Molecular dynamics investigation of a redox switch in the anti-HIV protein SAMHD1

HIV-1 is restricted in macrophages and certain quiescent myeloid cells due to a “Scorched Earth” dNTP starvation strategy attributed to the sterile alpha motif and HD domain protein—SAMHD1. Active SAMHD1 tetramers are assembled by GTP-Mg+2-dNTP cross bridges and cleave the triphosphate groups of dNTPs at a K _m of ~10 μM, which is consistent with dNTP concentrations in cycling cells, but far higher than the equivalent concentration in quiescent cells. Given the substantial disparity between the dNTP concentrations required to activate SAMHD1 tetramers (~10 μM) and the dNTP concentrations in noncycling cells (~10 nM), the possibility of alternate enzymatically active forms of SAMHD1, including monomers remains open. In particular, the possibility of redox regulation of such monomers is also an open question. There have been experimental studies on the regulation of SAMHD1 by Glutathione driven redox reactions recently. Therefore, in this work, we have performed all-atom molecular dynamics simulations to study the dynamics of monomeric SAMHD1 constructs in the context of the three redox-susceptible Cysteine residues and compared them to monomers assembled within a tetramer. Our results indicate that assembly into a tetramer causes ordering of the catalytic core and increased solvent accessibility of the Catalytic Site. We have also found that glutathionylation of surface exposed C522 causes long range allosteric disruptions extending into the protein core. Finally, we see evidence suggesting a transient interaction between C522 and C341. Such a disulfide linkage has been hypothesized by experimental models, but has never been observed in crystal structures before.     

A short review on creatine–creatine kinase system in relation to cancer and some experimental results on creatine as adjuvant in cancer therapy

The creatine/creatine kinase (CK) system plays a key role in cellular energy buffering and transport. In vertebrates, CK has four isoforms expressed in a tissue-specific manner. In the process of creatine biosynthesis several other important metabolites are formed. The anticancer effect of creatine had been reported in the past, and recent literature has reported low creatine content in several types of malignant cells. Furthermore, creatine can protect cardiac mitochondria from the deleterious effects of some anticancer compounds. Previous work from our laboratory showed progressive decrease of phosphocreatine, creatine and CK upon transformation of skeletal muscle into sarcoma. It was convincingly demonstrated that prominent expression of creatine-synthesizing enzymes l-arginine: glycine amidinotransferase and N-guanidinoacetate methyltransferase occurs in sarcoma, Ehrlich ascites carcinoma and sarcoma 180 cells; whereas, both these enzymes are virtually undetectable in skeletal muscle. Creatine transporter also remained unaltered in malignant cells. The anticancer effect of methylglyoxal had been known for a long time. The present work shows that this anticancer effect of methylglyoxal is significantly augmented in presence of creatine. On creatine supplementation the effect of methylglyoxal plus ascorbic acid was further augmented and there was no visible sign of tumor. Moreover, creatine and CK, which were very low in sarcoma tissue, were significantly elevated with the concomitant regression of tumor.     

Effects of essential oils on methane production and fermentation by, and abundance and diversity of, rumen microbial populations

Five essential oils (EOs), namely, clove oil (CLO), eucalyptus oil (EUO), garlic oil (GAO), origanum oil (ORO), and peppermint oil (PEO), were tested in vitro at 3 different doses (0.25, 0.50, and 1.0 g/liter) for their effect on methane production, fermentation, and select groups of ruminal microbes, including total bacteria, cellulolytic bacteria, archaea, and protozoa. All the EOs significantly reduced methane production with increasing doses, with reductions by 34.4%, 17.6%, 42.3%, 87%, and 25.7% for CLO, EUO, GAO, ORO, and PEO, respectively, at 1.0 g/liter compared with the control. However, apparent degradability of dry matter and neutral detergent fiber also decreased linearly with increasing doses by all EOs except GAO. The concentrations of total volatile fatty acids were not affected by GAO, EUO, or PEO but altered linearly and quadratically by CLO and ORO, respectively. All the EOs also differed in altering the molar proportions of acetate, propionate, and butyrate. As determined by quantitative real-time PCR, all the EOs decreased the abundance of archaea, protozoa, and major cellulolytic bacteria (i.e., Fibrobacter succinogenes, Ruminococcus flavefaciens, and R. albus) linearly with increasing EO doses. On the basis of denaturing gradient gel electrophoresis analysis, different EOs changed the composition of both archaeal and bacterial communities to different extents. The Shannon-Wiener diversity index (H') was reduced for archaea by all EOs in a dose-dependent manner but increased for bacteria at low and medium doses (0.25 and 0.50 g/liter) for all EOs except ORO. Due to the adverse effects on feed digestion and fermentation at high doses, a single EO may not effectively and practically mitigate methane emission from ruminants unless used at low doses in combinations with other antimethanogenic compounds.     

Curcumin-embedded DBPC liposomes coated with chitosan layer as a fluorescence nanosensor for the selective detection of ribonucleic acid

One of the limitations of fluorescence probe molecules during biomedical estimation is their lack of ability to selectively determine the targeted species. To overcome this there have been various approaches that involve attaching a functional group or aptamers to the fluorescence probe. However, encapsulating probe molecules in a matrix using nanotechnology can be a viable and easier method. Curcumin (Cur) as a fluorescence marker cannot distinguish DNA and RNA. This research reports a novel selective approach involving the use of nanocapsules composed of liposomal curcumin coated with chitosan for the selective detection of RNA molecules using a fluorescence method. The increase in RNA concentration enhanced the electrostatic interaction between the negatively charge surface of RNA and the positively charged nanocapsule, which was further verified by zeta potential measurement. This method had a low limit of detection (36 ng/ml) and higher linear dynamic ranges compared with other studies found in the literature. Moreover, the method was not affected by DNA and was selective for the detection of RNA molecules for which the site of interaction was confined only to uracil. The selectivity for RNA molecules towards other analogues species was also examined and recovery range found was between 99 and 100.33%.     

Performance of nested multiplex PCR assay targeting MTP40 and IS6110 gene sequences for the diagnosis of tubercular lymphadenitis

The conventional methods for diagnosis of tubercular lymphadenitis (TBLN) such as - fine needle aspiration cytology, Ziehl-Neelsen staining and culture have limitations of low sensitivity and/or specificity. So, it becomes essential to develop a rapid, sensitive, and specific method for an early diagnosis of TBLN. Therefore, the present study was conducted to evaluate nested multiplex polymerase chain reaction (nMPCR) targeting MTP40 and IS6110 gene sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex, respectively in 48 successive patients of TBLN and 20 random patients with non-tubercular lymph node lesions. Out of the 48 cases of TBLN, 14 (29.2%) were found to be positive by Ziehl-Neelsen staining, 15 (31.2%) were positive by culture and 43 (89.6%) cases were positive after first round of PCR while 48 (100%) cases were positive by nMPCR assay. The sensitivity and specificity of nMPCR was found to be 100% for the diagnosis of TBLN. The results thus obtained indicate that nMPCR assay is a highly sensitive and specific tool for the diagnosis of TBLN.     

Computational characterization of epitopic region within the outer membrane protein candidate in Flavobacterium columnare for vaccine development

Abstract

Gram-negative bacteria is the main causative agents for columnaris disease outbreak to finfishes. The outer membrane proteins (OMPs) candidate of Flavobacterium columnare bacterial cell served a critical component for cellular invasion targeted to the eukaryotic cell and survival inside the macrophages. Therefore, OMPs considered as the supreme element for the development of promising vaccine against F. columnare. Implies advanced in silico approaches, the predicted 3-D model of targeted OMPs were characterized by the Swiss model server and validated through Procheck programs and Protein Structure Analysis (ProSA) web server. The protein sequences having B-cell binding sites were preferred from sequence alignment; afterwards the B cell epitopes prediction was prepared using the BCPred and amino acid pairs (AAP) prediction algorithms modules of BCPreds. Consequently, the selected antigenic amino acids sequences (B-cell epitopic regions) were analyzed for T-cell epitopes determination (MHC I and MHC II alleles binding sequence) performing the ProPred 1 and ProPred server respectively. The epitopes (9 mer: IKKYEPAPV, YGPNYKWKF and YRGLNVGTS) within the OMPs binds to both of the MHC classes (MHC I and MHC II) and covered highest number of MHC alleles are characterized. OMPs of F. columnare being conserved across serotypes and highly immunogenic for their exposed epitopes on the cell surface as a potent candidate focus to vaccine development for combating the disease problems in commercial aquaculture. The portrayed epitopes might be beneficial for practical designing of abundant peptide-based vaccine development against the columnaris through boosting up the advantageous immune responses.

Communicated by Ramaswamy H. Sarma     

Induction of sister chromatid exchanges by heavy metal salts in root meristem cells ofAllium cepa L.

Four heavy metal salts, nickel sulphate, mercuric chloride, cadmium sulphate and zinc sulphate, were tested for induction of sister chromatid exchange (SCE) in root meristem cells ofAllium cepa. A simple modified Feulgen staining procedure was employed for SCE-analysis. Maleic hydrazide and paraquat were included for comparison. An evaluation of genotoxicity of the above test chemicals made on the basis of SCE-assay was found positive for all the test chemicals with exception of zinc sulphate which gave a weak positive result.