Abstract Background: Retinal neurodegeneration is induced by a variety of environmental insults and stresses, but the exact mechanisms are unclear. In the present study, we explored the involvement of cytosolic mitochondrial DNA (mtDNA), resulting in the cGAS-STING dependent inflammatory response and apoptosis in retinal damage in vivo. Methods: Retinal injury was induced with white light or intravitreal injection of lipopolysaccharide (LPS). After light-or LPS-induced injury, the amount of cytosolic mtDNA in the retina was detected by PCR. The mtDNA was isolated and used to transfect retinas in vivo. WB and real-time PCR were used to evaluate the activation of cGAS-STING path-way and the levels of apoptosis-associated protein at different times after mtDNA injection. Retinal cell apoptosis rate was detected by TUNEL staining. Full-field electroretinography (ERG) was used to assess the retinal function. Results: Light injury and the intravitreal injection of LPS both caused the leakage of mtDNA into the cytoplasm in retinal tissue. After the transfection of mtDNA in vivo, the levels of cGAS, STING, and IFN-β mRNAs and the protein levels of STING, phosph-TBK1, phospho-IRF3, and IFN-β were upregulated. mtDNA injection also induced the activation of caspase 3 and caspase 9. BAX and BAK were increased at both the mRNA and protein levels. The release of cytochrome c from the mitochondria to the cytosol was increased after mtDNA injection. The wave amplitudes on ERG decreased and retinal cell apoptosis was detected after mtDNA injection. Conclusions: Cytosolic mtDNA triggers an inflammatory response. It also promotes apoptosis and the dysfunction of the retina.

Abstract Our efforts are oriented to assess bovine Y-chromosome gene expression patterns. One set of genes that are of interest are the so-called X-degenerate Y-chromosome genes that are located in the male-specific region of the Y-chromosome (MSY). This region contains 95% of the DNA of the Y chromosome. These genes are single copy and have an X-chromosome homolog. Both, the Y-encoded and X-encoded homologs have ubiquitous expression profiles. However, some genes, like SRY that regulates male sex determination, have functions that are more specific. Identifying DNA sequence differences between these homologs will allow evaluation of their spatial and temporal expression patterns. Identification of the Y-encoded mRNAs and their isoforms will allow our understanding of tissue specific expression of isoforms in male tissues. The latter will facilitate our evaluation of gene function in male sex differentiation and fertility. Hence, we hypothesized that each of these X-degenerate gene homologs generate isoforms and that differential expression patterns exist between sexes and across tissues. To investigate the latter we used a new generation sequencing (NGS) technology that generates long sequencing reads with a range between 1000 to 10,000 base pairs in length. Single molecule real time (SMRT) isoform sequencing (IsoSeq) of several tissues (liver, lung, adipose, muscle, hypothalamus and testis) was carried out. Transcript sequences were used for bioinformatics analysis and isoform characterization. Given the focus of this manuscript the SMRT technology we are only presenting results obtained with the analysis of the bUTY and bUTX genes.

Resumen Nuestros esfuerzos están orientados a evaluar patrones de expresión génica del cromosoma Y bovino. Los genes de interés son los denominados genes X-degenerados que se encuentran en la región específica masculina del cromosoma Y (MSY). Esta región contiene el 95% del ADN del cromosoma Y. Estos genes son de copia única y tienen un homólogo en el cromosoma X. Ambos homólogos tienen perfiles amplios de expresión. Sin embargo, algunos genes, como el SRY que regula la determinación del sexo masculino, tienen funciones más específicas. La identificación de las diferencias de secuencia de ADN entre estos homólogos permitirá evaluar sus patrones de expresión espacial y temporal. La identificación de los ARNm codificados en el cromosoma Y y de sus isoformas permitirán analizar la expresión específica de sus isoformas en tejidos masculinos. Esto último facilitará nuestra evaluación de función génica en la diferenciación sexual masculina y la fertilidad. Por lo tanto, planteamos la hipótesis de que cada uno de estos genes homólogos degenerados del X genera isoformas y que existen patrones de expresión diferencial entre sexos y tejidos. Para investigar esto último, utilizamos una tecnología de secuenciación de nueva generación (NGS) que genera lecturas de secuenciación largas con un rango de longitud de 1000 a 10,000 pares de bases. Se secuenciaron los transcriptomas en varios tejidos (hígado, pulmón, adiposo, muscular, hipotálamo y testículo). Se utilizaron las secuencias generadas para el análisis bioinformático y la caracterización de isoformas. Siendo el foco de este manuscrito la tecnología SMRT, solo presentamos los resultados obtenidos con el análisis de los genes bUTY y bUTX.

Resumo Fundamentos: Embora uma proporção de pacientes com SCX tenha dilatação mediada por fluxo da artéria braquial (DMF) prejudicada em resposta à hiperemia, sugerindo que a disfunção endotelial nestes pacientes pode ser sistémica e não limitar-se à circulação coronariana, os mecanismos subjacentes que desencadeiam a disfunção endotelial nestes pacientes ainda não são completamente compreendidos. Objetivos: Avaliar a associação do índice de resistência microcirculatória (IMR) com a disfunção endotelial e a inflamação em pacientes com SCX. Métodos: Estudaram-se 20 pacientes com SCX e 20 sujeitos de controle emparelhados em idade e género. A reserva de fluxo coronariano derivada da termodiluição (RFC) e a IMR forma medidas usando um fio guia com ponta de sensor de temperatura e pressão. A DMF da artéria braquial foi medida utilizando imagens ultrassónicas bidimensionais de alta resolução obtidas com um aparelho de ultrassom Doppler (HDI-ATL 5000, EE.UU.) com transdutor linear de 5 MHz a 12 MHz. Resultados: Em comparação com os sujeitos de controle, a RFC foi significativamente menor (2,42 ± 0,78 vs 3,59 ± 0,79, p < 0,001); o IMR foi maior (32,2 ± 8,0 frente a 19,5 ± 5,5, p < 0,001); a concentração de PCR-as e DMF foi maior (4,75 ± 1,62 frente a 2,75 ± 1,50, 5,24 ± 2,41 diante de 8,57 ± 2,46, p < 0,001) em pacientes com SCX. A escore de Duke (ED) se correlacionou positivamente com RFC e DMF (0,489 e 0,661, p < 0,001), foi negativa para IMR e PCR-as (-0,761 e -0,087, p < 0,001) em pacientes com SCX. Conclusões: O principal achado neste estudo é que o ED medido em pacientes com SCX esteve associado a PCR-as e DMF. Por outra parte, os efeitos independentes da tolerância ao exercício podem piorar significativamente a DMF e a PCR-as em pacientes com SCX especialmente, é particularmente importante que a DMF se associou negativamente com a RIM.

Abstract Background: Although a proportion of CSX patients have impaired brachial artery flow-mediated dilatation (FMD) in response to hyperemia, suggesting that endothelial dysfunction in these patients may be systemic and not just confined to the coronary circulation; the underlying mechanisms triggering endothelial dysfunction in these patients are still incompletely understood. Objectives: To assess the association of the index of Microcirculatory Resistance (IMR) with endothelial dysfunction and inflammation in patients with CSX. Methods: We studied 20 CSX patients and 20 age and gender-matched control subjects. Thermodilution-derived coronary flow reserve (CFR) and IMR were measured using a pressure-temperature sensor-tipped guidewire. Brachial artery FMD was measured using high-resolution, two-dimensional ultrasound images obtained with a Doppler ultrasound device (HDI-ATL 5000, USA) with a 5 MHz to 12 MHz linear-array transducer. Results: Compared with in control subjects, CFR was significantly lower (2.42 ± 0.78 vs. 3.59 ± 0.79, p < 0.001); IMR was higher (32.2 ± 8.0 vs. 19.5 ± 5.5, p < 0.001); the concentration of hs-CRP and FMD was higher (4.75 ± 1.62 vs. 2.75 ± 1.50; 5.24 ± 2.41 vs. 8.57 ± 2.46, p < 0.001) in CSX patients. The Duke treadmill score (DTS) was correlated positively to CFR and FMD (0.489 and 0.661, p < 0.001), it was negative to IMR and hsCRP (-0.761 and -0.087, p < 0.001) in CSX patients. Conclusions: The main finding in this study is that the DTS measured in patients with CSX was associated to hsCRP and FMD. Moreover, the independent effects of exercise tolerance can significantly impair FMD and hsCRP in CSX patients; especially it is particularly important to whom where FMD was associated negatively with IMR.

The WUSCHEL (WUS)-related homeobox (WOX) gene family coordinates transcription during the early phases of embryogenesis. In this study, a putative WOX2 homolog was isolated and characterized from Aegilops tauschii, the donor of D genome of Triticum aestivum. The sequence consisted of 2045 bp, and contained an open reading frame (ORF), encoded 322 amino acids. The predicted protein sequence contained a highly conserved homeodomain and the WUS-box domain, which is present in some members of the WOX protein family. The full-length ORF was subcloned into prokaryotic expression vector pET-30a, and an approximately 34-kDa protein was expressed in Escherichia coli BL21 (DE3) cells with IPTG induction. The molecular mass of the expressed protein was identical to that predicted by the cDNA sequence. Phylogenetic analysis suggested that Ae. tauschii WOX2 is closely related to the rice and maize orthologs. Quantitative PCR analysis showed that WOX2 from Ae. tauschii was primarily expressed in the seeds; transcription increased during seed development and declined after the embryos matured, suggesting that WOX2 is associated with embryo development in Ae. tauschii.