RESUMEN
BACKGROUND: Modern high-throughput technologies enable the processing of a large number of samples simultaneously, while also providing rapid and accurate procedures. In recent years, automated liquid handling workstations have emerged as an established technology for reproducible sample preparation. They offer flexibility, making them suitable for an expanding range of applications. Commonly, such approaches are well-developed for experimental procedures primarily designed for cell-line processing and xenobiotics testing. Conversely, little attention is focused on the application of automated liquid handlers in the analysis of whole organisms, which often involves time-consuming laboratory procedures. RESULTS: Here, we present a fully automated workflow for all steps, from RNA extraction to real-time PCR processing, for gene expression quantification in the ascidian marine model Ciona robusta. For procedure validation, we compared the results obtained with the liquid handler with those of the classical manual procedure. The outcome revealed comparable results, demonstrating a remarkable time saving particularly in the initial steps of sample processing. CONCLUSIONS: This work expands the possible application fields of this technology to whole-body organisms, mitigating issues that can arise from manual procedures. By minimizing errors, avoiding cross-contamination, decreasing hands-on time and streamlining the procedure, it could be employed for large-scale screening investigations.
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Perfilación de la Expresión Génica , Manejo de Especímenes , Automatización , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis por Micromatrices , Manejo de Especímenes/métodosRESUMEN
The origins of a "pass-through" gut in early bilaterians facilitated the exploration of new habitats, motivated the innovation of feeding styles and behaviors, and helped drive the evolution of more complex organisms. The gastrointestinal tract has evolved to consist of a series of interwoven exchanges between nutrients, host immunity, and an often microbe-rich environmental interface. Not surprisingly, animals have expanded their immune repertoires to include soluble effectors that can be secreted into luminal spaces, e.g., in the gut, facilitating interactions with microbes in ways that influence their settlement dynamics, virulence, and their interaction with other microbes. The immunoglobulin (Ig) domain, which is also found in some non-immune molecules, is recognized as one of the most versatile recognition domains lying at the interface of innate and adaptive immunity; among vertebrates, secreted Igs are known to play crucial roles in the management of gut microbial communities. In this mini-review, we will focus on secreted immune effectors possessing Ig-like domains in invertebrates, such as the fibrinogen-related effector proteins first described in the gastropod Biomphalaria glabrata, the Down syndrome cellular adhesion molecule first described in the arthropod, Drosophila melanogaster, and the variable region-containing chitin-binding proteins of the protochordates. We will highlight our current understanding of their function and their potential role, if not yet recognized, in the establishment and maintenance of host-microbial interfaces and argue that these Igs are likely also essential to microbiome management.
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Microbioma Gastrointestinal , Animales , Drosophila melanogaster , Dominios de Inmunoglobulinas , Invertebrados , VertebradosRESUMEN
BACKGROUND: The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with "cancer hallmarks" and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. METHODS: Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. RESULTS: We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models. CONCLUSIONS: This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.
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Oxidorreductasas de Alcohol , Proteínas de Unión al ADN , Melanoma , Humanos , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/metabolismo , Oxidorreductasas de Alcohol/genética , Animales , Ratones , Melanoma/tratamiento farmacológico , Melanoma/patología , Melanoma/metabolismo , Melanoma/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proliferación Celular/efectos de los fármacos , Antineoplásicos/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Cordados no Vertebrados , Animales , Lipopolisacáridos , Proteómica , Zimosan , Inmunidad Innata , Citocinas , Anticuerpos , Lectinas Tipo CRESUMEN
The formation of biofilms is critical for the successful and stable colonization of mucosal surfaces by microbes, which often build three-dimensional environments by exuding exopolysaccharides and other macromolecules such as proteins, lipids, and even DNA. It is not just bacteria, but fungi such as yeast, that form these adherent interacting communities. Historically, biofilms have been studied in the context of pathogenesis, but only recently it has been recognized that important relationships among members of host-associated microbiomes are maintained within the context of biofilms. Host immune responses impact biofilm formation in various ways; for example, it is likely that formation of stable biofilms by non-pathogens improves barrier defenses by not just filling available niche spaces but also by helping to ward off pathogens directly. Recently, it was found that soluble immune effector molecules such as immunoglobulin A (IgA) in mammals serve essential roles in modulating complex biofilm communities in ways that benefit the host. Additional lines of evidence from other secreted immune effectors, such as the variable region-containing chitin-binding proteins (VCBPs) in protochordates, now suggest that this phenomenon is much more widespread than previously recognized. The activity of these immune molecules also likely serves roles beyond those of simple defense strategies; rather, they may be improving the outcome of symbiotic interactions benefiting the host. Thus, traditional immune assays that are aimed at studying the function of secreted immune effectors, such as agglutination assays, should take into account the possibility that the first observation may not be the last if the microbes under study are not directly killed. Here, we describe a series of simple approaches to characterize biofilm formation when bacteria (or yeast) are cultured in the presence of a secreted immune effector. To model this approach, we use microbes isolated from the gut of Ciona robusta, each grown in the presence or absence of VCBPs. The approaches defined here are amenable to diverse model systems and their microbes.
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Biopelículas , Microbiota , Animales , Bacterias/metabolismo , Proteínas Portadoras , Quitina/metabolismo , Hongos/metabolismo , Proteínas , Saccharomyces cerevisiae/metabolismoRESUMEN
The complete nucleotide sequence of the mitochondrial (mt) genome of the demersal zebra seabream Diplodus cervinus (Lowe, 1838) was determined for the first time. The double stranded circular molecule is 16,559 base pairs (bp) in length and encodes for the typical 37 metazoan mitochondrial genes, and 2 non-coding regions (D-loop and L-origin). The gene arrangement of the D. cervinus mt genome follows the usual one for fishes. The nucleotide sequences of the mt protein coding and ribosomal genes of D. cervinus mt genome were aligned with orthologous sequences from representatives of the Sparidae family and phylogenetic relationships were inferred. Maximum likelihood analyses placed D. cervinus as a sister species of Diplodus sargus (Linnaeus, 1758).
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The functional ecology of the gastrointestinal tract impacts host physiology, and its dysregulation is at the center of various diseases. The immune system, and specifically innate immunity, plays a fundamental role in modulating the interface of host and microbes in the gut. While humans remain a primary focus of research in this field, the use of diverse model systems help inform us of the fundamental principles legislating homeostasis in the gut. Invertebrates, which lack vertebrate-style adaptive immunity, can help define conserved features of innate immunity that shape the gut ecosystem. In this context, we previously proposed the use of a marine invertebrate, the protochordate Ciona robusta, as a novel tractable model system for studies of host-microbiome interactions. Significant progress, reviewed herein, has been made to fulfill that vision. We examine and review discoveries from Ciona that include roles for a secreted immune effector interacting with elements of the microbiota, as well as chitin-rich mucus lining the gut epithelium, the gut-associated microbiome of adults, and the establishment of a large catalog of cultured isolates with which juveniles can be colonized. Also discussed is the establishment of methods to rear the animals germ-free, an essential technology for dissecting the symbiotic interactions at play. As the foundation is now set to extend these studies into the future, broadening our comprehension of how host effectors shape the ecology of these microbial communities in ways that establish and maintain homeostasis will require full utilization of "multi-omics" approaches to merge computational sciences, modeling, and experimental biology in hypothesis-driven investigations.
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Ciona intestinalis/microbiología , Microbioma Gastrointestinal , Interacciones Microbiota-Huesped , Mucosa Intestinal/inmunología , Animales , Ciona intestinalis/inmunología , Ecotoxicología , Inmunidad Innata , Inmunidad MucosaRESUMEN
Natural storms are able to determine reworking of seabed up to considerable depths and favour suspension of sediment-associated chemicals. Yet, a direct link between exposure to resuspended contaminants and the biological effects on marine organisms have to be fully established. We exposed adults of a suspension feeder, the ascidian Ciona robusta, to polluted sediment (e.g., containing mixtures of polycyclic aromatic hydrocarbons and heavy metals) from the industrial area of Bagnoli-Coroglio under two temporal patterns ('aggregated' vs. 'spaced') of turbulence events. Then, we assessed the impact of resuspended pollutants on the ascidian gut environment via four broad categories: oxidative stress, innate immunity, host-microbiota interactions, and epithelium. An early oxidative stress response was seen after a week of exposure to static sediment. Instead, water turbulence had no effect on the antioxidant defence. The first episode of turbulent suspension induced a minimal pro-inflammatory response in the 'spaced' pattern. Mucus overproduction and a complete occlusion of the crypt lumen were found following sediment reworking. This study suggests a protective response of the gut environment in marine invertebrates exposed to environmental extremes, leading to increased susceptibility to disease and to concerns on the combined effects of chronic environmental contamination and acute disturbance events possibly associated with climate change.
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Microbioma Gastrointestinal , Metales Pesados , Hidrocarburos Policíclicos Aromáticos , Contaminantes Químicos del Agua , Animales , Monitoreo del Ambiente , Sedimentos Geológicos , Invertebrados , Metales Pesados/toxicidad , Hidrocarburos Policíclicos Aromáticos/toxicidad , Contaminantes Químicos del Agua/toxicidadRESUMEN
The gut microbiome of animals consists of diverse microorganisms that include both prokaryotes and eukaryotes. Complex interactions occur among these inhabitants, as well as with the immune system of the host, and profoundly influence the overall health of both the host and its microbial symbionts. Despite the enormous importance for the host to regulate its gut microbiome, the extent to which animals generate immune-related molecules with the capacity to directly influence polymicrobial interactions remains unclear. The urochordate, Ciona robusta, is a model organism that has been adapted to experimental studies of host/microbiome interactions. Ciona variable-region containing chitin-binding proteins (VCBPs) are innate immune effectors, composed of immunoglobulin (Ig) variable regions and a chitin-binding domain (CBD) and are expressed in high abundance in the gut. It was previously shown that VCBP-C binds bacteria and influences both phagocytosis by granular amoebocytes and biofilm formation via its Ig domains. We show here that the CBD of VCBP-C independently recognizes chitin molecules present in the cell walls, sporangia (spore-forming bodies), and spores of a diverse set of filamentous fungi isolated from the gut of Ciona. To our knowledge, this is the first description of a secreted Ig-containing immune molecule with the capacity to directly promote transkingdom interactions through simultaneous binding by independent structural domains and could have broad implications in modulating the establishment, succession, and homeostasis of gut microbiomes.
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Bacterias/inmunología , Hongos/inmunología , Factores Inmunológicos/inmunología , Factores Inmunológicos/metabolismo , Animales , Bacterias/metabolismo , Quitina/química , Quitina/metabolismo , Técnica del Anticuerpo Fluorescente , Hongos/metabolismo , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Inmunidad Mucosa , Factores Inmunológicos/sangre , Factores Inmunológicos/química , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
The gastrointestinal tract of Ciona intestinalis, a solitary tunicate that siphon-filters water, shares similarities with its mammalian counterpart. The Ciona gut exhibits other features that are unique to protochordates, including certain immune molecules, and other characteristics, e.g. chitin-rich mucus, which appears to be more widespread than considered previously. Exposure of Ciona to dextran sulphate sodium (DSS) induces a colitis-like phenotype similar to that seen in other systems, and is characterized by alteration of epithelial morphology and infiltration of blood cells into lamina propria-like regions. DSS treatment also influences the production and localization of a secreted immune molecule shown previously to co-localize to chitin-rich mucus in the gut. Resistance to DSS is enhanced by exposure to exogenous chitin microparticles, suggesting that endogenous chitin is critical to barrier integrity. Protochordates, such as Ciona, retain basic characteristics found in other more advanced chordates and can inform us of uniquely conserved signals shaping host-microbiota interactions in the absence of adaptive immunity. These simpler model systems may also reveal factors and processes that modulate recovery from colitis, the role gut microbiota play in the onset of the disease, and the rules that help govern the reestablishment and maintenance of gut homeostasis.
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Microbes associate with animal hosts, often providing shelter in a nutrient-rich environment. The gut, however, can be a harsh environment with members of the microbiome settling in distinct niches resulting in more stable, adherent biofilms. These diverse communities can provide orders of magnitude more gene products than the host genome; selection and maintenance of a functionally relevant and useful microbiome is now recognized to be an essential component of homeostasis. Germ-free (GF) model systems allow dissection of host-microbe interactions in a simple and direct way where each member of the symbiosis can be studied in isolation. In addition, because immune defenses in the gut are often naïve in GF animals, host immune recognition and responses during the process of colonization can be studied. Ciona intestinalis, a basal chordate, is a well-characterized developmental model system and holds promise for addressing some of these important questions. With transparent juveniles, Ciona can be exposed to distinct bacterial isolates by inoculating GF artificial seawater; concentrated bacteria can subsequently be visualized in vivo if fluorescent stains are utilized. Rearing GF Ciona is a first step in untangling the complex dialogue between bacteria and innate immunity during colonization.
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Protochordate variable region-containing chitin-binding proteins (VCBPs) consist of immunoglobulin-type V domains and a chitin-binding domain (CBD). VCBP V domains facilitate phagocytosis of bacteria by granulocytic amoebocytes; the function of the CBD is not understood. Here we show that the gut mucosa of Ciona intestinalis contains an extensive matrix of chitin fibrils to which VCBPs bind early in gut development, before feeding. Later in development, VCBPs and bacteria colocalize to chitin-rich mucus along the intestinal wall. VCBP-C influences biofilm formation in vitro and, collectively, the findings of this study suggest that VCBP-C may influence the overall settlement and colonization of bacteria in the Ciona gut. Basic relationships between soluble immunoglobulin-type molecules, endogenous chitin and bacteria arose early in chordate evolution and are integral to the overall function of the gut barrier.
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Proteínas Portadoras/inmunología , Quitina/metabolismo , Ciona intestinalis/inmunología , Microbioma Gastrointestinal/inmunología , Inmunidad Mucosa/inmunología , Región Variable de Inmunoglobulina/inmunología , Mucosa Intestinal/inmunología , Animales , Biopelículas , Proteínas Portadoras/metabolismo , Quitina Sintasa/genética , Quitina Sintasa/metabolismo , Inmunohistoquímica , Hibridación Fluorescente in Situ , MocoRESUMEN
A variety of germline and somatic immune mechanisms have evolved in vertebrate and invertebrate species to detect a wide array of pathogenic invaders. The gut is a particularly significant site in terms of distinguishing pathogens from potentially beneficial microbes. Ciona intestinalis, a filter-feeding marine protochordate that is ancestral to the vertebrate form, possesses variable region-containing chitin-binding proteins (VCBPs), a family of innate immune receptors, which recognize bacteria through an immunoglobulin-type variable region. The manner in which VCBPs mediate immune recognition appears to be related to the development and bacterial colonization of the gut, and it is likely that these molecules are critical elements in achieving overall immune and physiological homeostasis.
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Bacterias/inmunología , Ciona intestinalis/inmunología , Tracto Gastrointestinal/inmunología , Receptores Inmunológicos/inmunología , Vertebrados/inmunología , Animales , Evolución Biológica , Ciona intestinalis/genética , Ciona intestinalis/microbiología , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/microbiología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Receptores Inmunológicos/genética , Transcriptoma/genética , Transcriptoma/inmunología , Vertebrados/genética , Vertebrados/microbiologíaRESUMEN
Variable region-containing chitin-binding proteins (VCBPs) are secreted, immune-type molecules that have been described in both amphioxus, a cephalochordate, and sea squirt, Ciona intestinalis, a urochordate. In adult Ciona, VCBP-A, -B and -C are expressed in hemocytes and the cells of the gastrointestinal tract. VCBP-C binds bacteria in the stomach lumen and functions as an opsonin in vitro. In the present paper the expression of VCBPs has been characterized during development using in situ hybridization, immunohistochemical staining and quantitative polymerase chain reaction (qPCR) technologies. The expression of VCBP-A and -C is detected first in discrete areas of larva endoderm and becomes progressively localized during differentiation in the stomach and intestine, marking the development of gut tracts. In "small adults" (1-2 cm juveniles) expression of VCBP-C persists and VCBP-A gradually diminishes, ultimately replaced by expression of VCBP-B. The expression of VCBP-A and -C in stage 7-8 juveniles, at which point animals have already started feeding, is influenced significantly by challenge with either Gram-positive or -negative bacteria. A potential role for VCBPs in gut-microbiota interactions and homeostasis is indicated.