Noves troballes sobre diabetis tipus 2 a nivell cel·lular

Els autors d'aquest treball van analitzar una proteïna present en el teixit muscular: el transportador de glucosa 4 o GLUT4, molt important en la regulació dels nivells de glucosa de la sang. Les conclusions de l'estudi permeten conèixer amb més profunditat els mecanismes cel·lulars implicats en el desenvolupament de la diabetis tipus 2.Los autores de este trabajo analizaron una proteína presente en el tejido muscular: el transportador de glucosa 4 o GLUT4, de gran importancia en la regulación de los niveles de glucosa en la sangre. Las conclusiones del estudio permiten conocer con más profundidad los mecanismos celulares implicados en el desarrollo de la diabetes tipo 2

Vacunes per a peixos per administració oral

Actualment la major part del consum de peixos prové de l'aqüicultura. En aquest sentit, per millorar la condició de les espècies cultivades, el grup d'Immunologia evolutiva de la Unitat de Mecanismes de resposta a l'estrès i la malaltia de l'institut de Biotecnologia i Biomedicina (IBB- UAB) liderat per la Dra. Nerea Roher ha desenvolupat un format de vacuna oral per peixos d'interès comercial, en col·laboració amb el Instituto de Investigación y Formación Agraria y Pesquera (IFAPA) de Càdiz.Actualmente la mayor parte del consumo de peces proviene de la acuicultura. En este sentido, para mejorar la condición de las especies cultivadas, el grupo de Inmunología evolutiva de la Unidad de Mecanismos de respuesta al estrés y la enfermedad del Instituto de Biotecnologia y Biomedicina (IBB-UAB) liderado por la Dra. Nerea Roher ha desarrollado un formato de vacuna oral para peces de interés comercial, en colaboración con el Instituto de Investigación y Formación Agraria y Pesquera (IFAPA) de Cádiz

Interacció de la proteïna quinasa CK2 amb la xaperona GRP94 : implicacions funcionals /

Consultable des del TDXTítol obtingut de la portada digitalitzadaLa proteïna quinasa CK2 és una serina/treonina quinasa molt conservada i ubiqua en organismes eucariotes, implicada en diversos processos cel·lulars importants com proliferació o tumorigénesis. L'enzim de mamífers està format por dues subunitats catalítiques (a y/o a') y dues subunitats reguladores (b) encara que també s'ha descrit l'existència de subunitats catalítiques lliures. La regulació de l'activitat CK2 no és una regulació clàssica mitjançant segons missatgers o fosforilació, de fet la subunitat catalítica té activitat constitutiva. Es postula que la regulació es podria dur a terme per variació de la localització subcel·lular mitjançant interacció con proteïnes adscrites a compartiments subcel·lulares concrets. La proteïna xaperona grp94 (94 kDa-glucose regulated protein) és una proteïna d'estrès que pertany a la família de les hsp90 i que es localitza al Reticle Endoplásmic (RE) de totes les cèl·lules eucariotes. S'expressa constitutivament en condiciones normals y es sobreexpressa en situacions d'estrès com depleció de calci, inhibició de la glicosilació, infecció vírica, agents reductores etc. Utilitzant tècniques de Ressonància Plasmònica, Far Western y Pull down demostrem que el domini carboxi terminal de grp94 interacciona amb la subunitat catalítica de CK2 (CK2a) però no amb la subunitat reguladora ni amb l'holoenzim (a2b2). S'ha pogut mapejar la regió d'interacció entre ambdues proteïnes, utilizant diversos mutants de CK2a, així la regió s'ha delimitat a una regió de lisines (K74-77) molt conservada entre las CK2a de diferents espècies. D'altra banda s'ha demostrat que grp94 té activitat xaperona in vitro sobre CK2a i citrat sintasa (CS). Utilizant assajos d'agregació induïda por xoc tèrmic y tècniques de microscòpia electrònica s'ha determinat que grp94 es capaç d'inhibir l'agregación de CK2a i CS. L'activitat xaperona depén fortament de l'estat d'oligomerització de grp94, de forma que en condicions reductores en que grp94 perd la seva estructura quaternària, la capacitat d'inhibir agregació de proteïnes està disminuïda. S'ha estudiat també la distribució subcel·lular de CK2 i grp94 en un model animal de rates genèticament obeses (fa/fa). Aquestes rates són hiperinsulinèmiques i resistents a insulina degut a una down-regulation del receptor de insulina. S'había descrit en cèl·lules en cultiu que el tractament amb dosis altes d'insulina induïa la sínstesi de grp94. S'ha observat que no hi ha variació ni en els nivells ni en la distribució subcel·lular de grp94 però si hi ha variació tan en els nivells com en la distribució de CK2, que disminueix marcadament en citosol i augmenta en les fraccions membranoses.Protein kinase CK2 is a highly conserved and ubiquitously distributed serin/threonin kinase described in all eukaryotic organisms. CK2 has been implied in different cellular processes as proliferation or cancer. The mammalian enzyme is composed of two catalytic subunits (a and/or a') and two regulatory subunits (b), but have been described free catalytic and regulatory subunits. Regulation of CK2 is not a classic regulation through second messengers or phosphorylation, furthermore the catalytic subunit posses constitutive activity and it has been postulated that regulation could be done by changes in subcellular distribution due to interaction with different proteins from defined subcellular compartments. The protein chaperone grp94 (94 kDa-glucose regulated protein) belongs to the hsp90 family and is localized in the Endoplasmic Reticulum (ER) of all eukaryotic cells. Is constitutively expressed in absence of stress and is overexpressed under stress conditions as calcium depletion, virus infection, reducing agents etc. In the present work using techniques as Surface Plasmon Ressonance (SPR), Far Western and Pull down assays we have demonstrated that carboxy-terminal domain of grp94 interacts with the catalytic subunit of CK2 (CK2a) but not with the regulatory subunit or with the holoenzyme (a2b2). We mapped the interacting region using different mutants of CK2a, and we delimited the interacting domain to the cluster of lysines present in the helix aC(K74-77) on CK2a. This basic cluster is highly conserved in CK2a from different species but is not present in other kinases of the same family. Furthermore we demonstrated that grp94 has chaperone activity in vitro on CK2a and citrate synthase (CS). Light scattering of aggregated samples (aggregation promoted by heat shock) and electronic microscope imaging of aggregated samples have demonstrated that grp94 is able to protect CK2a and CS from aggregation. Chaperone activity depends on the maintenance of oligomeric state of grp94, then in reducing conditions when grp94 has lost its quaternary structure the ability to inhibit aggregation is diminished. In this work we also have been studied the subcellular distribution of CK2 and grp94 in an animal model of genetically obese rats (fa/fa). Zucker fa/fa rats are hyperinsulinemics and insulin resistant due to a down-regulation of insulin receptor. Additionally it has been described that in cultured cells insulin promoted the overexpression of grp94. In our model there is no variation of the levels or distribution of grp94 but exist differences in the levels and distribution of CK2 in liver of obese rats. In this rats CK2 is lower in the cytosolic fraction and increase in membranous fractions

Nanodelivery Systems as New Tools for Immunostimulant or Vaccine Administration : Targeting the Fish Immune System

Fish disease treatments have progressed significantly over the last few years and have moved from the massive use of antibiotics to the development of vaccines mainly based on inactivated bacteria. Today, the incorporation of immunostimulants and antigens into nanomaterials provide us with new tools to enhance the performance of immunostimulation. Nanoparticles are dispersions or solid particles designed with specific physical properties (size, surface charge, or loading capacity), which allow controlled delivery and therefore improved targeting and stimulation of the immune system. The use of these nanodelivery platforms in fish is in the initial steps of development. Here we review the advances in the application of nanoparticles to fish disease prevention including: the type of biomaterial, the type of immunostimulant or vaccine loaded into the nanoparticles, and how they target the fish immune system

Polystyrene nanoplastics accumulate in ZFL cell lysosomes and in zebrafish larvae after acute exposure, inducing a synergistic immune response in vitro without affecting larval survival in vivo

Altres ajuts: pre-doctoral scholarship from UAB (2018FI_B_00711)The presence of small-sized plastic particles in marine and freshwater environments is a global problem but their long-term impact on ecosystems and human health is still far from being understood. Nanoplastics (<1000 nm) could pose a real and uncontrolled ecological challenge due to their smaller size and sharp ability to penetrate living organisms at any trophic level. Few studies evaluate the impact of nanoplastics in vivo on the immune system of aquatic organisms, while most of them assessed the impact on indirect markers of immune response such as regulation of gene expression, ROS production or DNA genotoxicity, among others. Moreover, the study of the effects of nanoplastics on aquatic vertebrate species in vivo is still scarce. In this context, we seek to shed light on the underlying effects of polystyrene nanoplastics (PS-NPs) on the immune response in a model fish species (Danio rerio, zebrafish) after an acute exposure, with a combination of in vitro and in vivo experiments. Our results show that PS-NPs (65 nm) are efficiently taken up by zebrafish liver cells, accumulating mainly in lysosomes. Furthermore, the expression of immune genes presents a synergy when cells were simultaneously exposed to PS-NPs, at a low dose and early time point (12 h) and challenged with a viral stimulus (poly(I:C)). Moreover, zebrafish larvae also internalize PS-NPs, accumulating them in the gut and pancreas. However, at concentrations of up to 50 mg l-1 in an acute exposure (48 h), PS-NPs do not interfere with the survival of the larvae after a lethal bacterial challenge (Aeromonas hydrophila). This study addresses the relevant environmental question of whether a living organism exposed to PS-NPs can cope with a real immune threat. We show that, although PS-NPs can induce an immune response, the survival of zebrafish larvae challenged with a bacterial infection after an acute exposure to PS-NP is not decimated with respect to unexposed larvae

A Novel Liposome-Based Nanocarrier Loaded with an LPS-dsRNA Cocktail for Fish Innate Immune System Stimulation

Development of novel systems of vaccine delivery is a growing demand of the aquaculture industry. Nano- and micro- encapsulation systems are promising tools to achieve efficient vaccines against orphan vaccine fish diseases. In this context, the use of liposomal based-nanocarriers has been poorly explored in fish; although liposomal nanocarriers have successfully been used in other species. Here, we report a new ~125 nm-in-diameter unilamellar liposome-encapsulated immunostimulant cocktail containing crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analog of dsRNA virus, aiming to be used as a non-specific vaccine nanocarrier in different fish species. This liposomal carrier showed high encapsulation efficiencies and low toxicity not only in vitro using three different cellular models but also in vivo using zebrafish embryos and larvae. We showed that such liposomal LPS-dsRNA cocktail is able to enter into contact with zebrafish hepatocytes (ZFL cell line) and trout macrophage plasma membranes, being preferentially internalized through caveolae-dependent endocytosis, although clathrin-mediated endocytosis in ZFL cells and macropinocytocis in macrophages also contribute to liposome uptake. Importantly, we also demonstrated that this liposomal LPS-dsRNA cocktail elicits a specific pro-inflammatory and anti-viral response in both zebrafish hepatocytes and trout macrophages. The design of a unique delivery system with the ability to stimulate two potent innate immunity pathways virtually present in all fish species represents a completely new approach in fish health

Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout

Background: The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). Methods: To determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses. Results: LPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha. Conclusions In view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction

Decoding the genetic and functional diversity of the DSF Quorum-Sensing system in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia uses the Diffusible Signal Factor (DSF) quorum sensing (QS) system to mediate intra- and inter-specific signaling and regulate virulence-related processes. The components of this system are encoded by the rpf cluster, with genes rpfF and rpfC encoding for the DSF synthase RpfF and sensor RpfC, respectively. Recently, we have shown that there exist two variants of the rpf cluster (rpf-1 and rpf-2), distinguishing two groups of S. maltophilia strains. Surprisingly, only rpf-1 strains produce detectable DSF, correlating with their ability to control biofilm formation, swarming motility and virulence. The evolutive advantage of acquiring two different rpf clusters, the phylogenetic time point and mechanism of this acquisition and the conditions that activate DSF production in rpf-2 strains, are however not known. Examination of this cluster in various species suggests that its variability originated most probably by genetic exchange between rhizosphere bacteria. We propose that rpf-2 variant strains make use of a strategy recently termed as "social cheating." Analysis of cellular and extracellular fatty acids (FAs) of strains E77 (rpf-1) and M30 (rpf-2) suggests that their RpfFs have also a thioesterase activity that facilitates the release of unspecific FAs to the medium in addition to DSF. Production of DSF in rpf-1 strains appears in fact to be modulated by some of these extracellular FAs in addition to other factors such as temperature and nutrients, while in rpf-2 strains DSF biosynthesis is derepressed only upon detection of DSF itself, suggesting that they require cohabitation with DSF-producer bacteria to activate their DSF regulatory machinery. Finally, we show that the mixed rpf-1/rpf-2 population presents synergism in DSF production and virulence capacity in an in vivo infection model. Recovery and quantification of DSF from co-infected animals correlates with the observed mortality rate

Conseqüències fisiológiques d'una activació del sistema immunitari sobre la reproducción i el creixement en peixos d'interés en aqüicultura

En peixos teleostis, una activació del sistema immunitari comporta la producció de factors immunitaris que ajuden a combatre una infecció i estableixen una comunicació entre la resposta immunitària innata i l'adaptativa. A part d'aquesta funció estrictament immunitària, els factors immunitaris poden afectar el funcionament d'altres cèl·lules i teixits no immunitaris. Dos processos particularment importants, tant per l'estat biològic d'una població de peixos, com per aspectes més relacionats amb la productivitat d'espècies en cultiu, són la reproducció i el creixement. En aquest treball es descriuen estudis encaminats a investigar els efectes d'una activació del sistema immunitari, i dels factors produïts, sobre la reproducció i el creixement en peixos teleostis.In teleost fish, an activation of the immune system entails the production of immune factors that are key to fighting an infeccion and establish a bridge between the innate and the adaptive immune responses. In addition to this strictly immune function, immune factors can also affect the function of other non-immune cells and tissues. Reproduction and growth are two processes that are particularly important for the biological condition of a population of fish as well as for aspects more related to productivity of cultured species. In this paper, we describe studies designed to investigate the effects of an activation of the immune system, as well as of the factors produced, on reproduction and growth in teleost fish