antibodies were proved to be specific for HMGB1 since they did not bind to HMGB2 or to human serum albumin. Following IgG purification, it has shown that both patients secreted HMGB1-hydrolyzing autoantibodies in vitro. These findings suggested that proteolytic antibodies directed against HMGB1 can be produced in patients surviving septic shock.

Chloroplast and oxygen evolution changes in Symbiodinium sp. as a response to latrunculin and butanedione monoxime treatments under various light conditions.

The actin cytoskeleton is a dynamic structure that provides an interactive platform for organelles and cellular components. It also serves as track for membranes and vesicles that move via myosin. The actin cytoskeleton of Symbiodinium is a well-organized reticular structure suggestive of multiple membrane interactions, very likely including those of the chloroplast. The Symbiodinium chloroplast membrane network is, in turn, a highly organized structure, suggestive of being under the control of an organizing network. We visualized the chloroplast membranes of cultured Symbiodinium sp. under various light conditions and observed changes dependent on illumination intensity. Since we suspected interaction between these two organelles, and we knew that the Symbiodinium actin cytoskeleton collapses upon treatment with either latrunculin B, an actin microfilament-disrupting agent, or butanedione monoxime, a myosin function inhibitor, we tested the Symbiodinium sp. oxygen evolution in their presence. Upon latrunculin B addition, the oxygen production decreased compared to non-treated cells; however, this was not observed after a 24 h latrunculin treatment. On the contrary, butanedione monoxime treatment caused a non-recoverable dysfunction of the chloroplast causing a severe loss in oxygen production even after long-term exposure. Using electron microscopy, we observed an alteration of the Symbiodinium sp. chloroplast distribution after latrunculin B treatment, with respect to untreated cells. Furthermore, a thorough disorganization of the chloroplast grana was observed after butanedione monoxime treatment. These data suggest that an actomyosin system would be important for chloroplast organization and distribution, and critical for normal photosynthetic function of Symbiodinium sp.

Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis.

The temperate symbiotic sea anemone Anemonia viridis, a member of the Cnidaria phylum, is a relevant experimental model to investigate the molecular and cellular events involved in the preservation or in the rupture of the symbiosis between the animal cells and their symbiotic microalgae, commonly named zooxanthellae. In order to increase research tools for this model, we developed a primary culture from A. viridis animal cells. By adapting enzymatic dissociation protocols, we isolated animal host cells from a whole tentacle in regeneration state. Each plating resulted in a heterogeneous primary culture consisted of free zooxanthellae and many regular, small rounded and adherent cells (of 3-5 µm diameter). Molecular analyses conducted on primary cultures, maintained for 2 weeks, confirmed a specific signature of A. viridis cells. Further serial dilutions and micromanipulation allowed us to obtain homogenous primary cultures of the small rounded cells, corresponding to A. viridis "epithelial-like cells". The maintenance and the propagation over a 4 weeks period of primary cells provide, for in vitro cnidarian studies, a preliminary step for further investigations on cnidarian cellular pathways notably in regard to symbiosis interactions.

DsRed-mediated oligomerization stabilizes HMGB1 on chromatin in vivo and on DNA in vitro.

High-mobility group box-1 (HMGB1) is remarkably mobile in living cells, which reflects its ability to interact only transiently with both DNA and protein. This property is likely essential for HMGB1 nuclear activities. Nonetheless the weak interaction of HMGB1 with DNA and/or protein partners has also been a major limitation for investigating HMGB1 subnuclear localisation and for the identification of HMGB1 containing complexes by conventional biochemical approaches. In the present study, FRAP experiments demonstrated that DsRed-mediated oligomerization strongly reduces HMGB1 mobility due to an increased affinity for cellular chromatin. Moreover, oligomerized DsRed-HMGB1 exhibited a higher affinity for supercoiled DNA in vitro compared to its monomeric counterpart. These results indicate that DsRed-meditated oligomerization is prone to stabilize labile interactions involving HMGB1 both in vivo and in vitro.

PCA-ELISA: a sensitive method to quantify free and masked forms of HMGB1.

OBJECTIVE: HMGB1 concentration is currently regarded as an important biological marker in many inflammation-related conditions. Although ELISA has been proposed as a convenient way to quantify HMGB1 in biological fluids, various molecules have been shown to complex with HMGB1 and may interfere with HMGB1 detection by this technique. We describe here a simple technical improvement that dissociates HMGB1 containing complexes and therefore increases ELISA sensitivity. This procedure was validated in sera from patients with septic shock. METHODS: We prepared in vitro complexes containing HMGB1 protein. Recombinant human HMGB1 (rhHMGB1) was incubated in the presence of molecules that are known to form complexes with HMGB1 such as LPS, IL-1ß, or a rabbit antiserum directed against HMGB1. Then we tested the capacity of perchloric acid (PCA) to dissociate these complexes by quantifying rhHMGB1 by ELISA immediately or following PCA treatment. RESULTS: We demonstrated for the first time that incubation of rhHMGB1 with, IL-1ß, LPS or specific antibodies significantly reduce the amount of protein detected by conventional ELISA (p<0.05). Treating the samples with PCA prior ELISA efficiently reversed this inhibition. As expected, PCA-modified ELISA detected significantly higher amounts of HMGB1 in plasma samples from 40 patients with septic shock compared to conventional ELISA (p=0.0006). CONCLUSIONS: We designed a performing assay that allows the detection of masked and unmasked forms of HMGB1 with a high sensitivity and practicability.

Emergence of autoantibodies to HMGB1 is associated with survival in patients with septic shock.

PURPOSE: To assess the prevalence and predictive value of natural autoantibodies to high-mobility group box 1 (HMGB1) during sepsis. METHODS: Anti-HMGB1 and anti-human serum albumin (HSA) autoantibodies were detected by ELISA in 178 plasma samples longitudinally collected from 40 critically ill patients with septic shock. One hundred thirty-two plasma samples from healthy donors were used as control. RESULTS: IgGs to HMGB1 were detected in 15/40 patients (37.5%). The prevalence of anti-HMGB1 antibodies was significantly higher in the patients who survived (55%) compared to the patients who did not (20%) (p<0.0001). The detection of anti-HMGB1 antibodies during the course of the disease was significantly associated with patient survival (p=0.038). Moreover, there is a progressive and significant emergence of anti-HMGB1 antibodies during the course of the disease, mostly in patients who survived. CONCLUSIONS: This study shows that autoantibodies to HMGB1 are produced during sepsis and are associated with a favorable outcome in patients undergoing septic shock.

Stepwise release of biologically active HMGB1 during HSV-2 infection.

BACKGROUND: High mobility group box 1 protein (HMGB1) is a major endogenous danger signal that triggers inflammation and immunity during septic and aseptic stresses. HMGB1 recently emerged as a key soluble factor in the pathogenesis of various infectious diseases, but nothing is known of its behaviour during herpesvirus infection. We therefore investigated the dynamics and biological effects of HMGB1 during HSV-2 infection of epithelial HEC-1 cells. METHODOLOGY/PRINCIPAL FINDINGS: Despite a transcriptional shutdown of HMGB1 gene expression during infection, the intracellular pool of HMGB1 protein remained unaffected, indicating its remarkable stability. However, the dynamics of HMGB1 was deeply modified in infected cells. Whereas viral multiplication was concomitant with apoptosis and HMGB1 retention on chromatin, a subsequent release of HMGB1 was observed in response to HSV-2 mediated necrosis. Importantly, extracellular HMGB1 was biologically active. Indeed, HMGB1-containing supernatants from HSV-2 infected cells induced the migration of fibroblasts from murine or human origin, and reactivated HIV-1 from latently infected T lymphocytes. These effects were specifically linked to HMGB1 since they were blocked by glycyrrhizin or by a neutralizing anti-HMGB1 antibody, and were mediated through TLR2 and the receptor for Advanced Glycation End-products (RAGE). Finally, we show that genital HSV-2 active infections also promote HMGB1 release in vivo, strengthening the clinical relevance of our experimental data. CONCLUSIONS: These observations target HMGB1 as an important actor during HSV-2 genital infection, notably in the setting of HSV-HIV co-infection.

PTCH1 +/- dermal fibroblasts isolated from healthy skin of Gorlin syndrome patients exhibit features of carcinoma associated fibroblasts.

Gorlin's or nevoid basal cell carcinoma syndrome (NBCCS) causes predisposition to basal cell carcinoma (BCC), the commonest cancer in adult human. Mutations in the tumor suppressor gene PTCH1 are responsible for this autosomal dominant syndrome. In NBCCS patients, as in the general population, ultraviolet exposure is a major risk factor for BCC development. However these patients also develop BCCs in sun-protected areas of the skin, suggesting the existence of other mechanisms for BCC predisposition in NBCCS patients. As increasing evidence supports the idea that the stroma influences carcinoma development, we hypothesized that NBCCS fibroblasts could facilitate BCC occurence of the patients. WT (n = 3) and NBCCS fibroblasts bearing either nonsense (n = 3) or missense (n = 3) PTCH1 mutations were cultured in dermal equivalents made of a collagen matrix and their transcriptomes were compared by whole genome microarray analyses. Strikingly, NBCCS fibroblasts over-expressed mRNAs encoding pro-tumoral factors such as Matrix Metalloproteinases 1 and 3 and tenascin C. They also over-expressed mRNA of pro-proliferative diffusible factors such as fibroblast growth factor 7 and the stromal cell-derived factor 1 alpha, known for its expression in carcinoma associated fibroblasts. These data indicate that the PTCH1(+/-) genotype of healthy NBCCS fibroblasts results in phenotypic traits highly reminiscent of those of BCC associated fibroblasts, a clue to the yet mysterious proneness to non photo-exposed BCCs in NBCCS patients.

[Contribution of molecular biology in the identification of new viruses]. / Apport de la biologie moléculaire dans l'identification de nouveaux virus.

The existence of infectious agents smaller than bacteria was demonstrated already near the close of the 19th century by Martinus Beijerinck. After this discovery it took more than 60 years before a resilient definition of viruses could be given and an introduction to modern virology was established. Indeed, the major challenge was to conceive living submicrospic agents exclusively defined in opposition to the bacteriological criteria (non-observable, non-cultivable,). Progresses in biochemistry, electron microscopy, and in control of cell culture techniques have led to the conviction that the viruses were infectious agents entirely original. These last 20 years unrevealed molecular biology as a tool of choice for the discovery of new viral agents and analysis of pathologies of viral etiology.

[HMGB1: a link between innate and adaptive immunity]. / HMGB1: un lien entre inflammation septique et non septique.

HMGB1 (High Mobility group box 1) protein was originally identified as a DNA-binding protein that functions as a structural co-factor. Recent works demonstrated that HMGB1 can be released outside the cell, upon immune activation or primary cell necrosis. In the extracellular space, HMGB1 acts as a potent soluble factor that coordinates cellular events that are crucial for the amplification of inflammation, establishment of early immune responses and tissue repair. However, extracellular HMGB1 also acts as a potent pro-inflammatory cytokine that contributes to the pathogenesis of diverse inflammatory and infectious disorders.

New components of Yarrowia lipolytica Golgi multi-protein complexes containing the alpha-1,6-mannosyltransferases YlMnn9p and YlAnl1p.

This paper reports the identification and the characterization of two new components of Yarrowia lipolytica Golgi multi-protein complexes. Blast analysis on the Y. lipolytica complete genome allowed us to find a new alpha-1,6-mannosyltransferase, YlAnl2p, which displays an overall identity of 59% and shares a Golgi cellular localization with the previously described YlAnl1p. Moreover, YlAnl2p was shown to directly interact with YlMnn9p using the two-hybrid system suggesting that the two proteins form a second Golgi sub-complex. In order to further elucidate the composition of the Y. lipolytica Golgi complexes containing alpha-1,6-mannosyltransferases, as M-Pol complexes in Saccharomyces cerevisiae, two-hybrid screens were performed using either YlMnn9p or YlAnl1p as bait. A specific partner of YlAnl1p, named YlAni1p was identified. The two proteins were shown to co-localize and co-precipitate in Y. lipolytica. YlAni1p, which displays a coiled-coil domain as Golgin, and YlAnl1p could be involved in the Golgi apparatus maintenance in the yeast Y. lipolytica.

Identification and characterization of two alpha-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica.

In this study, the identification and characterization of the Yarrowia lipolytica homologues of Saccharomyces cerevisiae alpha-1,6-mannosyltransferases Anp1p and Och1p, designated YlAnl1p and YlOch1p, are described. In order to confirm the function of the Y. lipolytica proteins, including the previously isolated YlMnn9p, in the N-glycosylation pathway, a phenotypic analysis of the disrupted strains Delta Ylmnn9, Delta Ylanl1, Delta Yloch1, Delta Ylanl1 Delta Ylmnn9 and Delta Ylmnn9 Delta Yloch1 was performed. Disruption of the YlMNN9, YlANL1 and YlOCH1 genes caused an increased sensitivity to SDS, compatible with a glycosylation defect, and to Calcofluor White, characteristic of cell-wall defects. Moreover, Western-blot analysis of a heterologous glycosylated protein confirmed a direct role of YlMnn9p and YlAnl1p in the N-glycosylation process. These mutant strains, Delta Ylmnn9, Delta Ylanl1, Delta Yloch1, Delta Ylanl1 Delta Ylmnn9 and Delta Ylmnn9 Delta Yloch1 may thus be used to establish a model for the Y. lipolytica N-linked glycosylation pathway.