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Objective We sought to inspect the role of AMD3100, which acts as an antagonist of stromal cell–derived factor-1/CXC chemokine receptor 4 on the formation of neointima in rabbit saccular aneurysm after flow diverter (FD) treatment. Methods Twenty saccular aneurysm models were established by using porcine pancreatic elastase. Three weeks later, a Tubridge FD was implanted into the saccular aneurysm. All treated models were immediately divided into 2 groups: the AMD3100 group was subcutaneously injected with AMD3100 (5 mg/kg per day), while the control group received saline. Morphology and thickness of the neointima were investigated 2 and 4 weeks after FD treatment, using hard tissue section and masson trichrome staining. Scanning electron microscope was used to observe endothelial-like cells, and fluorescence quantitative polymerase chain reaction was used to determine mRNA expression of neointima biomarkers, such as kinase insert domain receptor, VE-cadherin, CD34, and Tie2. Results Two and 4 weeks after FD treatment, the AMD3100 group had more endothelial-like cells than the control group in the neointima. Masson trichrome staining showed that the neointima in the AMD3100 group was more intact and thicker than that in the control group. Furthermore, increased mRNA levels of kinase insert domain receptor, VE-cadherin, and Tie2 in the neointima were found in the AMD3100 group compared with the control group. Conclusions Interval use of AMD3100 promotes the formation of neointima in rabbit saccular aneurysm and facilitates the endothelialization of the neointima after FD treatment. [ABSTRACT FROM AUTHOR]

Although transmission of the gene expression program from mother to daughter cells has been suggested to be mediated by gene bookmarking, the precise mechanism by which bookmarking mediates post-mitotic transcriptional re-activation has been unclear. Here, we used a real-time gene expression system to quantitatively demonstrate that transcriptional activation of the same genetic locus occurs with a significantly more rapid kinetics in post-mitotic cells versus interphase cells. RNA polymerase II large subunit (Pol II) and bromodomain protein 4 (BRD4) were recruited to the locus in a different sequential order on interphase initiation versus post-mitotic re-activation resulting from the recognition by BRD4 of increased levels of histone H4 Lys 5 acetylation (H4K5ac) on the previously activated locus. BRD4 accelerated the dynamics of messenger RNA synthesis by de-compacting chromatin and hence facilitating transcriptional re-activation. Using a real-time quantitative approach, we identified differences in the kinetics of transcriptional activation between interphase and post-mitotic cells that are mediated by a chromatin-based epigenetic mechanism. [ABSTRACT FROM AUTHOR]

The expression of the N-methyl-D-aspartate receptor (NMDA-R) in astrocytes is controversial. The receptor is commonly considered neuron-specific. We showed that astrocytes in primary cultures differentially expressed mRNA of NMDA-R subunits, NR1, NR2A and NR2B, in development, ischemia and post-ischemia. One-week-old cultures expressed detectable NR1 mRNA, which fell significantly at 2 weeks and became barely detectable at 4 weeks. NR2A and NR2B mRNA were both significantly up-regulated from 1 to 2 weeks. In 4 weeks, 2 h of ischemia caused a significant up-regulation of NR1 and NR2B mRNA; while 6 h caused down-regulation of NR2A mRNA. Under 3 h of post-ischemia, only NR1 mRNA was increased. Ischemia induced the expression of major NMDA-R effecter, nitric oxide synthase 1, which was unaffected by AMPA-R antagonist CNQX, but dose-dependently inhibited by NMDA-R specific antagonist MK-801. These findings reflected that astrocyte could express inducible functional NMDA receptors without the presence of neurons. [ABSTRACT FROM AUTHOR]

Crystallography is a powerful tool to determine the atomic structures of proteins and RNAs. X-ray crystallography has been used to determine the structure of many splicing related proteins and RNAs, making major contributions to our understanding of the molecular mechanism and regulation of pre-mRNA splicing. Compared to other structural methods, crystallography has its own advantage in the high-resolution structural information it can provide and the unique biological questions it can answer. In addition, two new crystallographic methods – the serial femtosecond crystallography and 3D electron crystallography – were developed to overcome some of the limitations of traditional X-ray crystallography and broaden the range of biological problems that crystallography can solve. This review discusses the theoretical basis, instrument requirements, troubleshooting, and exciting potential of these crystallographic methods to further our understanding of pre-mRNA splicing, a critical event in gene expression of all eukaryotes. [ABSTRACT FROM AUTHOR]

RT-qPCR is a commonly used method for evaluating gene expression; however, its accuracy and reliability are dependent upon the choice of appropriate reference gene(s), and there is limited information available on suitable reference gene(s) that can be used in mouse testis at different stages. In this study, using the RT-qPCR method, we investigated the expression variations of six reference genes representing different functional classes ( Actb, Gapdh, Ppia, Tbp, Rps29, Hprt1) in mice testis during embryonic and postnatal development. The expression stabilities of putative reference genes were evaluated using five algorithms: geNorm, NormFinder, Bestkeeper, the comparative delta C method and integrated tool RefFinder. Analysis of the results showed that Ppia, Gapdh and Actb were identified as the most stable genes and the geometric mean of Ppia, Gapdh and Actb constitutes an appropriate normalization factor for gene expression studies. The mRNA expression of AT1 as a test gene of interest varied depending upon which of the reference gene(s) was used as an internal control(s). This study suggested that Ppia, Gapdh and Actb are suitable reference genes among the six genes used for RT-qPCR normalization and provide crucial information for transcriptional analyses in future studies of gene expression in the developing mouse testis. [ABSTRACT FROM AUTHOR]