LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy.

Long non-coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial-associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2gib005Δ8/+ ) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta-b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2-mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA-mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.

Genome and transcriptome analysis of the mealybug Maconellicoccus hirsutus: Correlation with its unique phenotypes.

Mealybugs are aggressive pests with world-wide distribution and are suitable for the study of different phenomena like genomic imprinting and epigenetics. Genomic approaches facilitate these studies in absence of robust genetics in this system. We sequenced, de novo assembled, annotated Maconellicoccus hirsutus genome. We carried out comparative genomics it with four mealybug and eight other insect species, to identify expanded, specific and contracted gene classes that relate to pesticide and desiccation resistance. We identified horizontally transferred genes adding to the mutualism between the mealybug and its endosymbionts. Male and female transcriptome analysis indicates differential expression of metabolic pathway genes correlating with their physiology and the genes for sexual dimorphism. The significantly lower expression of endosymbiont genes in males relates to the depletion of endosymbionts in males during development.

Maternal vitamin B12 deficiency in rats alters DNA methylation in metabolically important genes in their offspring.

Vitamin B12 deficiency is a critical problem worldwide and peri-conceptional deficiency of this vitamin is associated with the risk of complex cardio-metabolic diseases. Nutritional perturbations during these stages of development may lead to changes in the fetal epigenome. Using Wistar rat model system, we have earlier shown that low maternal B12 levels are associated with low birth weight, adiposity, insulin resistance, and increased triglyceride levels in the offspring, which might predispose them to the risk of cardio-metabolic diseases in adulthood. In this study, we have investigated the effects of maternal B12 deficiency on genome-wide DNA methylation profile of the offspring and the effect of rehabilitation of mothers with B12 at conception. We have performed methylated DNA immunoprecipitation sequencing of liver from pups in four groups of Wistar rats: Control (C), B12-restricted (B12R), B12-rehabilitated at conception (B12RC), and B12-rehabilitated at parturition (B12RP). We have analyzed differentially methylated signatures between the three groups as compared to controls. We have identified a total of 214 hypermethylated and 142 hypomethylated regions in the 10 kb upstream region of transcription start site in pups of B12-deficient mothers, which are enriched in genes involved in fatty acid metabolism and mitochondrial transport/metabolism. B12 rehabilitation at conception and parturition is responsible for reversal of methylation status of many of these regions to control levels suggesting a causal association with metabolic phenotypes. Thus, maternal B12 restriction alters DNA methylation of genes involved in important metabolic processes and influences the offspring phenotype, which is reversed by B12 rehabilitation of mothers at conception.

A new strain of white spot syndrome virus affecting Litopenaeus vannamei in Indian shrimp farms.

White spot syndrome virus (WSSV)-infected shrimp samples collected from grow-out ponds located at Nellore, Andhra Pradesh, India, showed WSSV negative and positive by PCR using primer sets specific to ORF119 and VP28 gene of WSSV, respectively. This indicated the deletion of genetic fragments in the genome of WSSV. The WSSV isolate along with lab strain of WSSV was subjected to next-generation sequencing. The sequence analysis revealed a deletion of 13,170 bp at five positions in the genome of WSSV-NS (new strain) relative to WSSV-TH and WSSV-LS (lab strain). The PCR analysis using the ORF's specific primer sets revealed the complete deletion of 10 ORFs in the genome of WSSV-NS strain. The primer set was designed based on sequence covering ORF161/162/163 to amplify a product of 2,748 bp for WSSV-LS and 402 bp for WSSV-NS. Our surveillance programme carried out since 2002 revealed the replacement of WSSV-LS by WSSV-NS in Indian shrimp culture system.

Investigating Coronary Artery Disease methylome through targeted bisulfite sequencing.

BACKGROUND: Gene environment interactions leading to epigenetic alterations play pivotal role in the pathogenesis of Coronary Artery Disease (CAD). Altered DNA methylation is one such epigenetic factor that could lead to altered disease etiology. In this study, we comprehensively identified methylation sites in several genes that have been previously associated with young CAD patients. METHODS: The study population consisted of 42 healthy controls and 33 young CAD patients (age group <50 years). We performed targeted bisulfite sequencing of promoter as well as gene body regions of several genes in various pathways like cholesterol synthesis and metabolism, endothelial dysfunction, apoptosis, which are implicated in the development of CAD. RESULTS: We observed that the genes like GALNT2, HMGCR were hypermethylated in the promoter whereas LDLR gene promoter was hypomethylated indicating that intracellular LDL uptake was higher in CAD patients. Although APOA1 did not show significant change in methylation but APOC3 and APOA5 showed variation in methylation in promoter and exonic regions. Glucokinase (GCK) and endothelial nitric oxide synthase 3 (NOS3) were hyper methylated in the promoter. Genes involved in apoptosis (BAX/BCL2/AKT2) and inflammation (PHACTR1/LCK) also showed differential methylation between controls and CAD patients. A combined analysis of the methylated CpG sites using machine learning tool revealed 14 CpGs in 11 genes that could discriminate CAD cases from controls with over 93% accuracy. CONCLUSIONS: This study is unique because it highlights important gene methylation alterations which might predict the risk of young CAD in Indian population. Large scale studies in different populations would be important for validating our findings and understanding the epigenetic events associated with CAD.

RNA sequencing of db/db mice liver identifies lncRNA H19 as a key regulator of gluconeogenesis and hepatic glucose output.

Liver plays a key role in maintaining glucose homeostasis and impaired hepatic glucose metabolism is associated with type 2 diabetes. In the present study, we used RNA sequencing to profile the transcriptome of the livers of diabetic db/db mice as compared to the normal db/+ mice and identified 218 differentially expressed genes. Amongst these, there were 3 lncRNAs that were significantly downregulated and H19 was the most altered lncRNA in the livers of db/db mice. H19 expression significantly correlated with the expression of genes of the glycolysis and gluconeogenesis pathways, which suggest that altered hepatic H19 levels can directly or indirectly modulate their expression. Inhibition of H19 using specific siRNA in HepG2 cells and primary mouse hepatocytes significantly increased the levels of gluconeogenic genes. This was subsequently accompanied by increased hepatic glucose output. Further,H19 depletion in HepG2 cells impaired insulin signaling and increased nuclear localization of FoxO1, an important transcriptional regulator of gluconeogenic gene expression. Our results reveal a novel link between decreased H19 levels and impaired gluconeogenesis via regulation of FoxO1 nuclear levels. These put forth interesting observations on the regulatory role of H19 in altering hepatic physiology during diabetes.

Draft Genome Sequence of Urease-Producing Sporosarcina pasteurii with Potential Application in Biocement Production.

We describe here the draft genome sequence of Sporosarcina pasteurii, a urease-producing bacterium with potential applications in biocement production.