Functional Promoter Polymorphisms Govern Differential Expression of HMG-CoA Reductase Gene in Mouse Models of Essential Hypertension

3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5′-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Plasmid Curing from an Acidophilic Bacterium of the Genus Acidocella

Preservation of the acidophilic heterotroph, Acidocella sp. strain GS19h, at 4³C in stab culture eliminated all indigenous plasmids from this bacterium. Growth at 42³C initially caused changes in the plasmid profile followed by total elimination of plasmids after 10 cycles of growth. Concomitant to this loss of all plasmids, the cured derivatives became sensitive to CdSO4 and ZnSO4, and the MIC value of the salts dropped from 1 M for each in the case of parental strain to 2 mM and 5 mM, respectively, suggesting plasmid-mediated inheritance of metal resistance in this bacterium. The cured derivatives could not utilise lactose, indicating this metabolic activity to be plasmid-associated in this strain. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved

Resistance to Cadmium and Zinc in Acidiphilium symbioticum KM2 Is Plasmid Mediated

The acidophilic heterotroph, Acidiphilium symbioticum KM2, is highly resistant to several metals and harbors three plasmids of 3.8, 7.1, and 56 kb in size. The bacterium becomes extremely sensitive to metals when it is cured of its plasmids. A mini-plasmid library was constructed by ligating the plasmid DNA fragments generated by MboI partial digestion into the BamHI site of pBluescriptII KS�. The Lac�Ampr transformants of Escherichia coli DH5�, isolated after transformation with the library, were counter-selected on Cu2�, Cd2�, Ni2�, and Zn2�-containing plates. Only Cd2�- and Zn2�-resistant colonies were developed, and, after screening, four types of recombinant plasmids designated as pNM201 (7.2 kb), pNM206 (3.4 kb), pNM208 (4.5 kb), and pNM215 (4.9 kb) were obtained. The DNA insert in pNM206 hybridized strongly with the 3.8-kb plasmid and weakly with the 7.1-kb plasmid of Acidiphilium symbioticum KM2. The DNA insert in pNM215 hybridized only with the 7.1-kb plasmid. These results strongly suggested that resistance to cadmium and zinc in A. symbioticum KM2 is mediated by these plasmids. The smallest insert of 422 bp in pNM206 conferring metal resistance in E. coli has no sequence similarity with the reported metal-resistant genes. All the putative ORFs are significantly rich (up to 37%) in basic amino acids, mainly arginine

Generation of Novel Plasmids in Escherichia coli S17-1(pSUP106)

When the highly metal-resistant acidophilic heterotrophic strain, Acidiphilium symbioticum KM2, was incubated with two Escherichia coli strains, viz. S17-1 (pSUP106) and K12, on a medium that supported growth of these two divergent species of different habitats, E. coli transconjugants were isolated that contained novel plasmids and were resistant to Zn2� (48 mM), Cu2� (12 mM), Ni2� (12 mM), chloramphenicol (50 �g/ml), and tetracycline (25 �g/ml). The transconjugant plasmids did not hybridize with any of the A. symbioticum KM2 plasmids. After curing of the plasmids, the transconjugants became sensitive to 12 mM Zn2�, 12 mM Cu2�, and 12 mM Ni2�, but remained chloramphenicol and tetracycline resistant—the phenotypic markers that were originally present in pSUP106. That a part of pSUP106 was integrated into the chromosome of the transconjugants was evident from the hybridization of pSUP106 with chromosomal DNA of the cured derivatives of the transconjugants. Further, the transconjugant plasmids hybridized only with the chromosomal DNA of E. coli S17-1 and not with the chromosomal DNA of A. symbioticum KM2 or E. coli K12, suggesting their host chromosomal origin. Thus, the present study describes a unique event of genetic rearrangements in the E. coli strain S17-1 (pSUP106), resulting in the formation of novel plasmids conferring metal-resistance phenotypes in the cell

Epigenetic modifier alpha-ketoglutarate modulates aberrant gene body methylation and hydroxymethylation marks in diabetic heart

Abstract Background Diabetic cardiomyopathy (DCM) is a leading cause of death in diabetic patients. Hyperglycemic myocardial microenvironment significantly alters chromatin architecture and the transcriptome, resulting in aberrant activation of signaling pathways in a diabetic heart. Epigenetic marks play vital roles in transcriptional reprogramming during the development of DCM. The current study is aimed to profile genome-wide DNA (hydroxy)methylation patterns in the hearts of control and streptozotocin (STZ)-induced diabetic rats and decipher the effect of modulation of DNA methylation by alpha-ketoglutarate (AKG), a TET enzyme cofactor, on the progression of DCM. Methods Diabetes was induced in male adult Wistar rats with an intraperitoneal injection of STZ. Diabetic and vehicle control animals were randomly divided into groups with/without AKG treatment. Cardiac function was monitored by performing cardiac catheterization. Global methylation (5mC) and hydroxymethylation (5hmC) patterns were mapped in the Left ventricular tissue of control and diabetic rats with the help of an enrichment-based (h)MEDIP-sequencing technique by using antibodies specific for 5mC and 5hmC. Sequencing data were validated by performing (h)MEDIP-qPCR analysis at the gene-specific level, and gene expression was analyzed by qPCR. The mRNA and protein expression of enzymes involved in the DNA methylation and demethylation cycle were analyzed by qPCR and western blotting. Global 5mC and 5hmC levels were also assessed in high glucose-treated DNMT3B knockdown H9c2 cells. Results We found the increased expression of DNMT3B, MBD2, and MeCP2 with a concomitant accumulation of 5mC and 5hmC, specifically in gene body regions of diabetic rat hearts compared to the control. Calcium signaling was the most significantly affected pathway by cytosine modifications in the diabetic heart. Additionally, hypermethylated gene body regions were associated with Rap1, apelin, and phosphatidyl inositol signaling, while metabolic pathways were most affected by hyperhydroxymethylation. AKG supplementation in diabetic rats reversed aberrant methylation patterns and restored cardiac function. Hyperglycemia also increased 5mC and 5hmC levels in H9c2 cells, which was normalized by DNMT3B knockdown or AKG supplementation. Conclusion This study demonstrates that reverting hyperglycemic damage to cardiac tissue might be possible by erasing adverse epigenetic signatures by supplementing epigenetic modulators such as AKG along with an existing antidiabetic treatment regimen

SARS-CoV-2 infection and its effects on the endocrine system

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing corona virus disease 2019 (COVID-19) can infect multiple tissues, including endocrine organs, such as the pancreas, adrenal, thyroid, and adipose tissue. The main receptor for SARS-CoV-2, ACE2, is ubiquitously expressed in the cells of the endocrine organs and accordingly, the virus has been detected in various amounts in all endocrine tissues in post-mortem samples from COVID-19 patients. The infection with SARS-CoV-2 may directly lead to organ damage or dysfunction, such as hyperglycaemia or in rare cases, new-onset diabetes. Furthermore, an infection with SARS-CoV-2 may have indirect effects affecting the endocrine system. The exact mechanisms are not yet completely understood and have to be further investigated. Conversely, endocrine diseases may affect the severity of COVID-19 and emphasis has to be laid on reducing the prevalence, or enhance the treatment, of these often non-communicable diseases in the future

Enhancement in the efficiency of polymerase chain reaction by TiO<SUB>2</SUB> nanoparticles: crucial role of enhanced thermal conductivity

Improvement of the specificity and efficiency of the polymerase chain reaction (PCR) by nanoparticles is an emerging area of research. We observed that TiO2 nanoparticles of ~ 25 nm diameter caused significant enhancement of PCR efficiency for various types of templates (namely plasmid DNA, genomic DNA and complementary DNA). By a series of experiments, the optimal TiO2 concentration was determined to be 0.4 nM, which resulted in up to a seven-fold increase in the amount of PCR product. As much as 50% reduction in overall reaction time (by reduction of the number of cycles and the time periods of cycles) was also achieved by utilizing TiO2 nanoparticles without compromising the PCR yield. Investigations of the mechanism of such PCR enhancement by simulations using the 'Fluent K epsilon turbulent model' provided evidence of faster heat transfer in the presence of TiO2 nanoparticles. Consistent with these findings, TiO2 nanoparticles were observed to augment the denaturation of genomic DNA, indicating more efficient thermal conductivity through the reaction buffer. TiO2 nanoparticle-assisted PCR may be useful for profound reduction of the overall PCR reaction period and for enhanced amplification of DNA amplicons from a variety of samples, including GC-rich templates that are often observed to yield unsatisfactory results