Reference Genes for Real-Time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity

Hepatotoxicity is associated with major changes in liver gene expression induced by xenobiotic exposure. Understanding the underlying mechanisms is critical for its clinical diagnosis and treatment. MicroRNAs are key regulators of gene expression that control mRNA stability and translation, during normal development and pathology. The canonical technique to measure gene transcript levels is Real-Time qPCR, which has been successfully modified to determine the levels of microRNAs as well. However, in order to obtain accurate data in a multi-step method like RT-qPCR, the normalization with endogenous, stably expressed reference genes is mandatory. Since the expression stability of candidate reference genes varies greatly depending on experimental factors, the aim of our study was to identify a combination of genes for optimal normalization of microRNA and mRNA qPCR expression data in experimental models of acute hepatotoxicity. Rats were treated with four traditional hepatotoxins: acetaminophen, carbon tetrachloride, D-galactosamine and thioacetamide, and the liver expression levels of two groups of candidate reference genes, one for microRNA and the other for mRNA normalization, were determined by RT-qPCR in compliance with the MIQE guidelines. In the present study, we report that traditional reference genes such as U6 spliceosomal RNA, Beta Actin and Glyceraldehyde-3P-dehydrogenase altered their expression in response to classic hepatotoxins and therefore cannot be used as reference genes in hepatotoxicity studies. Stability rankings of candidate reference genes, considering only those that did not alter their expression, were determined using geNorm, NormFinder and BestKeeper software packages. The potential candidates whose measurements were stable were further tested in different combinations to find the optimal set of reference genes that accurately determine mRNA and miRNA levels. Finally, the combination of MicroRNA-16/5S Ribosomal RNA and Beta 2 Microglobulin/18S Ribosomal RNA were validated as optimal reference genes for microRNA and mRNA quantification, respectively, in rat models of acute hepatotoxicity

In vitro and ex vivo effect of hyaluronic acid on erythrocyte flow properties

<p>Abstract</p> <p>Background</p> <p>Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed <it>in vitro </it>experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the <it>in vitro </it>experiment with <it>ex vivo </it>RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.</p> <p>Methods</p> <p>Erythrocyte deformability (by filtration through nucleopore membranes) and erythrocyte aggregability (EA) were tested on blood from healthy donors additioned with purified HA. EA was measured by transmitted light and analyzed with a mathematical model yielding two parameters, the aggregation rate and the size of the aggregates. Conformational changes of cytoskeleton proteins were estimated by electron paramagnetic resonance spectroscopy (EPR).</p> <p>Results</p> <p><it>In vitro</it>, erythrocytes treated with HA showed increased rigidity index (RI) and reduced aggregability, situation strongly related to the rigidization of the membrane cytoskeleton triggered by HA, as shown by EPR results. Also, a significant correlation (r: 0.77, p < 0.00001) was found between RI and serum HA in RA patients.</p> <p>Conclusions</p> <p>Our results lead us to postulate the hypothesis that HA interacts with the erythrocyte surface leading to modifications in erythrocyte rheological and flow properties, both <it>ex vivo </it>and <it>in vitro</it>.</p

Commercially approved vaccines for canine leishmaniosis: a review of available data on their safety and efficacy

Canine leishmaniosis is an important vector-borne zoonosis caused mainly by Leishmania infantum. Diagnosis and treatment of affected individuals can be particularly complex, hindering infection control in endemic areas. Methods to prevent canine leishmaniosis include the use of topical insecticides, prophylactic immunotherapy and vaccination. Four vaccines against canine leishmaniosis have been licensed since 2004, two in Brazil (Leishmune®, the production and marketing license of which was withdrawn in 2014, and Leish-Tec®) and two in Europe (CaniLeish® and LetiFend®). After several years of marketing, doubts remain regarding vaccine efficacy and effectiveness, potential infectiousness of vaccinated and infected animals or the interference of vaccine-induced antibodies in L. infantum serological diagnosis. This review summarizes the scientific evidence for each of the vaccines commercially approved for canine leishmaniosis, while discussing possible weaknesses of these studies. Furthermore, it raises the need to address important questions related to vaccination impact in Leishmania-endemic countries and the importance of post-marketing pharmacological surveillance

Estudio de diferentes formas de incorporación de inulina a una matriz quesera

La inulina es un carbohidrato de reserva que forma parte de la fibra alimentaria y que tiene efecto prebiótico. También se utiliza como reemplazante de grasa en diferentes alimentos. Con el fin de desarrollar quesos bajos en grasa con inulina, se realizaron diferentes pruebas para determinar la mejor forma de incorporar este carbohidrato para alcanzar una buena retención en la matriz del queso. Se empleó leche cruda proveniente de un tambo de San Luis, la cual se desnató por centrifugación. Se añadió 1% de fermento y CaCl₂ (1g). Después se incorporó 1% de coagulante en polvo y se siguieron las etapas habituales en la elaboración de queso. Se probaron diferentes alternativas para la adición de inulina analizando el contenido del carbohidrato en las muestras por HPLC y la morfología por imágenes de SEM. Las alternativas estudiadas fueron: a) agregado de 125g de inulina en polvo directamente a la leche a partir de la cual se elaboraron los quesos (M1); b) agregado de 37,5g de inulina en polvo a la leche (M2); c)elaboración de un gel de inulina al 30% con un tiempo de calentamiento de 20‟(encontrado como el de mejor consistencia) y adicionado luego de la incorporación del cuajo y antes de formada la cuajada (M3) y d) incorporación del gel luego de la etapa de desuerado, mixando y moldeando (M4); Los resultados en porcentajes (p/p) de retención de inulina en las diferentes muestras fueron: M1:1,04%; M2: 0,65%; M3: 1,8%; M4: 1,3%. Como era de esperar, debido a la hidrosolubilidad de la inulina, las muestra M1y M2 fueron las que menos porcentaje de retención presentaron, eliminándose el carbohidrato en la etapa de desuerado. Las imágenes de SEM mostraron glóbulos grasos integrados a la matriz proteica y puntos blancos que indican la presencia de inulina en los muestras. Este estudio permitió concluir que es conveniente realizar la formación de un gel de inulina, previo a su incorporación en la matriz del queso, la cual debe realizarse en la etapa de cuajado, para obtener un adecuado enriquecimiento del mismo por la adición de una fuente de fibra dietaria con propiedades prebióticas. Además las micrografías demuestran el efecto texturizante que desempeña la inulina en la microestructura de las muestras. Para optimizar la cantidad de inulina retenida en el queso, se requieren más experiencias variando la concentración y el momento de incorporación del polisacárido de modo que el producto sea una fuente de fibra.Centro de Investigación y Desarrollo en Criotecnología de Alimento

The local crystallization in nanoscale diamond-like carbon films during annealing

The local crystallization during annealing at 600° C in nanoscale diamond-like carbon coatings films grown by pulsed vacuum-arc deposition method was observed using modern techniques of high-resolution transmission electron microscopy. The crystallites formed by annealing have a face-centred cubic crystal structure and grow in the direction [011] as a normal to the film surface. The number and size of the crystallites depend on the initial values of the intrinsic stresses before annealing, which in turn depend on the conditions of film growth. The sizes of crystallites are 10 nm for films with initial compressive stresses of 3 GPa and 17 nm for films with initial compres- sive stresses of 12 GPa. Areas of local crystallization arising during annealing have a structure dif- ferent from the graphit

Non-Abelian Einstein-Born-Infeld Black Holes

We construct regular and black hole solutions in SU(2) Einstein-Born-Infeld theory. These solutions have many features in common with the corresponding SU(2) Einstein-Yang-Mills solutions. In particular, sequences of neutral non-abelian solutions tend to magnetically charged limiting solutions, related to embedded abelian solutions. Thermodynamic properties of the black hole solutions are addressed.Comment: LaTeX, 14 pages, 6 postscript figures; typos corrected in reference

Strain-induced partially flat band, helical snake states, and interface superconductivity in topological crystalline insulators

Topological crystalline insulators in IV-VI compounds host novel topological surface states consisting of multi-valley massless Dirac fermions at low energy. Here we show that strain generically acts as an effective gauge field on these Dirac fermions and creates pseudo-Landau orbitals without breaking time-reversal symmetry. We predict the realization of this phenomenon in IV-VI semiconductor heterostructures, due to a naturally occurring misfit dislocation array at the interface that produces a periodically varying strain field. Remarkably, the zero-energy Landau orbitals form a flat band in the vicinity of the Dirac point, and coexist with a network of snake states at higher energy. We propose that the high density of states of this flat band gives rise to interface superconductivity observed in IV-VI semiconductor multilayers at unusually high temperatures, with non-BCS behavior. Our work demonstrates a new route to altering macroscopic electronic properties to achieve a partially flat band, and paves the way for realizing novel correlated states of matter.Comment: Accepted by Nature Physic