The brain endothelium is an important therapeutic target for the inhibition of cerebrovascular dysfunction in ischemic stroke. Previously, we documented the important regulatory roles of microRNAs in the cerebral vasculature, in particular the cerebral vascular endothelium. However, the functional significance and molecular mechanisms of other classes of non-coding RNAs in the regulation of cerebrovascular endothelial pathophysiology after stroke are completely unknown. Using RNA sequencing (RNA-seq) technology, we profiled long non-coding RNA (lncRNA) expressional signatures in primary brain microvascular endothelial cells (BMECs) after oxygen-glucose deprivation (OGD), an in vitro mimic of ischemic stroke conditions. After 16h of OGD exposure, the expression levels for 362 of the 10,677 lncRNAs analyzed changed significantly, including a total of 147 lncRNAs increased and 70 lncRNAs decreased by more than 2-fold. Among them, the most highly upregulated lncRNAs include Snhg12, Malat1, and lnc-OGD 1006, whereas the most highly downregulated lncRNAs include 281008D09Rik, Peg13, and lnc-OGD 3916. Alteration of the most highly upregulated/downregulated ODG-responsive lncRNAs was further confirmed in cultured BMECs after OGD as well as isolated cerebral microvessels in mice following transient middle cerebral artery occlusion (MCAO) and 24h reperfusion by the quantitative real-time PCR approach. Moreover, promoter analysis of altered ODG-responsive endothelial lncRNA genes by bioinformatics showed substantial transcription factor binding sites on lncRNAs, implying potential transcriptional regulation of those lncRNAs. These findings are the first to identify OGD-responsive brain endothelial lncRNAs, which suggest potential pathological roles for these lncRNAs in mediating endothelial responses to ischemic stimuli. Endothelial-selective lncRNAs may function as a class of novel master regulators in cerebrovascular endothelial pathologies after ischemic stroke.

The neutral theory of molecular evolution predicts that important proteins evolve more slowly than unimportant ones. High-throughput gene-knockout experiments in model organisms have provided information on the dispensability, and therefore importance, of thousands of proteins in a genome. However, previous studies of the correlation between protein dispensability and evolutionary rate were equivocal, and it has been proposed that the observed correlation is due to the covariation with the level of gene expression or is limited to duplicate genes. We here analyzed the gene dispensability data of the yeast Saccharomyces cerevisiae and estimated protein evolutionary rates by comparing S. cerevisiae with nine species of varying degrees of divergence from S. cerevisiae. The correlation between gene dispensability and evolutionary rate, although low, is highly significant, even when the gene expression level is controlled for or when duplicate genes are excluded. Our results thus support the hypothesis of lower evolution rates for more important proteins, a widely used principle in the daily practice of molecular biology. When the evolutionary rate is estimated from closely related species, the ratio between the mean rate of nonessential proteins to that of essential proteins is 1.4. This ratio declines to 1.1 when the evolutionary rate is estimated from distantly related species, suggesting that the importance of a protein may change in evolution, so the dispensability data obtained from a model organism only predicts a short-term rate of protein evolution. A comparison of the fitness contributions of orthologous genes in yeast and nematode supports this conclusion.

Chemosensation (smell and taste) is important to the survival and reproduction of vertebrates and is mediated by specific bindings of odorants, pheromones, and tastants by chemoreceptors that are encoded by several large gene families. This review summarizes recent comparative genomic and evolutionary studies of vertebrate chemoreceptor genes. It focuses on the remarkable diversity of chemoreceptor gene repertoires in terms of gene number and gene sequence across vertebrates and the evolutionary mechanisms that are responsible for generating this diversity. We argue that the great among-species variation of chemoreceptor gene repertoires is a result of adaptations of individual species to their environments and diets.

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Nuclear reprogramming is dependent upon a number of factors, including chromatin organization and modification. Trychostatin A (TSA), a histone deacetylase inhibitor, was used to increase histone acetylation and to improve reprogrammability in both cattle and mice. The objective of the study was to determine whether TSA could improve the pre-implantational development potential of rabbit nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of FSH and hCG. Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in 10% FBS M199 and confirmed under fluorescence microscopy. Cumulus cells were prepared as nuclear donors for NT; a donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte, and subsequently fused with the oocyte recipient by application of 3 direct current pulses at 3.2 kV cm−1 for a duration of 20 µs/pulse. Fused embryos were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg mL−1 cycloheximide for 1 h. Rabbit NT embryos were cultured in 5 nM TSA-2.5% FBS-B2 medium for 10 h before being transferred into regular medium (FBS-B2). The TSA-treated embryos (5 nM vs. 0 nM) were cultured in 400 µL FBS-B2 medium for 5 days in 5% CO2 in a humidified atmosphere at 38.5°C (initiation of activation = Day 0). NT embryo development to cleaved (2 to 4 cell), morula, and blastocyst stages was evaluated on Day 2, Day 3, and Day 5, respectively. The selected NT blastocysts were counted for cell numbers following Hoechst 33342 epifluorescenin staining. The results (Table 1) showed that there was no difference on pre-implantational development of cloned embryos between TSA-added and control groups (P > 0.05). However, a significantly higher cell number per NT blastocyst was found in the TSA-added group (357 vs. 113; P < 0.05). This indicated that the blastocyst quality in NT embryos was improved with the addition of TSA by increasing histone acetylation activity. The developmental potential of TSA-treated NT embryos to term is under investigation. Table 1.Effects of TSA on the pre-implantational development of cloned rabbit embryos This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261-01.