Methionine- and Choline-Deficient Diet Identifies an Essential Role for DNA Methylation in Plasmacytoid Dendritic Cell Biology.

Diet plays an important role in lifestyle disorders associated with the disturbed immune system. During the study of methionine- and choline-deficient diet-induced nonalcoholic fatty liver disease, we observed a specific decrease in the plasmacytoid dendritic cell (pDC) fraction from murine spleens. While delineating the role for individual components, we identified that l-methionine supplementation correlates with representation of the pDC fraction. S-adenosylmethionine (SAM) is a key methyl donor, and we demonstrate that supplementation of methionine-deficient medium with SAM but not homocysteine reverses the defect in pDC development. l-Methionine has been implicated in maintenance of methylation status in the cell. Based on our observed effect of SAM and zebularine on DC subset development, we sought to clarify the role of DNA methylation in pDC biology. Whole-genome bisulfite sequencing analysis from the splenic DC subsets identified that pDCs display differentially hypermethylated regions in comparison with classical DC (cDC) subsets, whereas cDC1 and cDC2 exhibited comparable methylated regions, serving as a control in our study. We validated differentially methylated regions in the sorted pDC, CD8α+ cDC1, and CD4+ cDC2 subsets from spleens as well as FL-BMDC cultures. Upon analysis of genes linked with differentially methylated regions, we identified that differential DNA methylation is associated with the MAPK pathway such that its inhibition guides DC development toward the pDC subtype. Overall, our study identifies an important role for methionine in pDC biology.

The Signature Amino Acid Residue Serine 31 of HIV-1C Tat Potentiates an Activated Phenotype in Endothelial Cells.

The natural cysteine to serine variation at position 31 of Tat in HIV-1C disrupts the dicysteine motif attenuating the chemokine function of Tat. We ask if there exists a trade-off in terms of a gain of function for HIV-1C Tat due to this natural variation. We constructed two Tat-expression vectors encoding Tat proteins discordant for the serine 31 residue (CS-Tat vs. CC-Tat), expressed the proteins in Jurkat cells under doxycycline control, and performed the whole transcriptome analysis to compare the early events of Tat-induced host gene expression. Our analysis delineated a significant enrichment of pathways and gene ontologies associated with the angiogenic signaling events in CS-Tat stable cells. Subsequently, we validated and compared angiogenic signaling events induced by CS- vs. CC-Tat using human umbilical vein endothelial cells (HUVEC) and the human cerebral microvascular endothelial cell line (hCMEC/D3). CS-Tat significantly enhanced the production of CCL2 from HUVEC and induced an activated phenotype in endothelial cells conferring on them enhanced migration, invasion, and in vitro morphogenesis potential. The ability of CS-Tat to induce the activated phenotype in endothelial cells could be of significance, especially in the context of HIV-associated cardiovascular and neuronal disorders. The findings from the present study are likely to help appreciate the functional significance of the SAR (signature amino acid residues) influencing the unique biological properties.

Differential transcriptome analysis in HPV-positive and HPV-negative cervical cancer cells through CRISPR knockout of miR-214.

In this study we have investigated the effects of a tumour suppressor microRNA, miR-214, on gene expression in HPV-positive (CaSki) and HPV-negative cervical cancer cells (C33A) by RNA sequencing using next generation sequencing. The HPV-positive and HPV-negative cervical cancer cells were either miR-214- knocked-out or miR-214-overexpressed. Gene expression analysis showed that a total of 904 genes were upregulated and 365 genes were downregulated between HPV-positive and HPV-negative cervical cancer cells with a fold change of +/- 2. Furthermore, 11 differentially expressed and relevant genes (TNFAIP3, RAB25, MET, CYP1B1, NDRG1, CD24, LOXL2, CD44, PMS2, LATS1 and MDM1) which showed a fold change of +/-5 were selected to confirm by real-time PCR. This study represents the first report of miR-214 on global gene expression in the context of HPV.

Transcriptomic analyses of gene expression by CRISPR knockout of miR-214 in cervical cancer cells.

In this study, we investigate the effect of one such micro RNA, miR-214 which is frequently down-regulated in cervical cancer. In this study, we either CRISPR knocked out or overexpressed miR-214 in cervical cancer cells and analyzed the global mRNA expression by Next Generation Sequencing (NGS) It was observed that a total of 108 genes were upregulated and 178 downregulated between the samples, above and below the baseline respectively. Gene Ontology and KEGG pathway analysis reveal distinct biological processes and pathways. Analysis of gene regulatory networks also gave different network patterns in the two samples. We confirmed the RNA sequencing data for 10 genes; IFIF27, SMAD3, COX11, TP53INP1, ABL2, FGF8, TNFAIP3, NRG1, SP3 and MDM4 by Real-time PCR. This is the first report on the effect of miR-214 on global mRNA profile in cervical cancer cells. This study also reports new biomarkers for cervical cancer prognosis.

Meloidogyne incognita (Nematoda: Meloidogynidae) sterol-binding protein Mi-SBP-1 as a target for its management.

Meloidogyne incognita is a polyphagous plant-parasitic nematode that causes considerable yield loss in agricultural and horticultural crops. The management options available for M. incognita are extremely limited. Here we identified and characterised a M. incognita homolog of Caenorhabditis elegans sterol-binding protein (Mi-SBP-1), a transcriptional regulator of several lipogenesis pathway genes, and used RNA interference-mediated gene silencing to establish its utility as a target for the management of M. incognita. Mi-sbp-1 is predicted to be a helix-loop-helix domain containing DNA binding transcription factor, and is present in the M. incognita genome in three copies. The RNA-Seq analysis of Mi-sbp-1 silenced second stage juveniles confirmed the key role of this gene in lipogenesis regulation in M. incognita. In vitro and host-induced gene silencing of Mi-sbp-1 in M. incognita second stage juveniles resulted in loss of nematodes' ability to utilise the stored fat reserves, slower nematode development, and reduced parasitism on adzuki bean and tobacco plants. The multiplication factor for the Mi-sbp-1 silenced nematodes on adzuki bean plants was reduced by 51% compared with the control nematodes in which Mi-sbp-1 was not silenced. Transgenic expression of the double-stranded RNA construct of the Mi-sbp-1 gene in tobacco plants caused 40-45% reduction in M. incognita multiplication, 30-43.8% reduction in the number of egg masses, and 33-54% reduction in the number of eggs per egg mass compared with the wild type control plants. Our results confirm that Mi-sbp-1 is a key regulator of lipogenesis in M. incognita and suggest that it can be used as an effective target for its management. The findings of this study can be extended to develop methods to manage other economically important parasitic nematodes.

Lipoic acid prevents Cr(6+) induced cell transformation and the associated genomic dysregulation.

Investigation of the transcription profile of cells transformed by Cr(6+) in vivo was undertaken. The objective was to elucidate genomic changes underlying the mechanism of action of the carcinogenic dose of Cr(6+)and their prevention using metabolic antioxidant lipoic acid (LA). Cr(6+) was administered intraperitoneally to LPS+TPA challenged Swiss albino mice in host mediated cell transformation assay using peritoneal macrophages in vivo. The cell transforming potential of Cr(6+) test doses was validated by gain of anchorage independent growth potential in soft agar and loss of Fc receptor on target cells. LA was administered in equimolar doses. Compared to non-transformed cells, the gene expression profile of transformed cells was found to be dysregulated substantially and in dose dependent manner. Genes showing down regulation were found to be involved in tumour suppression, apoptosis, DNA repair, and cell-cycle. A similar response was noted in the genes pertaining to immune system, morphogenesis, cell-communication, energy-metabolism, and biosynthesis. The co-administration of lipoic acid prevented the transcription dysregulation and cell transformation by Cr(6+) in vivo. The influenced pathways seem to be crucial for progression as well as mitigation of Cr toxicity; and their response to LA indicated their critical role in mechanism of anti-carcinogenic action of LA. Results are of importance to mitigate Cr(6+) induced occupational cancer hazard.

Kelch repeat proteins control yeast PKA activity in response to nutrient availability.

Regulation of protein kinase A (PKA) by binding of cAMP to the regulatory subunit and the resulting release of the active catalytic subunit is a very well established mechanism of kinase activation. We have shown recently that PKA in budding yeast is also subject to an additional level of regulation that that modulates its activity in response to nutrient availability. Nutrient regulation of PKA activity requires a pair of proteins, Gpb1 and Gpb2, that contain several kelch repeats, a sequence motif that predicts that they fold into a ß-propeller structure. The regulatory process mediated by Gpb1 and Gpb2 causes an increase in the stability and phosphorylation of the PKA regulatory subunit Bcy1 in response to low extracellular glucose concentrations. Phosphorylation of serine-145 of Bcy1 controls its stability, and other phosphorylation events at the cluster of serines at positions 74-84 correlate with changes in nutrient availability. Here we present data consistent with a model in which the effects of Gpb1 and Gpb2 on Bcy1 are an indirect consequence of their primary effects on the PKA catalytic subunits.

Nutrient control of yeast PKA activity involves opposing effects on phosphorylation of the Bcy1 regulatory subunit.

GPB1 and GPB2 encode kelch repeat-containing proteins that regulate protein kinase A (PKA) in yeast by a cAMP-independent process. Here we show that Gpb1 and Gpb2 stimulate phosphorylation of PKA regulatory subunit Bcy1 in low glucose concentrations, thereby promoting the inhibitory function of Bcy1 when nutrients are scarce and PKA activity is expected to be low. Gpb1 and Gpb2 stimulate Bcy1 phosphorylation at an unknown site, and this modification stabilizes Bcy1 that has been phosphorylated by PKA catalytic subunits at serine-145. The BCY1(S145A) mutation eliminates the effect of gpb1Δ gpb2Δ on Bcy1 stability but maintains their effect on phosphorylation and signaling, indicating that modulation of PKA activity by Gpb1 and Gpb2 is not solely due to increased levels of Bcy1. Inhibition of PKA catalytic subunits that are ATP analog-sensitive causes increased Bcy1 phosphorylation at the unknown site in high glucose. When PKA is inhibited, gpb1Δ gpb2Δ mutations have no effect on Bcy1 phosphorylation. Therefore, Gpb1 and Gpb2 oppose PKA activity by blocking the ability of PKA to inhibit Bcy1 phosphorylation at a site other than serine-145. Stimulation of Bcy1 phosphorylation by Gpb1 and Gpb2 produces a form of Bcy1 that is more stable and is a more effective PKA inhibitor.

Cytoprotection against Cr(6+)-induced DNA damage by alpha-lipoic acid: implications in reducing occupational cancer risk.

Alpha-lipoic acid (LA), the metabolic antioxidant, was evaluated for its potential to protect against Cr(6+)-induced DNA damage. Potassium dichromate was administered to Swiss albino mice orally ad libitum at the doses of 5, 10 or 25 mg/kg body weight in drinking water to set DNA damage in cells, which was characterized in mouse peripheral blood mononuclear cells and bone marrow cells using single-cell gel electrophoresis and analyses of generated comets for Tail moment, Tail DNA and Tail length. DNA damage was dose dependent. Cytoprotection by LA was remarkable. LA (5, 10 and 25 mg/kg body weight intraperitoneally) in pre-, co- and post-toxicant administration schedule abrogated DNA damage substantially in both cell types. Protection by LA was also dose dependent. LA annulled DNA damage by Cr(6+) in plasmid relaxation assay. A negligible DNA damage resulted during interaction of Cr(6+) and LA. Compared to ascorbate, LA emerged as a better antioxidant and least DNA damaging. In conclusion, our study advocated an experimental therapeutic research potential in LA against Cr(6+)-induced DNA damage for reduction of occupational cancer risk in humans.

Prevention of chromate induced oxidative stress by alpha-lipoic acid.

The parenteral administration of alpha-lipoic acid (LA) protected against chromate induced oxidative stress in mouse liver. A shift in Cr induced pro-oxidant state to antioxidant-state by LA was noteworthy. The degree of protection was significant and similar in different LA administration regimens (prior-, co- and post- parenteral Cr exposure) explored. An improved status of the tissue antioxidants by LA appeared to be the mechanism of mitigation. The results are of chemopreventive value and suggest a possible alternative to ascorbic acid for abrogation of Cr toxicity.

Substantial inhibition of chromate induced DNA-protein crosslink formation in vivo by α-lipoic acid.

Cellular DNA-protein crosslink (DPCs) are the biologically active nucleoprotein complexes. Chromate compounds induce its formation. We have found that intraperitoneal-administration of α-lipoic acid (LA, a metabolic antioxidant) inhibited substantially the chromate induced DPCs formation in mouse peripheral blood lymphocytes (PBL). Change in LA administration schedule (i.e. pre-, co- or post-toxin) did not influence its effect. Results are of significance in two aspects viz. chemoprevention of chromate toxicity and exposure/effect estimation of chromate handling subjects. The antioxidant diet/therapy may mask the exposure/effect assessments and yield high frequency of false-negatives in study subjects.

DNA-protein crosslinks as a biomarker of exposure to solar radiation: a preliminary study in brick-kiln workers.

In India, fired clay bricks are produced in small-scale factories. There are 60, 000 active brick kilns, providing employment to nearly 12 million people in different suboccupations. This industry is largely non-mechanized and operates from November to June. Almost all the workers are exposed to direct sunlight for 8-10 h a day. Cellular DNA-protein crosslinks (DPCs) are the biologically active nucleoprotein complexes formed between DNA and proteins. Ultraviolet light and gamma-rays, and other suspected carcinogens in humans, induce DPC formation in blood cells. DPCs have therefore been identified as a biomarker for monitoring exposure to these hazardous agents. Here we report steady-state levels of DPCs in human peripheral lymphocytes from 46 brick-kiln workers exposed occupationally for 8-10 h a day to solar radiation in brickfields and 25 unexposed controls. A significant increase (p <0.05) in DPC content and DPC coefficients in peripheral lymphocytes was observed in the brick-kiln workers compared with the controls. The data suggest that the DPC content of lymphocytes could be a possible biomarker of exposure to solar radiation. However, further work is necessary to confirm this.