A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system.

BACKGROUND: Toll-like receptor 4 (TLR4) plays a pivotal role in the pathophysiology of stroke-induced inflammation. Both astroglia and microglia express TLR4, and endogenous ligands produced in the ischemic brain induce inflammatory responses. Reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines produced by TLR4 activation play harmful roles in neuronal damage after stroke. Although astroglia exhibit pro-inflammatory responses upon TLR4 stimulation by lipopolysaccharide (LPS), they may also play cytoprotective roles via the activation of the pentose phosphate pathway (PPP), reducing oxidative stress by glutathione peroxidase. We investigated the mechanisms by which astroglia reduce oxidative stress via the activation of PPP, using TLR4 stimulation and hypoxia in concert with microglia. METHODS: In vitro experiments were performed using cells prepared from Sprague-Dawley rats. Coexisting microglia in the astroglial culture were chemically eliminated using L-leucine methyl ester (LME). Cells were exposed to LPS (0.01 µg/mL) or hypoxia (1 % O2) for 12-15 h. PPP activity was measured using [1-(14)C]glucose and [6-(14)C]glucose. ROS and NO production were measured using 2',7'-dichlorodihydrofluorescein diacetate and diaminofluorescein-FM diacetate, respectively. The involvement of nuclear factor-erythroid-2-related factor 2 (Nrf2), a cardinal transcriptional factor under stress conditions that regulates glucose 6-phosphate dehydrogenase, the rate-limiting enzyme of PPP, was evaluated using immunohistochemistry. RESULTS: Cultured astroglia exposed to LPS elicited 20 % increases in PPP flux, and these actions of astroglia appeared to involve Nrf2. However, the chemical depletion of coexisting microglia eliminated both increases in PPP and astroglial nuclear translocation of Nrf2. LPS induced ROS and NO production in the astroglial culture containing microglia but not in the microglia-depleted astroglial culture. LPS enhanced astroglial ROS production after glutathione depletion. U0126, an upstream inhibitor of mitogen-activated protein kinase, eliminated LPS-induced NO production, whereas ROS production was unaffected. U0126 also eliminated LPS-induced PPP activation in astroglial-microglial culture, indicating that microglia-derived NO mediated astroglial PPP activation. Hypoxia induced astroglial PPP activation independent of the microglia-NO pathway. Elimination of ROS and NO production by sulforaphane, a natural Nrf2 activator, confirmed the astroglial protective mechanism. CONCLUSIONS: Astroglia in concert with microglia may play a cytoprotective role for countering oxidative stress in stroke.

Effective use of sequence information to predict CRISPR-Cas9 off-target.

The CRISPR/Cas9 gene-editing system is the third-generation gene-editing technology that has been widely used in biomedical applications. However, off-target effects occurring CRISPR/Cas9 system has been a challenging problem it faces in practical applications. Although many predictive models have been developed to predict off-target activities, current models do not effectively use sequence pair information. There is still room for improved accuracy. This study aims to effectively use sequence pair information to improve the model's performance for predicting off-target activities. We propose a new coding scheme for coding sequence pairs and design a new model called CRISPR-IP for predicting off-target activity. Our coding scheme distinguishes regions with different functions in the sequence pairs through the function channel. Moreover, it distinguishes between bases and base pairs using type channels, effectively representing the sequence pair information. The CRISPR-IP model is based on CNN, BiLSTM, and the attention layer to learn features of sequence pairs. We performed performance verification on two data sets and found that our coding scheme can represent sequence pair information effectively, and the CRISPR-IP model performance is better than others. Data and source codes are available at https://github.com/BioinfoVirgo/CRISPR-IP.

Analysis of genomic tRNA revealed presence of novel genomic features in cyanobacterial tRNA.

Transfer RNAs (tRNA) are important molecules that involved in protein translation machinery and acts as a bridge between the ribosome and codon of the mRNA. The study of tRNA is evolving considerably in the fields of bacteria, plants, and animals. However, detailed genomic study of the cyanobacterial tRNA is lacking. Therefore, we conducted a study of cyanobacterial tRNA from 61 species. Analysis revealed that; cyanobacteria contain thirty-six to seventy-eight tRNA gens per genome that encodes for 20 tRNA isotypes. The number of iso-acceptors (anti-codons) ranged from thirty-two to forty-three per genome. tRNAIle with anti-codon AAU, GAU, and UAU was reported to be absent from the genome of Gleocapsa PCC 73,106 and Xenococcus sp. PCC 7305. Instead, they were contained anti-codon CAU that is common to tRNAMet and tRNAIle as well. The iso-acceptors ACA (tRNACys), ACC (tRNAGly), AGA, ACU (tRNASer), AAA (tRNAPhe), AGG (tRNAPro), AAC (tRNAVal), GCG (tRNAArg), AUG (tRNAHis), and AUC (tRNAAsp) were absent from the genome of cyanobacterial lineages studied so far. A few of the cyanobacterial species encode suppressor tRNAs, whereas none of the species were found to encode a selenocysteine iso-acceptor. Cyanobacterial species encode a few putative novel tRNAs whose functions are yet to be elucidated.

Genome-wide analysis revealed novel molecular features and evolution of Anti-codons in cyanobacterial tRNAs.

Transfer RNAs (tRNAs) play important roles to decode the genetic information contained in mRNA in the process of translation. The tRNA molecules possess conserved nucleotides at specific position to regulate the unique function. However, several nucleotides at different position of the tRNA undergo modification to maintain proper stability and function. The major modifications include the presence of pseudouridine (Ψ) residue instead of uridine and the presence of m5-methylation sites. We found that, Ψ13 is conserved in D-stem, whereas Ψ38 & Ψ39 were conserved in the anti-codon loop (AL) and anti-codon arm (ACA), respectively. Furthermore, Ψ55 found to be conserved in the Ψ loop. Although, fourteen possible methylation sites can be found in the tRNA, cyanobacterial tRNAs were found to possess conserved G9, m3C32, C36, A37, m5C38 and U54 methylation sites. The presence of multiple conserved methylation sites might be responsible for providing necessary stability to the tRNA. The evolutionary study revealed, tRNAMet and tRNAIle were evolved earlier than other tRNA isotypes and their evolution is date back to at least 4000 million years ago. The presence of novel pseudouridination and m5-methylation sites in the cyanobacterial tRNAs are of particular interest for basic biology. Further experimental study can delineate their functional significance in protein translation.

Gene interactions observed with the HDL-c blood lipid, intakes of protein, sugar and biotin in relation to circulating homocysteine concentrations in a group of black South Africans.

BACKGROUND: Elevated homocysteine (Hcy) is associated with several pathologies. Gene-diet interactions related to Hcy might be used to customize dietary advice to reduce disease incidence. To explore this possibility, we investigated interactions between anthropometry, biochemical markers and diet and single-nucleotide polymorphisms (SNPs) in relation to Hcy concentrations. Five SNPs of Hcy-metabolizing enzymes were analyzed in 2010 black South Africans. RESULTS: Hcy was higher with each additional methylenetetrahydrofolate reductase (MTHFR) C677T minor allele copy, but was lower in methionine synthase (MTR) 2756AA homozygotes than heterozygotes. Individuals harboring cystathionine ß synthase (CBS) 833 T/844ins68 had lower Hcy concentrations than others. No interactive effects were observed with any of the anthropometrical markers. MTHFR C677T and CBS T833C/844ins68 homozygote minor allele carriers presented with lower Hcy as high density lipoprotein cholesterol (HDL-c) increased. Hcy concentrations were negatively associated with dietary protein and animal protein intake in the TT and TC genotypes, but positively in the CC genotype of CBS T833C/844ins68. Hcy was markedly higher in TT homozygotes of MTHFR C677T as added sugar intake increased. In CBS T833C/844ins68 major allele carriers, biotin intake was negatively associated with Hcy; but positively in those harboring the homozygous minor allele. CONCLUSIONS: The Hcy-SNP associations are modulated by diet and open up the possibility of invoking dietary interventions to treat hyperhomocysteinemia. Future intervention trials should further explore the observed gene-diet and gene-blood lipid interactions.

Mammalian Glutamyl Aminopeptidase Genes (ENPEP) and Proteins: Comparative Studies of a Major Contributor to Arterial Hypertension.

Glutamyl aminopeptidase (ENPEP) is a member of the M1 family of endopeptidases which are mammalian type II integral membrane zinc-containing endopeptidases. ENPEP is involved in the catabolic pathway of the renin-angiotensin system forming angiotensin III, which participates in blood pressure regulation and blood vessel formation. Comparative ENPEP amino acid sequences and structures and ENPEP gene locations were examined using data from several mammalian genome projects. Mammalian ENPEP sequences shared 71-98% identities. Five N-glycosylation sites were conserved for all mammalian ENPEP proteins examined although 9-18 sites were observed, in each case. Sequence alignments, key amino acid residues and predicted secondary and tertiary structures were also studied, including transmembrane and cytoplasmic sequences and active site residues. Highest levels of human ENPEP expression were observed in the terminal ileum of the small intestine and in the kidney cortex. Mammalian ENPEP genes contained 20 coding exons. The human ENPEP gene promoter and first coding exon contained a CpG island (CpG27) and at least 6 transcription factor binding sites, whereas the 3'-UTR region contained 7 miRNA target sites, which may contribute to the regulation of ENPEP gene expression in tissues of the body. Phylogenetic analyses examined the relationships of mammalian ENPEP genes and proteins, including primate, other eutherian, marsupial and monotreme sources, using chicken ENPEP as a primordial sequence for comparative purposes.

Local chromatin microenvironment determines DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase.

Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known function in catalyzing methylation. In situations of extremely low levels of S-adenosyl methionine (SAM), DNMT-3A and -3B might demethylate C-5 methyl cytosine (5mC) via deamination to thymine, which is subsequently replaced by an unmodified cytosine through the base excision repair (BER) pathway. Alternatively, 5mC when converted to 5- hydroxymethylcytosine (5hmC) by TET enzymes, might be further modified to an unmodified cytosine by DNMT-3A and -3B under oxidized redox conditions, although exact pathways are yet to be elucidated. Interestingly, even direct conversion of 5mC to cytosine might be catalyzed by DNMTs. Here, we summarize the evidence on the DNA dehydroxymethylase and demethylase activity of DNMT-3A and -3B. Although physiological relevance needs to be demonstrated, the current indications on the 5mC- and 5hmC-modifying activities of de novo DNA C-5 methyltransferases shed a new light on these enzymes. Despite the extreme circumstances required for such unexpected reactions to occur, we here put forward that the chromatin microenvironment can be locally exposed to extreme conditions, and hypothesize that such waves of extremes allow enzymes to act in differential ways.

Distribution of CYP17α polymorphism and selected physiochemical factors of uterine leiomyoma in Barbados.

Uterine leiomyoma is a major reproductive health disease among women and in particular Black women. The present study sought to determine whether a single nucleotide polymorphism (SNP) of CYP17 (rs743572) was associated with the risk of developing uterine leiomyoma (UL) in affected women in Barbados; a majority Black population. It also sought to determine if BMI, waist circumference and oestradiol levels were associated with UL in this group. A total of 96 random persons were assessed in a case-control study using a PCR-RFLP assay, and measurements of body mass index, waist circumference, and oestradiol levels were also assessed. Our results showed no genetic association with the risk of UL and this gene. The genetic distribution of CYP 17α- alleles resembled a normal Hardy-Weinberg distribution, and a relatively low risk of 0.25 at a confidence interval at 95%, of UL disease development. However, a significant association was found between oestradiol levels and fibroids, as well as oestradiol levels and BMI, at P < 0.05 among cases. Therefore our study indicates that significant associations between physiochemical factors comprising BMI, waist circumference, and oestrogen levels are disease indicators in this population. In conclusion, our findings suggest that obesity and its associated risk factors are important in a majority Black Caribbean population, although the sample size needs to be increased.