Over-expression of NAD kinase in Corynebacterium crenatum and its Impact on L-Arginine Biosynthesis

Purpose: To improve the biosynthesis of L-arginine by overexpressing homologous NAD kinase (ppnk) in Corynebacterium crenatum SYPA5-5 and to study its impact in presence of high (HOS) and low oxygen supply (LOS).Methods: A recombinant plasmid (pJC1-tac-ppnK) harboring homologous NAD kinase (ppnk) was constructed in a shuttle vector pJC1 and transferred in L-arginine producing strain Corynebacterium crenatum SYPA5-5. Furthermore, fermentation was performed by shake flask method with consecutive determination of cell growth and glucose concentration. NAD+ kinase activity was studied by stop method and NADP(H) concentrations were determined by spectrophotometric enzymatic cycling method. To check the biosynthesis of amino acids, HPLC method was used to determine extracellular amino acid concentrations.Results: In HOS condition, NAD+ kinase activity increased by 116 %, while intracellular concentrations of NADP+ and NADPH increased by 7.3 and 36.8 %, respectively. Whereas, in LOS condition , NAD+ kinase activity increased 49 % , with intracellular 14.67 and 15 % increases in NADP+ and NADPHrespectively. More importantly, recombinant strain could produce 26.47 and 11.36 g/L L-arginine in HOS and LOS respectively, which is higher than control strain value of 24.29 and 7.58 g/L respectively.Conclusion: These results suggest that altering the concentration of co-enzymes by NAD kinase in Corynebacterium crenatum is an effective way to increase NADP+ with concurrent production of NADPH for further enhanced L-arginine biosynthesis in Corynebacterium crenatum in both conditions of high and low oxygen supply.Keywords: NAD kinase, PpnK, L-arginine, Corynebacterium crenatu

First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Nepal

BACKGROUND: Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown. METHODS AND FINDINGS: We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42-4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43-5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status. CONCLUSIONS: We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian reg

Strategies in a metallophyte species to cope with manganese excess

The effect of exposure to high Mn concentration was studied in a metallophyte species, Erica andevalensis, using hydroponic cultures with a range of Mn concentrations (0.06, 100, 300, 500, and 700 mg L-1). At harvest, biomass production, element uptake, and biochemical indicators of metal stress (leaf pigments, organic acids, amino acids, phenols, and activities of catalase, peroxidase, superoxide dismutase) were determined in leaves and roots. Increasing Mn concentrations led to a decrease in biomass accumulation, and tip leaves chlorosis was the only toxicity symptom detected. In a similar way, photosynthetic pigments (chlorophylls a and b, and carotenoids) were affected by high Mn levels. Among organic acids, malate and oxalate contents in roots showed a significant increase at the highest Mn concentration, while in leaves, Mn led to an increasing trend in citrate and malate contents. An increase of Mn also induced an increase in superoxide dismutase activity in roots and catalase activity in leaves. As well, significant changes in free amino acids were induced by Mn concentrations higher than 300 mg L-1, especially in roots. No significant changes in phenolic compounds were observed in the leaves, but root phenolics were significantly increased by increasing Mn concentrations in treatments. When Fe supply was increased 10 and 20 times (7–14 mg Fe L-1 as Fe-EDDHA) in the nutrient solutions at the highest Mn concentration (700 mg Mn L-1), it led to significant increases in photosynthetic pigments and biomass accumulation. Manganese was mostly accumulated in the roots, and the species was essentially a Mn excluder. However, considering the high leaf Mn concentration recorded without toxicity symptoms, E. andevalensis might be rated as a Mn-tolerant speciesinfo:eu-repo/semantics/publishedVersio

Attrition among Human Immunodeficiency Virus (HIV)- Infected Patients Initiating Antiretroviral Therapy in China, 2003–2010

BACKGROUND: Mortality and morbidity from HIV have dramatically decreased in both high- and low-income countries. However, some patients may not benefit from combination antiretroviral therapy (cART) because of inadequate access to HIV care, including attrition after care initiation. METHODOLOGY/PRINCIPAL FINDINGS: The study population included all HIV-infected patients receiving cART through the Chinese National Free Antiretroviral Treatment Program from 1 January 2003 to 31 December 2010 (n = 106,542). We evaluated retention in HIV care and used multivariable Cox proportional hazard models to identify independent factors predictive of attrition. The cumulative probability of attrition from cART initiation was 9% at 12 months, 13% at 18 months, 16% at 24 months and 24% at 60 months. A number of factors were associated with attrition, including younger age, male gender, and being single or divorced. Patients with higher CD4 cell counts at cART initiation were more likely to drop out of HIV care. The proportion of patients remaining in HIV care increased in more recent calendar years and among patients who initiated modern cART regimens. CONCLUSIONS/SIGNIFICANCE: Retention in HIV care is essential for optimizing individual and public health outcomes. Attrition, even the degree observed in our study, can lead to premature morbidity and mortality, and possibly affect further transmission of HIV and HIV resistant drug variants. Effective strategies to promote retention in HIV care programs are needed. In China these strategies may include focusing particularly on younger male patients and those with higher CD4 cell counts at therapy initiation

Modulation of Sn concentration in ZnO nanorod array: intensification on the conductivity and humidity sensing properties

Tin (Sn)-doped zinc oxide (ZnO) nanorod arrays (TZO) were synthesized onto aluminum-doped ZnO-coated glass substrate via a facile sonicated sol–gel immersion method for humidity sensor applications. These nanorod arrays were grown at different Sn concentrations ranging from 0.6 to 3 at.%. X-ray diffraction patterns showed that the deposited TZO arrays exhibited a wurtzite structure. The stress/strain condition of the ZnO film metamorphosed from tensile strain/compressive stress to compressive strain/tensile stress when the Sn concentrations increased. Results indicated that 1 at.% Sn doping of TZO, which has the lowest tensile stress of 0.14 GPa, generated the highest conductivity of 1.31 S cm− 1. In addition, 1 at.% Sn doping of TZO possessed superior sensitivity to a humidity of 3.36. These results revealed that the optimum performance of a humidity-sensing device can be obtained mainly by controlling the amount of extrinsic element in a ZnO film

Fine Mapping of Posttranslational Modifications of the Linker Histone H1 from Drosophila melanogaster

The linker histone H1 binds to the DNA in between adjacent nucleosomes and contributes to chromatin organization and transcriptional control. It is known that H1 carries diverse posttranslational modifications (PTMs), including phosphorylation, lysine methylation and ADP-ribosylation. Their biological functions, however, remain largely unclear. This is in part due to the fact that most of the studies have been performed in organisms that have several H1 variants, which complicates the analyses. We have chosen Drosophila melanogaster, a model organism, which has a single H1 variant, to approach the study of the role of H1 PTMs during embryonic development. Mass spectrometry mapping of the entire sequence of the protein showed phosphorylation only in the ten N-terminal amino acids, mostly at S10. For the first time, changes in the PTMs of a linker H1 during the development of a multicellular organism are reported. The abundance of H1 monophosphorylated at S10 decreases as the embryos age, which suggests that this PTM is related to cell cycle progression and/or cell differentiation. Additionally, we have found a polymorphism in the protein sequence that can be mistaken with lysine methylation if the analysis is not rigorous

Fgfr3 Is a Transcriptional Target of Ap2δ and Ash2l-Containing Histone Methyltransferase Complexes

Polycomb (PcG) and trithorax (trxG) proteins play important roles in establishing lineage-specific genetic programs through induction of chromatin modifications that lead to gene silencing or activation. Previously, we described an association between the MLL/SET1 complexes and a highly restricted, gene-specific DNA-binding protein Ap2δ that is required for recruitment of the MLL/SET1 complex to target Hoxc8 specifically. Here, we reduced levels of Ap2δ and Ash2l in the neuroblastoma cell line, Neuro2A, and analyzed their gene expression profiles using whole-genome mouse cDNA microarrays. This analysis yielded 42 genes that are potentially co-regulated by Ap2δ and Ash2l, and we have identified evolutionarily conserved Ap2-binding sites in 20 of them. To determine whether some of these were direct targets of the Ap2δ-Ash2l complex, we analyzed several promoters for the presence of Ap2δ and Ash2l by chromatin immunoprecipitation (ChIP). Among the targets we screened, we identified Fgfr3 as a direct transcriptional target of the Ap2δ-Ash2l complex. Additionally, we found that Ap2δ is necessary for the recruitment of Ash2l-containing complexes to this promoter and that this recruitment leads to trimethylation of lysine 4 of histone H3 (H3K4me3). Thus, we have identified several candidate targets of complexes containing Ap2δ and Ash2l that can be used to further elucidate their roles during development and showed that Fgfr3 is a novel direct target of these complexes

Beta functions of topologically massive supergravity

We compute the one-loop beta functions of the cosmological constant, Newton's constant and the topological mass in topologically massive supergravity in three dimensions. We use a variant of the proper time method supplemented by a simple choice of cutoff function. We also employ two different analytic continuations of AdS3 and consider harmonic expansions on the 3-sphere as well as a 3-hyperboloid, and then show that they give the same results for the beta functions. We find that the dimensionless coefficient of the Chern-Simons term, 28, has vanishing beta function. The flow of the cosmological constant and Newton's constant depends on 28; we study analytically the structure of the flow and its fixed points in the limits of small and large ?. Open Access, \ua9 2014 The Authors

Intrinsically Disordered Proteins Display No Preference for Chaperone Binding In Vivo

Intrinsically disordered/unstructured proteins (IDPs) are extremely sensitive to proteolysis in vitro, but show no enhanced degradation rates in vivo. Their existence and functioning may be explained if IDPs are preferentially associated with chaperones in the cell, which may offer protection against degradation by proteases. To test this inference, we took pairwise interaction data from high-throughput interaction studies and analyzed to see if predicted disorder correlates with the tendency of chaperone binding by proteins. Our major finding is that disorder predicted by the IUPred algorithm actually shows negative correlation with chaperone binding in E. coli, S. cerevisiae, and metazoa species. Since predicted disorder positively correlates with the tendency of partner binding in the interactome, the difference between the disorder of chaperone-binding and non-binding proteins is even more pronounced if normalized to their overall tendency to be involved in pairwise protein–protein interactions. We argue that chaperone binding is primarily required for folding of globular proteins, as reflected in an increased preference for chaperones of proteins in which at least one Pfam domain exists. In terms of the functional consequences of chaperone binding of mostly disordered proteins, we suggest that its primary reason is not the assistance of folding, but promotion of assembly with partners. In support of this conclusion, we show that IDPs that bind chaperones also tend to bind other proteins

Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells

INTRODUCTION: A physiological feature of many tumor tissues and cells is the tendency to accumulate high concentrations of copper. While the precise role of copper in tumors is cryptic, copper, but not other trace metals, is required for angiogenesis. We have recently reported that organic copper-containing compounds, including 8-hydroxyquinoline-copper(II) and 5,7-dichloro-8-hydroxyquinoline-copper(II), comprise a novel class of proteasome inhibitors and tumor cell apoptosis inducers. In the current study, we investigate whether clioquinol (CQ), an analog of 8-hydroxyquinoline and an Alzheimer's disease drug, and pyrrolidine dithiocarbamate (PDTC), a known copper-binding compound and antioxidant, can interact with copper to form cancer-specific proteasome inhibitors and apoptosis inducers in human breast cancer cells. Tetrathiomolybdate (TM), a strong copper chelator currently being tested in clinical trials, is used as a comparison. METHODS: Breast cell lines, normal, immortalized MCF-10A, premalignant MCF10AT1K.cl2, and malignant MCF10DCIS.com and MDA-MB-231, were treated with CQ or PDTC with or without prior interaction with copper, followed by measurement of proteasome inhibition and cell death. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity and ubiquitinated proteins in protein extracts of the treated cells. Apoptotic cell death was measured by morphological changes, Hoechst staining, and poly(ADP-ribose) polymerase cleavage. RESULTS: When in complex with copper, both CQ and PDTC, but not TM, can inhibit the proteasome chymotrypsin-like activity, block proliferation, and induce apoptotic cell death preferentially in breast cancer cells, less in premalignant breast cells, but are non-toxic to normal/non-transformed breast cells at the concentrations tested. In contrast, CQ, PDTC, TM or copper alone had no effects on any of the cells. Breast premalignant or cancer cells that contain copper at concentrations similar to those found in patients, when treated with just CQ or PDTC alone, but not TM, undergo proteasome inhibition and apoptosis. CONCLUSION: The feature of breast cancer cells and tissues to accumulate copper can be used as a targeting method for anticancer therapy through treatment with novel compounds such as CQ and PDTC that become active proteasome inhibitors and breast cancer cell killers in the presence of copper