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Abstract The resources and platforms available on the internet for collecting and sharing information and performing genomic sequence analysis have made it possible to follow closely the evolution the evolution of SARS-CoV-2. However, the current monkeypox outbreak in the world brings us back to the need to use these resources to appraise the extent of this outbreak. The objective of this work was an analysis of the information presented so far in the genomic database GISAID EpiPox™, using various tools available on the web. The results indicate that the monkeypox outbreak is referred as MPXV clade II B.1 lineage and sub-lineages, isolated from male patients mainly from the European and American continents. In the current scenario, the access to genomic sequences, epidemiological information, and tools available to the scientific community is of great importance for global public health in order to follow the evolution of pathogens.

Resumen Los recursos y plataformas disponibles en Internet para recopilar, compartir información y realizar análisis de secuencias genómicas han permitido seguir de cerca la evolución del SARS-CoV-2. El actual brote global de viruela del mono en el mundo, requiere de nuevo utilizar estos recursos para conocer el alcance de este brote. El objetivo de este trabajo fue un análisis de la información presentada hasta el momento en la base de datos genómica EpiPox™ de GISAID, utilizando diversas herramientas disponibles en la web. Los resultados indican que el brote de la viruela del mono o símica está referido al linaje y sub-linajes B.1 del clado II de MPXV, aislado principalmente de pacientes hombres de Europa y América. En el escenario actual, el acceso a las secuencias genómicas, la información epidemiológica, y las herramientas disponibles para la comunidad científica son de gran importancia para la salud pública mundial con el fin de seguir la evolución de los patógenos.

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Rotavirus infection modifies Ca(2+) homeostasis provoking an increase in Ca(2+) permeation, cytoplasmic Ca(2+) concentration ([Ca(2+)](cyto)), total Ca(2+) pools and, a decrease of Ca(2+) response to agonists. These effects are mediated by NSP4. The mechanism by which NSP4 deranges Ca(2+) homeostasis is not yet known. It has been proposed that the increase in [Ca(2+)](cyto) is the result of Ca(2+) release from intracellular stores, thereby activating store-operated Ca(2+) entry (SOCE). We studied the mechanisms involved in the changes of Ca(2+) permeability of the plasma membrane elicited by rotavirus infection and NSP4 expression in Cos-7 cells loaded with fura-2 or fluo-4, using inhibitors and activators of different pathways. Total depletion of ER Ca(2+) stores induced by thapsigargin or ATP was not able to elicit Ca(2+) entry in mock-infected cells to the level attained with infection or NSP4-EGFP expression. The pathway induced by NSP4-EGFP expression or infection shows properties shared by SOCE: it can be inactivated by high [Ca(2+)](cyto), is permeable to Mn(2+) and inhibited by La(3+) and the SOC inhibitor 2-aminoethoxydiphenyl borate (2-APB). Contribution of the agonist-operated channels (AOCs) to Ca(2+) entry is small and not modified by infection. The plasma membrane permeability to Ca(2+) in rotavirus infected or NSP4-EGFP expressing cells is also blocked by KB-R7943, an inhibitor of the plasma membrane Na(+)/Ca(2+) exchanger (NCX), operating in its reverse mode. In conclusion, the expression of NSP4 in infected Cos-7 cells appears to activate the NCX in reverse mode and the SOCE pathway to induce increased Ca(2+) entry.

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Due to poor sanitary conditions, total global diarrhea mortality occurs almost exclusively in underdeveloped countries. Since, in these countries, sanitation is greatly overlooked, in the case of diarrheal disease, the emphasis must be oriented to treatment rather than prevention. An important aspect in the treatment of diarrheal disease is to avoid the malnutrition it causes and therefore, proper feeding during and after the diarrheal episode is a crucial issue. Accordingly, we studied a diarrhea rat model, based on the replacement of part of the dietary corn starch by lactose which produces diarrhea of increasing severity that reproduces the negative nutritional effects of diarrhea. Five levels of replacement (0, 30, 50, 70 and 90%) were studied and feces were collected on day 1-3, 4-6, 11-13 and 18-20 of the experiment. This resulted in an increment in fecal mass (diarrhea) which was proportional to the replacement level; it reached its maximum during the first week and declined thereafter. During the same period, reductions on the apparent digestibility of the dietary protein, fat, carbohydrates and energy were detected. These reductions were proportional to the severity of diarrhea and occurred in conjunction with a reduction in food intake. These two conditions resulted in a deficient growth which was also proportional to the severity of the lactose induced diarrhea. This model reproduces the negative effects that diarrhea has on infantile nutrition and it may be useful in preselecting the most appropriated foods to be tested in human studies.

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La mortalidad por diarrea ocurre casi en su totalidad en los países subdesarrollados. Esto, se debe fundamentalmente a las malas condiciones sanitarias que los caracterizan. En consecuencia, mientras estas condiciones no cambien, en estos países, el enfoque en el caso de las enfermedades diarreicas, debe concentrarse en el tratamiento. Un aspecto importante del tratamiento de la diarrea infantil es evitar la desnutrición que causa y en consecuencia, la alimentación apropiada durante la diarrea es de vital importancia. De acuerdo con esto, se estudió un modelo de diarrea en ratas que se basa en reemplazar parte del almidón de la dieta por lactosa lo cual permite obtener diarreas de severidad variable, que reproducen los efectos nutricionales de la diarrea infantil. Se estudiaron 5 niveles de reemplazo: 0, 30, 50, 70 y 90 por ciento y se realizaron colecciones fecales entre los días 1-3, 4-6, 11-13 y 18-20 del experimento. El reemplazo causó un aumento en la masa fecal que fue proporcional al nivel de sustitución. La masa fecal fue máxima durante la primera semana y disminuyó en las semanas subsiguientes. Se observó una reducción en la digestibilidad aparente de la proteína, grasa, carbohidratos y energía que fue proporcional a la severidad. La diarrea además estuvo asociada con una reducción en el consumo de alimento que en conjunto con la disminución en la absorción causaron un importante déficit de crecimiento. Este modelo reproduce los efectos negativos de la diarrea sobre la nutrición infantil y puede ser servir en la preselección de los alimentos más apropiados para la alimentación durante la diarrea.

Due to poor sanitary conditions, total global diarrhea mortality occurs almost exclusively in underdeveloped countries. Since, in these countries, sanitation is greatly overlooked, in the case of diarrheal disease, the emphasis must be oriented to treatment rather than prevention. An important aspect in the treatment of diarrheal disease is to avoid the malnutrition it causes and therefore, proper feeding during and after the diarrheal episode is a crucial issue. Accordingly, we studied a diarrhea rat model, based on the replacement of part of the dietary corn starch by lactose which produces diarrhea of increasing severity that reproduces the negative nutritional effects of diarrhea. Five levels of replacement (0, 30, 50, 70 y 90 percent) were studied and feces were collected on day 1-3, 4-6, 11-13 and 18-20 of the experiment. This resulted in an increment in fecal mass (diarrhea) which was proportional to the replacement level; it reached its maximum during the first week and declined thereafter. During the same period, reductions on the apparent digestibility of the dietary protein, fat, carbohydrates and energy were detected. These reductions were proportional to the severity of diarrhea and occurred in conjunction with a reduction in food intake. These two conditions resulted in a deficient growth which was also proportional to the severity of the lactose induced diarrhea. This model reproduces the negative effects that diarrhea has on infantile nutrition and it may be useful in pre-selecting the most appropriated foods to be tested in human studies.

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Rotavirus infection modifies Ca(2+) homeostasis, provoking an increase in Ca(2+) permeation, the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyto)), and total Ca(2+) pools and a decrease in Ca(2+) response to agonists. A glycosylated viral protein(s), NSP4 and/or VP7, may be responsible for these effects. HT29 or Cos-7 cells were infected by the SA11 clone 28 strain, in which VP7 is not glycosylated, or transiently transfected with plasmids coding for NSP4-enhanced green fluorescent protein (EGFP) or NSP4. The permeability of the plasma membrane to Ca(2+) and the amount of Ca(2+) sequestered in the endoplasmic reticulum released by carbachol or ATP were measured in fura-2-loaded cells at the single-cell level under a fluorescence microscope or in cell suspensions in a fluorimeter. Total cell Ca(2+) pools were evaluated as (45)Ca(2+) uptake. Infection with SA11 clone 28 induced an increase in Ca(2+) permeability and (45)Ca(2+) uptake similar to that found with the normally glycosylated SA11 strain. These effects were inhibited by tunicamycin, indicating that inhibition of glycosylation of a viral protein other than VP7 affects the changes of Ca(2+) homeostasis induced by infection. Expression of NSP4-EGFP or NSP4 in transfected cells induced the same changes observed with rotavirus infection, whereas the expression of EGFP or EGFP-VP4 showed the behavior of uninfected and untransfected cells. Increased (45)Ca(2+) uptake was also observed in cells expressing NSP4-EGFP or NSP4, as evidenced in rotavirus infection. These results indicate that glycosylated NSP4 is primarily responsible for altering the Ca(2+) homeostasis of infected cells through an initial increase of cell membrane permeability to Ca(2+).

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The mechanism by which rotavirus and other nonenveloped viruses enter the cell is still not clear. We have proposed an endocytosis model where the critical step for virus uncoating and membrane permeabilization is the decrease in Ca(2+) concentration in the endosome. In this paper, we monitored rotavirus entry by measuring alpha-sarcin-rotavirus coentry and infectivity in MA104 cells. The participation of endocytosis, acidification, and endosomal Ca(2+) concentration on virus entry was studied by inhibiting the endosomal H(+)-ATPase with bafilomycin A1 and/or increasing the extracellular calcium reservoir by addition of 10 mM CaEGTA. Rotavirus-alpha-sarcin coentry was inhibited by bafilomycin A1 and by addition of 10 mM CaEGTA. These effects were additive. These substances induced a significant inhibition of infectivity without affecting virus binding and postentry steps. These results are compatible with the interpretation that bafilomycin A1 and CaEGTA block rotavirus penetration from the endosome into the cytoplasm and support our hypothesis of a Ca(2+)-dependent endocytosis model.

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