Stable in vitro fluorescence for enhanced live imaging of infection models for Batrachochytrium dendrobatidis.

Realistic and modifiable infection models are required to study the pathogenesis of amphibian chytridiomycosis. Understanding the mechanism by which Batrachochytrium dendrobatidis (Bd) can infect and kill diverse amphibians is key to mitigating this pathogen and preventing further loss of biodiversity. In vitro studies of Bd typically rely on a tryptone based growth media, whereas the recent development of a kidney cell-line infection model has provided a more realistic alternative, without the need for live animals. Here we use expression of a fluorescent reporter to enhance the in vitro cell-line based growth assay, and show that transformed Bd cells are able to invade and grow in an amphibian kidney epithelial cell line (A6) as well as in a new system using a lung fibroblast cell line (DWJ). Both Bd and host cells were modified to express reporter fluorescent proteins, enabling immediate and continuous observation of the infection process without the need for destructive sampling for fixation and staining. Plasmid DNA conferring hygromycin resistance and TdTomato (RFP) expression was delivered to Bd zoospores via electroporation, and continuous antibiotic selection after recovery produced stable fluorescent Bd transformants. Host cells (A6 and DWJ) were transfected before each assay using lipofection to deliver plasmid DNA conferring green fluorescent protein (GFP) and containing an empty shRNA expression cassette. Bd RFP expression allowed easy localisation of fungal cells and identification of endobiotic growth was assisted by host GFP expression, by allowing visualization of the space in the host cell occupied by the invading fungal body. In addition to enabling enhanced live imaging, these methods will facilitate future genetic modification and characterisation of specific genes and their effect on Bd virulence.

The Amphibian Genomics Consortium: advancing genomic and genetic resources for amphibian research and conservation.

Amphibians represent a diverse group of tetrapods, marked by deep divergence times between their three systematic orders and families. Studying amphibian biology through the genomics lens increases our understanding of the features of this animal class and that of other terrestrial vertebrates. The need for amphibian genomics resources is more urgent than ever due to the increasing threats to this group. Amphibians are one of the most imperiled taxonomic groups, with approximately 41% of species threatened with extinction due to habitat loss, changes in land use patterns, disease, climate change, and their synergistic effects. Amphibian genomics resources have provided a better understanding of ontogenetic diversity, tissue regeneration, diverse life history and reproductive modes, antipredator strategies, and resilience and adaptive responses. They also serve as critical models for understanding widespread genomic characteristics, including evolutionary genome expansions and contractions given they have the largest range in genome sizes of any animal taxon and multiple mechanisms of genetic sex determination. Despite these features, genome sequencing of amphibians has significantly lagged behind that of other vertebrates, primarily due to the challenges of assembling their large, repeat-rich genomes and the relative lack of societal support. The advent of long-read sequencing technologies, along with computational techniques that enhance scaffolding capabilities and streamline computational workload is now enabling the ability to overcome some of these challenges. To promote and accelerate the production and use of amphibian genomics research through international coordination and collaboration, we launched the Amphibian Genomics Consortium (AGC) in early 2023. This burgeoning community already has more than 282 members from 41 countries (6 in Africa, 131 in the Americas, 27 in Asia, 29 in Australasia, and 89 in Europe). The AGC aims to leverage the diverse capabilities of its members to advance genomic resources for amphibians and bridge the implementation gap between biologists, bioinformaticians, and conservation practitioners. Here we evaluate the state of the field of amphibian genomics, highlight previous studies, present challenges to overcome, and outline how the AGC can enable amphibian genomics research to "leap" to the next level.

Exploring the Role of a Putative Secondary Metabolite Biosynthesis Pathway in Mycobacterium abscessus Pathogenesis Using a Xenopus laevis Tadpole Model.

Mycobacterium abscessus (Mab) is an emerging human pathogen that has a high rate of incidence in immunocompromised individuals. We have found a putative secondary metabolite pathway within Mab, which may be a key factor in its pathogenesis. This novel pathway is encoded in a gene cluster spanning MAB_0284c to 0305 and is related to Streptomyces pathways, producing the secondary metabolites streptonigrin and nybomycin. We constructed an in-frame deletion of the MAB_0295 (phzC) gene and tested it in our Xenopus laevis animal model. We have previously shown that X. laevis tadpoles, which have functional lungs and T cells, can serve as a reliable comparative model for persistent Mab infection and pathogenesis. Here, we report that tadpoles intraperitoneally infected with the ∆phzC mutant exhibit early decreased bacterial loads and significantly increased survival compared with those infected with WT Mab. ∆phzC mutant Mab also induced lower transcript levels of several pro-inflammatory cytokines (IL-1ß, TNF-α, iNOS, IFN-γ) than those of WT Mab in the liver and lungs. In addition, there was impaired macrophage recruitment and decreased macrophage infection in tadpoles infected with the ∆phzC mutant, by tail wound inoculation, compared to those infected with the WT bacteria, as assayed by intravital confocal microscopy. These data underline the relevance and usefulness of X. laevis tadpoles as a novel comparative animal model to identify genetic determinants of Mab immunopathogenesis, suggesting a role for this novel and uncharacterized pathway in Mab pathogenesis and macrophage recruitment.

Ingestion of polyethylene terephthalate microplastic water contaminants by Xenopus laevis tadpoles negatively affects their resistance to ranavirus infection and antiviral immunity.

Small plastic debris (0.1 µm-5 mm) or microplastics (MPs) have become major pollutants of aquatic ecosystems worldwide and studies suggest that MPs exposure can pose serious threats to human and wildlife health. However, to date the potential biological impacts of MPs accumulating in low amount in tissues during early life remains unclear. Here, for a more realistic assessment, we have used environmentally representative, mildly weathered, polyethylene terephthalate microplastics (PET MPs), cryomilled (1-100 µm) and fluorescently labelled. We leveraged the amphibian Xenopus laevis tadpoles as an animal model to define the biodistribution of PET MPs and determine whether exposure to PET MPs induce perturbations of antiviral immunity. Exposure to PET MPs for 1-14 days resulted in detectable PET MPs biodistribution in intestine, gills, liver, and kidney as determined by fluorescence microscopy on whole mount tissues. PET MPs accumulation rate in tissues was further evaluated via a novel in situ enzymatic digestion and subsequent filtration using silicon nanomembranes, which shows that PET MPs rapidly accumulate in tadpole intestine, liver and kidneys and persist over a week. Longer exposure (1 month) of tadpoles to relatively low concentration of PET MPs (25 µg/ml) significantly increased susceptibility to viral infection and altered innate antiviral immunity without inducing overt inflammation. This study provides evidence that exposure to MPs negatively impact immune defenses of aquatic vertebrates.

The Binding, Infection, and Promoted Growth of Batrachochytrium dendrobatidis by the Ranavirus FV3.

Increasing reports suggest the occurrence of co-infection between Ranaviruses such as Frog Virus 3 (FV3) and the chytrid fungus Batrachochytrium dendrobatidis (Bd) in various amphibian species. However, the potential direct interaction of these two pathogens has not been examined to date. In this study, we investigated whether FV3 can interact with Bd in vitro using qPCR, conventional microscopy, and immunofluorescent microscopy. Our results reveal the unexpected ability of FV3 to bind, promote aggregation, productively infect, and significantly increase Bd growth in vitro. To extend these results in vivo, we assessed the impact of FV3 on Xenopus tropicalis frogs previously infected with Bd. Consistent with in vitro results, FV3 exposure to previously Bd-infected X. tropicalis significantly increased Bd loads and decreased the host's survival.

[Mechanisms of cancer drug resistance]. / Mécanismes de résistance aux traitements des cancers.

Despite decades of research into the molecular mechanisms of cancer and the development of new treatments, drug resistance persists as a major problem. This is in part due to the heterogeneity of cancer, including the diversity of tumor cell lineage and cell plasticity, the spectrum of somatic mutations, the complexity of microenvironments, and immunosuppressive characteristic, then necessitating the use of many different therapeutic approaches. We summarize here the biological causes of resistance, thus offering new perspectives for tackle drug resistance.

Larval T Cells Are Functionally Distinct from Adult T Cells in Xenopus laevis.

The amphibian Xenopus laevis tadpole provides a unique comparative experimental organism for investigating the roles of innate-like T (iT) cells in tolerogenic immunity during early development. Unlike mammals and adult frogs, where conventional T cells are dominant, tadpoles rely mostly on several prominent distinct subsets of iT cells interacting with cognate nonpolymorphic MHC class I-like molecules. In the present study, to investigate whole T cell responsiveness ontogenesis in X. laevis, we determined in tadpoles and adult frogs the capacity of splenic T cells to proliferate in vivo upon infection with two different pathogens, ranavirus FV3 and Mycobacterium marinum, as well as in vitro upon PHA stimulation using the thymidine analogous 5-ethynyl-2'-deoxyuridine and flow cytometry. We also analyzed by RT-quantitative PCR T cell responsiveness upon PHA stimulation. In vivo tadpole splenic T cells showed limited capacity to proliferate, whereas the in vitro proliferation rate was higher than adult T cells. Gene markers for T cell activation and immediate-early genes induced upon TCR activation were upregulated with similar kinetics in tadpole and adult splenocytes. However, the tadpole T cell signature included a lower amplitude in the TCR signaling, which is a hallmark of mammalian memory-like T cells and iT or "preset" T cells. This study suggests that reminiscent of mammalian neonatal T cells, tadpole T cells are functionally different from their adult counterpart.

Raising concerns on questionable ethics approvals - a case study of 456 trials from the Institut Hospitalo-Universitaire Méditerranée Infection.

BACKGROUND: The practice of clinical research is strictly regulated by law. During submission and review processes, compliance of such research with the laws enforced in the country where it was conducted is not always correctly filled in by the authors or verified by the editors. Here, we report a case of a single institution for which one may find hundreds of publications with seemingly relevant ethical concerns, along with 10 months of follow-up through contacts with the editors of these articles. We thus argue for a stricter control of ethical authorization by scientific editors and we call on publishers to cooperate to this end. METHODS: We present an investigation of the ethics and legal aspects of 456 studies published by the IHU-MI (Institut Hospitalo-Universitaire Méditerranée Infection) in Marseille, France. RESULTS: We identified a wide range of issues with the stated research authorization and ethics of the published studies with respect to the Institutional Review Board and the approval presented. Among the studies investigated, 248 were conducted with the same ethics approval number, even though the subjects, samples, and countries of investigation were different. Thirty-nine (39) did not even contain a reference to the ethics approval number while they present research on human beings. We thus contacted the journals that published these articles and provide their responses to our concerns. It should be noted that, since our investigation and reporting to journals, PLOS has issued expressions of concerns for several publications we analyze here. CONCLUSION: This case presents an investigation of the veracity of ethical approval, and more than 10 months of follow-up by independent researchers. We call for stricter control and cooperation in handling of these cases, including editorial requirement to upload ethical approval documents, guidelines from COPE to address such ethical concerns, and transparent editorial policies and timelines to answer such concerns. All supplementary materials are available.

The amphibian immune system.

Amphibians are at the forefront of bridging the evolutionary gap between mammals and more ancient, jawed vertebrates. Currently, several diseases have targeted amphibians and understanding their immune system has importance beyond their use as a research model. The immune system of the African clawed frog, Xenopus laevis, and that of mammals is well conserved. We know that several features of the adaptive and innate immune system are very similar for both, including the existence of B cells, T cells and innate-like T cells. In particular, the study of the immune system at early stages of development is benefitted by studying X. laevis tadpoles. The tadpoles mainly rely on innate immune mechanisms including pre-set or innate-like T cells until after metamorphosis. In this review we lay out what is known about the innate and adaptive immune system of X. laevis including the lymphoid organs as well as how other amphibian immune systems are similar or different. Furthermore, we will describe how the amphibian immune system responds to some viral, bacterial and fungal insults. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Introduction to the special issue Amphibian immunity: stress, disease and ecoimmunology.

Amphibian populations have been declining worldwide, with global climate changes and infectious diseases being among the primary causes of this scenario. Infectious diseases are among the primary drivers of amphibian declines, including ranavirosis and chytridiomycosis, which have gained more attention lately. While some amphibian populations are led to extinction, others are disease-resistant. Although the host's immune system plays a major role in disease resistance, little is known about the immune mechanisms underlying amphibian disease resistance and host-pathogen interactions. As ectotherms, amphibians are directly subjected to changes in temperature and rainfall, which modulate stress-related physiology, including immunity and pathogen physiology associated with diseases. In this sense, the contexts of stress, disease and ecoimmunology are essential for a better understanding of amphibian immunity. This issue brings details about the ontogeny of the amphibian immune system, including crucial aspects of innate and adaptive immunity and how ontogeny can influence amphibian disease resistance. In addition, the papers in the issue demonstrate an integrated view of the amphibian immune system associated with the influence of stress on immune-endocrine interactions. The collective body of research presented herein can provide valuable insights into the mechanisms underlying disease outcomes in natural populations, particularly in the context of changing environmental conditions. These findings may ultimately enhance our ability to forecast effective conservation strategies for amphibian populations. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Advances in the Xenopus immunome: Diversification, expansion, and contraction.

Xenopus is a genus of African clawed frogs including two species, X. tropicalis and X. laevis that are extensively used in experimental biology, immunology, and biomedical studies. The availability of fully sequenced and annotated Xenopus genomes is strengthening genome-wide analyses of gene families and transgenesis to model human diseases. However, inaccuracies in genome annotation for genes involved in the immune system (i.e., immunome) hamper immunogenetic studies. Furthermore, advanced genome technologies (e.g., single-cell and RNA-Seq) rely on well-annotated genomes. The annotation problems of Xenopus immunome include a lack of established orthology across taxa, merged gene models, poor representation in gene pages on Xenbase, misannotated genes and missing gene IDs. The Xenopus Research Resource for Immunobiology in collaboration with Xenbase and a group of investigators are working to resolve these issues in the latest versions of genome browsers. In this review, we summarize the current problems of previously misannotated gene families that we have recently resolved. We also highlight the expansion, contraction, and diversification of previously misannotated gene families.

Amphibians as a model to study the role of immune cell heterogeneity in host and mycobacterial interactions.

Mycobacterial infections represent major concerns for aquatic and terrestrial vertebrates including humans. Although our current knowledge is mostly restricted to Mycobacterium tuberculosis and mammalian host interactions, increasing evidence suggests common features in endo- and ectothermic animals infected with non-tuberculous mycobacteria (NTMs) like those described for M. tuberculosis. Importantly, most of the pathogenic and non-pathogenic NTMs detected in amphibians from wild, farmed, and research facilities represent, in addition to the potential economic loss, a rising concern for human health. Upon mycobacterial infection in mammals, the protective immune responses involving the innate and adaptive immune systems are highly complex and therefore not fully understood. This complexity results from the versatility and resilience of mycobacteria to hostile conditions as well as from the immune cell heterogeneity arising from the distinct developmental origins according with the concept of layered immunity. Similar to the differing responses of neonates versus adults during tuberculosis development, the pathogenesis and inflammatory responses are stage-specific in Xenopus laevis during infection by the NTM M. marinum. That is, both in human fetal and neonatal development and in tadpole development, responses are characterized by hypo-responsiveness and a lower capacity to contain mycobacterial infections. Similar to a mammalian fetus and neonates, T cells and myeloid cells in Xenopus tadpoles and axolotls are different from the adult immune cells. Fetal and amphibian larval T cells, which are characterized by a lower T cell receptor (TCR) repertoire diversity, are biased toward regulatory function, and they have distinct progenitor origins from those of the adult immune cells. Some early developing T cells and likely macrophage subpopulations are conserved in adult anurans and mammals, and therefore, they likely play an important role in the host-pathogen interactions from early stages of development to adulthood. Thus, we propose the use of developing amphibians, which have the advantage of being free-living early in their development, as an alternative and complementary model to study the role of immune cell heterogeneity in host-mycobacteria interactions.

Scientific Integrity Requires Publishing Rebuttals and Retracting Problematic Papers.

Recently, an article by Seneff et al. entitled "Innate immunosuppression by SARS-CoV-2 mRNA vaccinations: The role of G-quadruplexes, exosomes, and MicroRNAs" was published in Food and Chemical Toxicology (FCT). Here, we describe why this article, which contains unsubstantiated claims and misunderstandings such as "billions of lives are potentially at risk" with COVID-19 mRNA vaccines, is problematic and should be retracted. We report here our request to the editor of FCT to have our rebuttal published, unfortunately rejected after three rounds of reviewing. Fighting the spread of false information requires enormous effort while receiving little or no credit for this necessary work, which often even ends up being threatened. This need for more scientific integrity is at the heart of our advocacy, and we call for large support, especially from editors and publishers, to fight more effectively against deadly disinformation.

Environmental endocrine disruptors and amphibian immunity: A bridge between the thyroid hormone axis and T cell development.

Immunity is susceptible to reprogramming by environmental chemical and endocrine signals. Notably, numerous thyroid disrupting chemicals (TDCs) have the potential to perturb immune endpoints, but data are lacking on the mechanisms by which TDCs can influence the development of the immune system. T cell immunity is particularly vulnerable to modulation by TDCs during thymic education, differentiation, and selection. The following review discusses the ways in which thyroid hormones may influence T cell development, as well as emerging TDCs with potential to impact both thyroid hormone physiology and immune outcomes. To overcome the challenges of studying TDC impacts on immune toxicological endpoints, a comparative approach using the amphibian Xenopus laevis is recommended. X. laevis are ideally suited to studying TDC impacts on immunity due to the importance of thyroid hormones for metamorphosis, and the wealth of immunological models to measure immune endpoints in both tadpoles and adult frogs.

[A 2022 inventory in oncology news]. / Que retenir dans la prise en charge des cancers en 2022 ?

The Cancer Bulletin continues its tradition. At the beginning of 2023, the members of the editorial committee would like to share with you their analyses of the highlights of 2022. The objective remains to highlight what will change our practices and lead to different diagnostic or therapeutic options. Our synthesis will therefore focus on published data. They have been analyzed and placed in the more general context of the management of each type of cancer to deduce the practical consequences for our patients. This synthesis exercise will concern almost all tumor pathologies, most often on the therapeutic level, and will, however, exclude the evolution of techniques, whether they are diagnostic or used for the follow-up of our patients. The final objective is to allow you to have a thoughtful, didactic and practical reading. Our goal is to provide our readers with the rational bases that can lead to a different approach for treatments in 2023.

Developmental exposure to thyroid disrupting chemical mixtures alters metamorphosis and post-metamorphic thymocyte differentiation.

While there is some evidence to suggest that disruption of the thyroid hormone (TH)-axis during perinatal development may weaken T cell immunity later in life, data are currently lacking on whether environmentally relevant thyroid disrupting chemicals (TDCs) can induce similar outcomes. To fill this gap in knowledge, X. laevis tadpoles were exposed to an environmentally relevant mixture of TDCs, either during early tadpole development, or immediately before and during metamorphosis, to assess T cell differentiation and anti-viral immune response against FV3 infection after metamorphosis. Extending our previous study showing a delay in metamorphosis completion, here we report that TDC exposure prior to metamorphosis reduced the frequency of surface MHC-II + splenic lymphocytes and weakened some aspects of the anti-viral immune response. TDC exposure during metamorphosis slowed post-metamorphic migration of the thymus reduced the renewal of cortical thymocytes and splenic CD8 + T cells. The results indicate that TDC exposure during perinatal development may perturb the formation of T cell immunity later in life.