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Hepatic steatosis is characterized by triglyceride accumulation within hepatocytes in response to a high calorie intake, and it may be related to intestinal microbiota disturbances. The prebiotic inulin is a naturally occurring polysaccharide with a high dietary fiber content. Here, we evaluate the effect of inulin on the intestinal microbiota in a non-alcoholic fatty liver disease model. Mice exposed to a standard rodent diet or a fat-enriched diet, were supplemented or not, with inulin. Liver histology was evaluated with oil red O and H&E staining and the intestinal microbiota was determined in mice fecal samples by 16S rRNA sequencing. Inulin treatment effectively prevents liver steatosis in the fat-enriched diet group. We also observed that inulin re-shaped the intestinal microbiota at the phylum level, were Verrucomicrobia genus significantly increased in the fat-diet group; specifically, we observed that Akkermansia muciniphila increased by 5-fold with inulin supplementation. The family Prevotellaceae was also significantly increased in the fat-diet group. Overall, we propose that inulin supplementation in liver steatosis-affected animals, promotes a remodeling in the intestinal microbiota composition, which might regulate lipid metabolism, thus contributing to tackling liver steatosis.

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INTRODUCTION: SARS-CoV-2 infection in Mexico has caused ~2.7 million confirmed cases; around 20%-25% of health workers will be infected by the virus at their workplace, with approximately 4.4% of mortality. High infectivity of SARS-CoV-2 is related with cell entry mechanism, through the ACE receptor. SARS-CoV-2 requires transmembrane protease serine 2 to cleave its spike glycoprotein and ensure fusion of host cell and virus membrane. We propose studying prophylactic treatment with hydroxychloroquine (HCQ) and bromhexine (BHH), which have been shown to be effective in preventing SARS-CoV-2 infection progression when administered in early stages. The aim of this study is to assess the efficacy of HCQ and BHH as prophylactic treatments for SARS-CoV-2 infection in healthy health workers exposed to the virus. METHODS AND ANALYSIS: Double-blind randomised clinical trial, with parallel allocation at a 1:1 ratio with placebo, of low doses of HCQ plus BHH, for 60 days. Study groups will be defined as follows: (1) HCQ 200 mg/day+BHH 8 mg/8 hours versus (2) HCQ placebo plus BHH placebo. Primary endpoint will be efficacy of both interventions for the prevention of SARS-CoV-2 infection, determined by the risk ratio of infected personnel and the absolute risk. At least a 16% reduction in absolute risk is expected between the intervention and placebo groups; a minimum of 20% infection is expected in the placebo group. The sample size calculation estimated a total of 214 patients assigned: two groups of 107 participants each. ETHICS AND DISSEMINATION: This protocol has been approved by the local Medical Ethics Committee (National Institute of Rehabilitation 'Luis Guillermo Ibarra Ibarra', approval number INRLGII/25/20) and by the Federal Commission for Protection against Sanitary Risks (COFEPRIS, approval number 203 300 410A0058/2020). The results of the study will be submitted for publication in peer-reviewed journals and disseminated through conferences. TRIAL REGISTRATION NUMBER: NCT04340349.

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AIMS: The retinal pigment epithelium (RPE) is a highly specialized cell monolayer, that plays a key role in the maintenance of photoreceptor function and the blood-retina barrier (BRB). In this study, we found that a myristoylated pseudosubstrate of PKC-ζ (PKCζ PS), considered as a PKC-ζ inhibitor, plays a distinct role in RPE. MAIN METHODS: We demonstrated that PKCζ PS stimulates the release of Glutamate (Glu) using in vitro3H-Glutamate release experiments. By western blot, kinase assays, and Fluoresence Ca+2 Concentration Measurements, we determined the cellular mechanisms involved in such release. KEY FINDINGS: Surprisingly, PKCζ PS has no effect on either phosphorylation of T560, essential for catalytic activity, nor it has an effect on kinase activity. It induces the dose-dependent release of Glu by increasing intracellular Ca+2 levels. Interestingly, this release was not observed upon stimulation by other non-competitive PKC-ζ inhibitors. We here demonstrated that the PKCζ PS stimulates the release of Glutamate from RPE by activating the Ca2+-dependent Cl channel Bestrophin 1 (Best1). SIGNIFICANCE: These results question PKCζ PS specificity as an inhibitor of this enzyme. Furthermore, the present results underline the relevance of clarifying the molecular mechanisms involved in glutamate release from the retina under conditions derived from excitotoxic stimuli.

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The common opossum, Didelphis marsupialis thrives near human settlements. Understanding its ecology could help planning management decisions about this species, especially in anthropogenic landscapes. Yet, there are no density estimations for this species throughout its distribution range in Bolivia. We estimated the density of D. marsupialis in a rural agricultural community, where agroforestry plantations and fallows cover most of the land. We counted individuals in line transects and used DISTANCE software to calculate density. We covered a total of 70.21 km in 143 night counts, obtaining 38 records of D. marsupialis. We estimated a density of 0.30 individuals ha-1 (SE = 0.062; range: 0.20 - 0.45 individuals ha-1), with a mean encounter rate of 0.54 individuals km-1. Encounter rate varied between habitats, with mean values of 0.20 in secondary forests and 0.64 in agroforestry plantations. Our density estimate is near the lower range of previously reported values for the common opossum in other countries. We argue that our results may reflect the response to the availability of food resources and predation pressure in agroforestry plantations. (AU)

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PAR1 activation by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration, characteristic of fibroproliferative eye diseases. Due to the cleavage of PAR1 N-terminal domain, carried by thrombin, the arrest of PAR1 signaling is achieved by transport into lysosomes and degradation. Recent findings suggest that the GTPase Rab11a in conjunction with its effector RCP may direct PAR1 to lysosomes. Hereby we demonstrate that thrombin-induced PAR1 internalization and lysosomal targeting requires the disassembly of the Rab11a/RCP complex, and that this process depends on thrombin-induced intracellular calcium increase and calpain activation. These findings unveil a novel mechanism that regulates thrombin activated PAR1 internalization and degradation.

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La carachupa, Didelphis marsupialis prospera cerca de asentamientos humanos. Entender la ecología de una población puede ayudar a tomar decisiones de manejo de esta especie, especialmente en paisajes antropogénicos. Sin embargo, no existen estimaciones de la densidad de esta especie en todo su rango de distribución en Bolivia. Este estudio estimó la densidad de D. marsupialis en una comunidad agrícola, donde los sistemas agroforestales y barbechos cubren la mayoría de la tierra. Empleamos conteos en transectos lineales y usamos el programa DISTANCE para calcular la densidad. En total recorrimos 70.21 km en 143 noches de conteo y obtuvimos 38 registros de D. marsupialis. Estimamos una densidad de 0.30 individuos ha-1 (SE = 0.062; rango: 0.20 - 0.45 individuos ha-1) y una tasa de encuentro promedio de 0.54 individuos km-1. La tasa de encuentro varió entre hábitats, con valores promedio de 0.2 para bosque secundario y 0.64 para sistemas agroforestales. Nuestra estimación de densidad se encuentra cerca de los valores más bajos antes reportados para la especie en otros países. Creemos que nuestros resultados reflejan la respuesta a la disponibilidad de recursos en sistemas agroforestales y la presión de depredación en nuestro sitio de estudio.(AU)

The common opossum, Didelphis marsupialis thrives near human settlements. Understanding its ecology could help planning management decisions about this species, especially in anthropogenic landscapes. Yet, there are no density estimations for this species throughout its distribution range in Bolivia. We estimated the density of D. marsupialis in a rural agricultural community, where agroforestry plantations and fallows cover most of the land. We counted individuals in line transects and used DISTANCE software to calculate density. We covered a total of 70.21 km in 143 night counts, obtaining 38 records of D. marsupialis. We estimated a density of 0.30 individuals ha-1 (SE = 0.062; range: 0.20 - 0.45 individuals ha-1), with a mean encounter rate of 0.54 individuals km-1. Encounter rate varied between habitats, with mean values of 0.20 in secondary forests and 0.64 in agroforestry plantations. Our density estimate is near the lower range of previously reported values for the common opossum in other countries. We argue that our results may reflect the response to the availability of food resources and predation pressure in agroforestry plantations.(AU)

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Purpose: We analyzed the molecular mechanisms leading to glutamate release from rat primary cultures of RPE cells, under isosmotic conditions. Thrombin has been shown to stimulate glutamate release from astrocytes and retinal glia; however, the effect of thrombin on glutamate release from RPE cells has not been examined. Our previous work showed that upon the alteration of the blood-retina barrier, the serine protease thrombin could contribute to the transformation, proliferation, and migration of RPE cells. In this condition, elevated extracellular glutamate causes neuronal loss in many retinal disorders, including glaucoma, ischemia, diabetic retinopathy, and inherited photoreceptor degeneration. Methods: Primary cultures of rat RPE cells were preloaded with 1 µCi/ml 3H-glutamate in Krebs Ringer Bicarbonate (KRB) buffer for 30 min at 37 °C. Cells were rinsed and super-perfused with 1 ml/min KRB for 15 min. Stable release was reached at the 7th minute, and on the 8th minute, fresh KRB containing stimuli was added. Results: This study showed for the first time that thrombin promotes specific, dose-dependent glutamate release from RPE cells, induced by the activation of protease-activated receptor 1 (PAR-1). This effect was found to depend on the Ca2+ increase mediated by the phospholipase C-ß (PLC-ß) and protein kinase C (PKC) pathways, as well as by the reverse activity of the Na+/Ca2+ exchanger. Conclusions: Given the intimate contact of the RPE with the photoreceptor outer segments, diffusion of RPE-released glutamate could contribute to the excitotoxic death of retinal neurons, and the development of thrombin-induced eye pathologies.

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The serine protease thrombin activates Protease-Activated Receptors (PARs), a family of G-protein-coupled receptors (GPCRs) activated by the proteolytic cleavage of their extracellular N-terminal domain. Four members of this family have been identified: PAR1-4. The activation of Protease-Activated Receptor 1(PAR1), the prototype of this receptor family, leads to an increase in intracellular Ca+2 concentration ([Ca+2]i) mediated by Gq11α coupling and phospholipase C (PLC) activation. We have previously shown that the stimulation of PAR1 by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration which characterize fibroproliferative eye diseases leading to blindness. Within this context, the elucidation of the mechanisms involved in PAR1 inactivation is of utmost importance. Due to the irreversible nature of PAR1 activation, its inactivation must be efficiently regulated in order to terminate signaling. Using ARPE-19 human RPE cell line, we characterized thrombin-induced [Ca+2]i increase and demonstrated the calcium-dependent activation of µ-calpain mediated by PAR1. Calpains are a family of calcium-activated cysteine proteases involved in multiple cellular processes including the internalization of membrane proteins through clathrin-coated vesicles. We demonstrated that PAR1-induced calpain activation results in the degradation of α-spectrin by calpain, essential for receptor endocytosis, and the consequent decrease in PAR1 membrane expression. Collectively, the present results identify a novel µ-calpain-dependent mechanism for PAR1 inactivation following exposure to thrombin.

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We studied the home range and habitat use of paca (Cuniculus paca) in a rural agricultural community in the Alto Beni region, in La Paz department, Bolivia, where game meat is the main source of protein for a large portion of local inhabitants. We captured and radio collared five individual paca (4 females and 1 male), which were followed for six months in 2012 to estimate home range of the individuals and assess their habitat use and selection. The total mean home range (95% Minimum Convex Polygon) was 2.26 ha (SD = 0.80), and ranged from 1.5 to 2.96 ha. The mean female home range (2.04 ha) was smaller than the males (2.95 ha). Pacas used two (crops and fallows) of the three available habitats (secondary forest patches), and appeared to positively select crops over the other two. The importance of crops may be linked to the type of agriculture conducted in the area (i.e. mainly cacao in agroforestry systems), which may offer more food and shelter for pacas. Given that, the importance of habitats may change seasonally; this topic should be examined in future studies. Our results suggest that agroforestry systems may be important for the species since they offer a variety of food resources throughout the year.(AU)

Hemos estudiado el ámbito de hogar y uso de hábitat del jochi pintado (Cuniculus paca) en una comunidad de Alto Beni en el Norte de La Paz-Bolivia, donde la carne obtenida por cacería es, como en muchas regiones de la Amazonia, una fuente principal de proteína para muchas poblaciones indígenas y campesinas. Hemos capturado y seguido con radio-collar a cinco individuos (4 hembras y 1 macho) por seis meses en 2012. El ámbito de hogar promedio de los individuos fue de 2,26 ha (ds=0,80), usando el estimador Mínimo Polígono Convexo (MPC al 95%) y los valores se encuentran entre 1,5 a 2,96 ha. El ámbito de hogar promedio de las hembras fue más pequeño (2,04 ha) que del macho (2,95 ha). Los jochis estudiados usaron 2 (cultivos y barbechos) de los 3 hábitats disponibles (parches de bosque secundario), aunque los cultivos parecen ser seleccionados positivamente respecto a los otros dos. La importancia de los cultivos podría estar ligada al tipo de cultivo que se realiza en la zona (sistemas agroforestales), que podrían estar ofreciendo mejores condiciones para ser usados por los jochis. Adicionalmente, la importancia de los hábitats podría cambiar de acuerdo a la época del año, lo cual podría ser abordado en siguientes estudios. Este trabajo es el primero en dar detalles sobre la ecología de C. paca en vida silvestre en Bolivia; sin embargo sería necesario contar con más individuos para tener más detalle sobre la ecología espacial de esta especie.(AU)

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ABSTRACT We studied the home range and habitat use of paca (Cuniculus paca) in a rural agricultural community in the Alto Beni region, in La Paz department, Bolivia, where game meat is the main source of protein for a large portion of local inhabitants. We captured and radio collared five individual paca (4 females and 1 male), which were followed for six months in 2012 to estimate home range of the individuals and assess their habitat use and selection. The total mean home range (95% Minimum Convex Polygon) was 2.26 ha (SD = 0.80), and ranged from 1.5 to 2.96 ha. The mean female home range (2.04 ha) was smaller than the male´s (2.95 ha). Pacas used two (crops and fallows) of the three available habitats (secondary forest patches), and appeared to positively select crops over the other two. The importance of crops may be linked to the type of agriculture conducted in the area (i.e. mainly cacao in agroforestry systems), which may offer more food and shelter for pacas. Given that, the importance of habitats may change seasonally; this topic should be examined in future studies. Our results suggest that agroforestry systems may be important for the species since they offer a variety of food resources throughout the year.

RESUMEN Hemos estudiado el ámbito de hogar y uso de hábitat del jochi pintado (Cuniculus paca) en una comunidad de Alto Beni en el Norte de La Paz-Bolivia, donde la carne obtenida por cacería es, como en muchas regiones de la Amazonia, una fuente principal de proteína para muchas poblaciones indígenas y campesinas. Hemos capturado y seguido con radio-collar a cinco individuos (4 hembras y 1 macho) por seis meses en 2012. El ámbito de hogar promedio de los individuos fue de 2,26 ha (ds=0,80), usando el estimador Mínimo Polígono Convexo (MPC al 95%) y los valores se encuentran entre 1,5 a 2,96 ha. El ámbito de hogar promedio de las hembras fue más pequeño (2,04 ha) que del macho (2,95 ha). Los jochis estudiados usaron 2 (cultivos y barbechos) de los 3 hábitats disponibles (parches de bosque secundario), aunque los cultivos parecen ser seleccionados positivamente respecto a los otros dos. La importancia de los cultivos podría estar ligada al tipo de cultivo que se realiza en la zona (sistemas agroforestales), que podrían estar ofreciendo mejores condiciones para ser usados por los jochis. Adicionalmente, la importancia de los hábitats podría cambiar de acuerdo a la época del año, lo cual podría ser abordado en siguientes estudios. Este trabajo es el primero en dar detalles sobre la ecología de C. paca en vida silvestre en Bolivia; sin embargo sería necesario contar con más individuos para tener más detalle sobre la ecología espacial de esta especie.

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The breakdown of the blood-retina barrier exposes retinal pigment epithelium (RPE) to serum components, thrombin among them. In addition to coagulation, thrombin acting through Protease-Activated Receptors (PARs 1-4) participates in a number of processes including cell proliferation, transformation, and migration. The purpose of this study was to identify interacting signaling pathways by which the activation of PAR1 by thrombin triggers cyclin D1 gene (Ccnd1) expression and the proliferation of RPE cells, characteristic of proliferative vitreoretinopathy (PVR). Our results demonstrate that thrombin induces the expression of the c-fos gene (c-fos), the activation of the (fos/jun) AP-1 site and the expression of Ccnd1, in precise correlation with the activation of CREB. Although the expression of both, c-fos and Ccnd1 requires the activation of conventional PKC isoforms and PI3K, downstream signaling from PI3K differs for both genes. Whereas the expression of c-fos requires PI3K-induced PDK1/Akt activity, that of Ccnd1 is mediated by PDK1-independent PKCζ signaling. Additionally, CREB activation may contribute to the induction of Ccnd1 expression through binding to the Ca/CRE element in the Ccnd1 gene promoter. Since cyclin D1 is a key regulator of cell cycle G1/S phase progression essential for proliferation, these findings further strengthen the critical involvement of thrombin in the development of proliferative retinopathies and may provide pharmacologic targets for the prevention or treatment of these diseases.

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Tricarbonyl[9-(triphenylphosphonio)fluorenylidene]ruthenium, [Ru(C31H21P)(CO)3], (I), is mononuclear, consisting of a single Ru centre, to which three carbonyl units and a chelating µ3-9-(triphenylphosphonio)fluorenide ylide bind to generate a distorted octahedral RuC6 core. Nonacarbonyl-µ3-fluorenylidene-µ2-hydrido-triangulo-triosmium(III), [Os3H(C13H7)(CO)9], (II), is trinuclear and presents a triangular triosmium core, nine carbonyl ligands and one fluorenylidene ligand. Two of the Os(III) centres present a highly distorted hexacoordinated Os(Os2C4) core and are in turn bridged by a hydride ligand. The remaining Os(III) cation is octacoordinated, with an Os(Os2C6) nucleus. The crystal structures of both compounds are the result of nondirectional forces, much resembling the packing of weakly interacting quasi-spherical units, viz. the molecules themselves in (I) and centrosymmetric π-π-bonded dimers in (II).