Conserved Signaling Pathways in the Ciona robusta Gut.

The urochordate Ciona robusta exhibits numerous functional and morphogenetic traits that are shared with vertebrate models. While prior investigations have identified several analogies between the gastrointestinal tract (i.e., gut) of Ciona and mice, the molecular mechanisms responsible for these similarities remain poorly understood. This study seeks to address this knowledge gap by investigating the transcriptional landscape of the adult stage gut. Through comparative genomics analyses, we identified several evolutionarily conserved components of signaling pathways of pivotal importance for gut development (such as WNT, Notch, and TGFß-BMP) and further evaluated their expression in three distinct sections of the gastrointestinal tract by RNA-seq. Despite the presence of lineage-specific gene gains, losses, and often unclear orthology relationships, the investigated pathways were characterized by well-conserved molecular machinery, with most components being expressed at significant levels throughout the entire intestinal tract of C. robusta. We also showed significant differences in the transcriptional landscape of the stomach and intestinal tract, which were much less pronounced between the proximal and distal portions of the intestine. This study confirms that C. robusta is a reliable model system for comparative studies, supporting the use of ascidians as a model to study gut physiology.

Transcriptional and proteomic analysis of the innate immune response to microbial stimuli in a model invertebrate chordate.

Inflammatory response triggered by innate immunity can act to protect against microorganisms that behave as pathogens, with the aim to restore the homeostatic state between host and beneficial microbes. As a filter-feeder organism, the ascidian Ciona robusta is continuously exposed to external microbes that may be harmful under some conditions. In this work, we used transcriptional and proteomic approaches to investigate the inflammatory response induced by stimuli of bacterial (lipopolysaccharide -LPS- and diacylated lipopeptide - Pam2CSK4) and fungal (zymosan) origin, in Ciona juveniles at stage 4 of metamorphosis. We focused on receptors, co-interactors, transcription factors and cytokines belonging to the TLR and Dectin-1 pathways and on immune factors identified by homology approach (i.e. immunoglobulin (Ig) or C-type lectin domain containing molecules). While LPS did not induce a significant response in juvenile ascidians, Pam2CSK4 and zymosan exposure triggered the activation of specific inflammatory mechanisms. In particular, Pam2CSK4-induced inflammation was characterized by modulation of TLR and Dectin-1 pathway molecules, including receptors, transcription factors, and cytokines, while immune response to zymosan primarily involved C-type lectin receptors, co-interactors, Ig-containing molecules, and cytokines. A targeted proteomic analysis enabled to confirm transcriptional data, also highlighting a temporal delay between transcriptional induction and protein level changes. Finally, a protein-protein interaction network of Ciona immune molecules was rendered to provide a wide visualization and analysis platform of innate immunity. The in vivo inflammatory model described here reveals interconnections of innate immune pathways in specific responses to selected microbial stimuli. It also represents the starting point for studying ontogeny and regulation of inflammatory disorders in different physiological conditions.

Environmental stress and nanoplastics' effects on Ciona robusta: regulation of immune/stress-related genes and induction of innate memory in pharynx and gut.

In addition to circulating haemocytes, the immune system of the solitary ascidian Ciona robusta relies on two organs, the pharynx and the gut, and encompasses a wide array of immune and stress-related genes. How the pharynx and the gut of C. robusta react and adapt to environmental stress was assessed upon short or long exposure to hypoxia/starvation in the absence or in the presence of polystyrene nanoplastics. We show that the immune response to stress is very different between the two organs, suggesting an organ-specific immune adaptation to the environmental changes. Notably, the presence of nanoplastics appears to alter the gene modulation induced by hypoxia/starvation in both organs, resulting in a partial increase in gene up-regulation in the pharynx and a less evident response to stress in the gut. We have also assessed whether the hypoxia/starvation stress could induce innate memory, measured as gene expression in response to a subsequent challenge with the bacterial agent LPS. Exposure to stress one week before challenge induced a substantial change in the response to LPS, with a general decrease of gene expression in the pharynx and a strong increase in the gut. Co-exposure with nanoplastics only partially modulated the stress-induced memory response to LPS, without substantially changing the stress-dependent gene expression profile in either organ. Overall, the presence of nanoplastics in the marine environment seems able to decrease the immune response of C. robusta to stressful conditions, hypothetically implying a reduced capacity to adapt to environmental changes, but only partially affects the stress-dependent induction of innate memory and subsequent responses to infectious challenges.

Methodological Approaches To Assess Innate Immunity and Innate Memory in Marine Invertebrates and Humans.

Assessing the impact of drugs and contaminants on immune responses requires methodological approaches able to represent real-life conditions and predict long-term effects. Innate immunity/inflammation is the evolutionarily most widespread and conserved defensive mechanism in living organisms, and therefore we will focus here on immunotoxicological methods that specifically target such processes. By exploiting the conserved mechanisms of innate immunity, we have examined the most representative immunotoxicity methodological approaches across living species, to identify common features and human proxy models/assays. Three marine invertebrate organisms are examined in comparison with humans, i.e., bivalve molluscs, tunicates and sea urchins. In vivo and in vitro approaches are compared, highlighting common mechanisms and species-specific endpoints, to be applied in predictive human and environmental immunotoxicity assessment. Emphasis is given to the 3R principle of Replacement, Refinement and Reduction of Animals in Research and to the application of the ARRIVE guidelines on reporting animal research, in order to strengthen the quality and usability of immunotoxicology research data.

Mejorar la calidad de las evaluaciones de riesgos psicosociales mediante el control de sesgos / Systematic Review of the Reduction of Negative Emotional Effects in Emergency and Disaster Response Workers Through Catharsis Technique

La evaluación de riesgos psicosociales de origen laboral constituye un fenómeno complejo y controvertido. Sus principales críticas están centradas en la validez de las herramientas que se emplean para la recogida de información, fundamentalmente autoinformes sensibles a diversas fuentes de error. Si bien se ha incrementado el esfuerzo por proporcionar evidencias sobre la fiabilidad y la validez de los instrumentos de evaluación disponibles, existen importantes fuentes de error, sesgos, que con frecuencia son ignoradas. La detección y control de sesgos es un tópico abordado desde varios ámbitos, entre ellos el de la psicología del trabajo, sin embargo, es desconocido por muchos técnicos de prevención de riesgos laborales. En este sentido, este artículo intenta acercar a los profesionales de la prevención de riesgos laborales, una mejor comprensión de este concepto, así como diversas técnicas para su identificación y control, en aras de mejorar la calidad de las evaluaciones de riesgo psicosocial

The assessment of workplace psychosocial risk factors is complex and controversial. The main criticism is focused on the validity of the tools used in their evaluation; basically, self-report measures which are subject to several sources of error. While there has been an increase in efforts to provide evidence on the reliability and the validity of the available assessment instruments, bias is often ignored as an important source of error. Although bias may be a novel topic to many prevention technicians, their detection and control is a traditional and well-studied theme in the field of work psychology. This article gives lay practitioners a better understanding of the concept of bias, as well as the related modes of identification and control, to improve the quality of psychosocial risk assessments

[Mejorar la calidad de las evaluaciones de riesgos psicosociales mediante el control de sesgos]. / Improving the quality of psychosocial risk assessments based on the identification and control of bias.

The assessment of workplace psychosocial risk factors is complex and controversial. The main criticism is focused on the validity of the tools used in their evaluation; basically, self-report measures which are subject to several sources of error. While there has been an increase in efforts to provide evidence on the reliability and the validity of the available assessment instruments, bias is often ignored as an important source of error. Although bias may be a novel topic to many prevention technicians, their detection and control is a traditional and well-studied theme in the field of work psychology. This article gives lay practitioners a better understanding of the concept of bias, as well as the related modes of identification and control, to improve the quality of psychosocial risk assessments.

La evaluación de riesgos psicosociales de origen laboral constituye un fenómeno complejo y controvertido. Sus principales críticas están centradas en la validez de las herramientas que se emplean para la recogida de información, fundamentalmente autoinformes sensibles a diversas fuentes de error. Si bien se ha incrementado el esfuerzo por proporcionar evidencias sobre la fiabilidad y la validez de los instrumentos de evaluación disponibles, existen importantes fuentes de error, sesgos, que con frecuencia son ignoradas. La detección y control de sesgos es un tópico abordado desde varios ámbitos, entre ellos el de la psicología del trabajo, sin embargo, es desconocido por muchos técnicos de prevención de riesgos laborales. En este sentido, este artículo intenta acercar a los profesionales de la prevención de riesgos laborales, una mejor comprensión de este concepto, así como diversas técnicas para su identificación y control, en aras de mejorar la calidad de las evaluaciones de riesgo psicosocial.

Possible role of BMP-4 in the hyper-pigmentation of psoriatic plaques after anti-TNF-α treatment.

Psoriasis over-expresses several inflammatory mediators, which impacts the activity of melanocytes. Tyrosinase (Tyr) and microphthalmia-associated transcription factor (MITF) are the primary regulators of melanogenesis. Furthermore, bone morphogenetic proteins (BMPs) modulate various pathobiologic processes including inflammation, melanogenesis and melanomagenesis. To determine the association between psoriasis and melanogenesis, psoriatic lesional skin was screened through gene expression, immunohistochemistry, immunogold staining and melanin content assays. The present study detected a decreased expression of Tyr, MITF and BMP-4 in psoriatic lesional skin compared with healthy skin. Tyr, BMP-4 and melanin content were also evaluated in the psoriatic lesional skin of patients receiving adalimumab therapy, before and after 16 weeks of treatment. TNF-α blockade modulated the Tyr, BMP-4 and melanin content of the patient skin lesions, which supported the hypothesis that hyper-pigmentation may occur in areas of psoriatic plaque after biological treatment. The present study confirmed the influence of the psoriatic pro-inflammatory network on melanogenesis, exerting an inhibitory effect mediated by TNF-α. Furthermore, the results regarding BMP-4 in the present study add another important element to the mechanism of psoriasis.

Assessing Immunological Memory in the Solitary Ascidian Ciona robusta.

The immune defensive mechanisms active in the solitary ascidian Ciona robusta include phagocytic and encapsulating activity, largely brought about by phagocytic cells within the haemocyte population, the presence of complement components, which have been molecularly and functionally identified, and expression of a number of immune-related genes and pathways, identified by genome-based homology with vertebrate counterparts. Since C. robusta only displays highly conserved innate immune mechanisms, being devoid of an adaptive immune system, this organism is an excellent model for studying the features of innate memory, i.e., the capacity of the innate immune system to re-programming its responsiveness to potentially dangerous agents upon repeated exposure. In this study, we have developed an in vivo model for assessing the establishment and molecular/functional features of innate memory, by sequentially exposing C. robusta to a priming stimulus (microbial molecules), followed by a period of resting to return to basal conditions, and a challenge with microbial agents in homologous or cross-stimulation. The endpoints of immune activation were a functional activity (phagocytosis) and the molecular profiles of immune-related gene expression. The results show that exposure of C. robusta to microbial agents induces a reaction that primes animals for developing a different (expectedly more protective) response to subsequent challenges, showing the effective establishment of an immune memory. This immune memory relies on the modulation of a number of different mechanisms, some of which are priming-specific, others that are challenge-specific, and others that are non-specific, i.e., are common to all priming/challenge combinations (e.g., up-regulation of the Tnf and Lbp genes). Memory-dependent expression of the humoral immunity-related gene C3ar inversely correlates with memory-dependent variations of phagocytic rate, suggesting that complement activation and phagocytosis are alternative defensive mechanisms in C. robusta. Conversely, memory-dependent expression of the cellular immunity-related gene Cd36 directly correlates with variations of phagocytic rate, suggesting a direct involvement of this gene in the functional regulation of phagocytosis.

Characterization of paralogous uncx transcription factor encoding genes in zebrafish.

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.

Characterization of paralogous uncx transcription factor encoding genes in zebrafish.

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.

Immunological response to bacterial infection in a pelagic tunicate: Inflammation in the salp Thalia democratica.

Thaliaceans are pelagic tunicates that play a key role in trophic chains of the oceans. In the field of tunicate immunity, a notable gap is the lack of data on their inflammatory response. The common salp, Thalia democratica, possesses scant immunocytes, represented by a phagocytic line (hyaline amebocytes) and a mast cell-like line (granular cells). We aimed to provide the first investigation of defense reactions upon exposure to a large amount of bacteria (Bacillus clausii). We detected (i) bacterial phagocytosis by hyaline amebocytes, (ii) degradation of phagocytizing hyaline amebocytes in the tunic after transcellular diapedesis from the hemocoel, and (iii) release of heparin, histamine, and TNF-α by granular cells. Cell degranulation and phagocytosis occurred in epidermal cells lining the hemocoel, and an excess of mucus was observed in the post-branchial gut, causing a functional inhibition of cilia and microvilli. These findings indicate multi-step events comparable to an inflammation involving responses at both tissue and organismal levels.

Innate Immune Memory in Invertebrate Metazoans: A Critical Appraisal.

The ability of developing immunological memory, a characteristic feature of adaptive immunity, is clearly present also in innate immune responses. In fact, it is well known that plants and invertebrate metazoans, which only have an innate immune system, can mount a faster and more effective response upon re-exposure to a stimulus. Evidence of immune memory in invertebrates comes from studies in infection immunity, natural transplantation immunity, individual, and transgenerational immune priming. These studies strongly suggest that environment and lifestyle take part in the development of immunological memory. However, in several instances the formal correlation between the phenomenon of immune memory and molecular and functional immune parameters is still missing. In this review, we have critically examined the cellular and humoral aspects of the invertebrate immune memory responses. In particular, we have focused our analysis on studies that have addressed immune memory in the most restrictive meaning of the term, i.e., the response to a challenge of a quiescent immune system that has been primed in the past. These studies highlight the central role of an increase in the number of immune cells and of their epigenetic re-programming in the establishment of sensu stricto immune memory in invertebrates.

Comparative localization of serotonin-like immunoreactive cells in Thaliacea informs tunicate phylogeny.

BACKGROUND: Thaliaceans is one of the understudied classes of the phylum Tunicata. In particular, their phylogenetic relationships remain an issue of debate. The overall pattern of serotonin (5-HT) distribution is an excellent biochemical trait to interpret internal relationships at order level. In the experiments reported here we compared serotonin-like immunoreactivity at different life cycle stages of two salpid, one doliolid, and one pyrosomatid species. This multi-species comparison provides new neuroanatomical data for better resolving the phylogeny of the class Thaliacea. RESULTS: Adults of all four examined thaliacean species exhibited serotonin-like immunoreactivity in neuronal and non-neuronal cell types, whose anatomical position with respect to the nervous system is consistently identifiable due to α-tubulin immunoreactivity. The results indicate an extensive pattern that is consistent with the presence of serotonin in cell bodies of variable morphology and position, with some variation within and among orders. Serotonin-like immunoreactivity was not found in immature forms such as blastozooids (Salpida), tadpole larvae (Doliolida) and young zooids (Pyrosomatida). CONCLUSIONS: Comparative anatomy of serotonin-like immunoreactivity in all three thaliacean clades has not been reported previously. These results are discussed with regard to studies of serotonin-like immunoreactivity in adult ascidians. Lack of serotonin-like immunoreactivity in the endostyle of Salpida and Doliolida compared to Pyrosomella verticillata might be the result of secondary loss of serotonin control over ciliary beating and mucus secretion. These data, when combined with other plesiomorphic characters, support the hypothesis that Pyrosomatida is basal to these clades within Phlebobranchiata and that Salpida and Doliolida constitute sister-groups.

Mutation studies in ascidians: a review.

Historically, mutations have had a significant impact on the study of developmental processes and phenotypic evolution. Lesions in DNA are created by artificial methods or detected by natural genetic variation. Random mutations are then ascribed to genetic change by direct sequencing or positional cloning. Tunicate species of the ascidian genus Ciona represent nearly fully realized model systems in which gene function can be investigated in depth. Additionally, tunicates are valuable organisms for the study of naturally occurring mutations due to the capability to exploit genetic variation down to the molecular level. Here, we summarize the available information about how mutations are studied in ascidians with examples of insights that have resulted from these applications. We also describe notions and methodologies that might be useful for the implementation of easy and tight procedures for mutations studies in Ciona.

Telomerase expression in amyotrophic lateral sclerosis (ALS) patients.

Telomerase and telomeric complex have been linked to a variety of disease states related to neurological dysfunction. In amyotrophic lateral sclerosis (ALS) patients, telomerase activity, as human telomerase reverse transcriptase (hTERT) expression, has not been characterized yet. Here, for the first time, we characterized telomerase and related pathway in blood sample and spinal cord from ALS patients compared with healthy controls. We found that hTERT expression level was significantly lower in ALS patients and was correlated either to p53 mRNA expression or p21 expression, pointing out the hypothesis that telomerase inhibition could be a pathogenetic contributor to neurodegeneration in ALS. As a consequence of the reduced telomerase activity, we identified shorter telomeres in leukocytes from sporadic ALS patients compared with healthy control group.

IL-33 is secreted by psoriatic keratinocytes and induces pro-inflammatory cytokines via keratinocyte and mast cell activation.

IL-33 is a novel pro-inflammatory cytokine and ligand for the orphan receptor ST2. Although originally defined as an inducer of Th2-mediated responses, IL-33 was recently found to be involved in arthritis, a Th1/Th17-mediated disease. Here, we assessed the ability of IL-33 to promote inflammation via mast cells (MCs) and keratinocytes (KCs) activation in psoriasis. IL-33 resulted elevated in the skin but not in the serum of psoriasis patients. IL-33 was secreted by psoriasis KCs and HaCaT cells after TNF-α stimulation. In HMC-1, TNF-α, but not IL-17, could induce a robust increase in IL-33 expression. In HaCaT cells, TNF-α was able to induce IL-6, MCP-1 and VEGF, and the addition of IL-33 reinforced these increases. TNF-α + IL-33 combination showed similar results in primary KCs and ex vivo skin organ culture. In conclusion, our study suggests that IL-33 may be involved in psoriasis biology via MCs and KCs.

ciCD94-1, an ascidian multipurpose C-type lectin-like receptor expressed in Ciona intestinalis hemocytes and larval neural structures.

C-type lectins play an important role in the immune system and are part of a large superfamily that includes C-type lectin-like domain (CTLD)-containing proteins. Divergent evolution, acting on the CTLD fold, has generated the Ca2+-dependent carbohydrate-binding lectins and molecules, as the lectin-like natural killer (NK) receptors that bind proteins, rather than sugars, in a Ca(2+)-independent manner. We have studied ciCD94-1, a CTLD-containing protein from the tunicate Ciona intestinalis, which is a homolog of the CD94 vertebrate receptor that is expressed on NK cells and modulates their cytotoxic activity by interacting with MHC class I molecules. ciCD94-1 shares structural features with the CTLD-containing molecules that recognize proteins, suggesting that it could be located along the evolutionary pathway leading to the NK receptors. ciCD94-1 was up-regulated in response to inflammation induced by lipopolysaccharide (LPS) acting on a blood cell type present in both the tunic and circulating blood. Furthermore, an anti-ciCD94-1 antibody specifically inhibited the phagocytic activity of these cells. ciCD94-1 was also expressed during development in the larva and in the early stages of metamorphosis in structures related to the nervous system, and loss of its function affected the correct differentiation of these territories. These findings suggest that ciCD94-1 has different roles in immunity and in development, thus strengthening the concept of gene co-option during evolution and of an evolutionary relationship between the nervous and the immune systems.

Potential role of Nanos3 in maintaining the undifferentiated spermatogonia population.

Nanos gene encodes for zinc-finger protein with putative RNA-binding activity which shows an evolutionary conserved function in germ cell development. In the mouse, three Nanos homologs have been identified: Nanos1, Nanos2 and Nanos3. The Nanos3 ortholog is expressed in both male and female gonads of early embryo and, after birth, it is found only in the testis. Nanos3 targeted disruption results in the complete loss of germ cells in both sexes; however the role of Nanos3 in the testis during the postnatal period has not been explored yet. In this study, we show that, in prepuberal testis, Nanos3 is expressed in undifferentiated spermatogonia and that its up-regulation causes accumulation of cells in the G1 phase, indicating that this protein is able to delay the cell cycle progression of spermatogonial cells. This is in line with the observation that the cell cycle length of the undifferentiated germ cells is longer than in differentiating spermatogonia. We also demonstrate a conserved mechanism of action of Nanos3, involving the interaction with the murine RNA-binding protein Pumilio2 and consisting of a potential translational repressor activity. According to the possible role of Nanos3 in inhibiting spermatogonia cell differentiation, we show that treatment with the differentiating factor all-trans retinoic acid induces a dramatic down-regulation of its expression. These results allow to conclude that, in the prepuberal testis, Nanos3 is important to maintain undifferentiated spermatogonia via the regulation of their cell cycle.

Ammonium channel expression is essential for brain development and function in the larva of Ciona intestinalis.

Ammonium uptake into the cell is known to be mediated by ammonium transport (Amt) proteins, which are present in all domains of life. The physiological role of Amt proteins remains elusive; indeed, loss-of-function experiments suggested that Amt proteins do not play an essential role in bacteria, yeast, and plants. Here we show that the reverse holds true in the tunicate Ciona intestinalis. The genome of C. intestinalis contains two AMT genes, Ci-AMT1a and Ci-AMT1b, which we show derive from an ascidian-specific gene duplication. We analyzed Ci-AMT expression during embryo development. Notably, Ci-AMT1a is expressed in the larval brain in a small number of cells defining a previously unseen V-shaped territory; these cells connect the brain cavity to the external environment. We show that the knockdown of Ci-AMT1a impairs the formation of the brain cavity and consequently the function of the otolith, the gravity-sensing organ contained in it. We speculate that the normal mechanical functioning (flotation and free movement) of the otolith may require a close regulation of ammonium salt(s) concentration in the brain cavity, because ammonium is known to affect both fluid density and viscosity; the cells forming the V territory may act as a conduit in achieving such a regulation.