Direct regulation of topoisomerase activity by a nucleoid-associated protein

The topological homeostasis of bacterial chromosomes is maintained by the balance between compaction and the topological organization of genomes. Two classes of proteins play major roles in chromosome organization: the nucleoid-associated proteins (NAPs) and topoisomerases. The NAPs bind DNA to compact the chromosome, whereas topoisomerases catalytically remove or introduce supercoils into the genome. We demonstrate that HU, a major NAP of Mycobacterium tuberculosis specifically stimulates the DNA relaxation ability of mycobacterial topoisomerase I (TopoI) at lower concentrations but interferes at higher concentrations. A direct physical interaction between M. tuberculosis HU (MtHU) and TopoI is necessary for enhancing enzyme activity both in vitro and in vivo. The interaction is between the amino terminal domain of MtHU and the carboxyl terminal domain of TopoI. Binding of MtHU did not affect the two catalytic trans-esterification steps but enhanced the DNA strand passage, requisite for the completion of DNA relaxation, a new mechanism for the regulation of topoisomerase activity. An interaction-deficient mutant of MtHU was compromised in enhancing the strand passage activity. The species-specific physical and functional cooperation between MtHU and TopoI may be the key to achieve the DNA relaxation levels needed to maintain the optimal superhelical density of mycobacterial genomes.     

Detection and validation of stay-green QTL in post-rainy sorghum involving widely adapted cultivar,M35-1 and a popular stay-green genotype B35

Background

Sorghum [Sorghum bicolor (L.) Moench] is an important dry-land cereal of the world providing food, fodder, feed and fuel. Stay-green (delayed-leaf senescence) is a key attribute in sorghum determining its adaptation to terminal drought stress. The objective of this study was to validate sorghum stay-green quantitative trait loci (QTL) identified in the past, and to identify new QTL in the genetic background of a post-rainy adapted genotype M35-1.

Results

A genetic linkage map based on 245 F₉ Recombinant Inbred Lines (RILs) derived from a cross between M35-1 (more senescent) and B35 (less senescent) with 237 markers consisting of 174 genomic, 60 genic and 3 morphological markers was used. The phenotypic data collected for three consecutive post-rainy crop seasons on the RIL population (M35-1 × B35) was used for QTL analysis. Sixty-one QTL were identified for various measures of stay-green trait and each trait was controlled by one to ten QTL. The phenotypic variation explained by each QTL ranged from 3.8 to 18.7%. Co-localization of QTL for more than five traits was observed on two linkage groups i.e. on SBI-09-3 flanked by S18 and Xgap206 markers and, on SBI-03 flanked by XnhsbSFCILP67 and Xtxp31. QTL identified in this study were stable across environments and corresponded to sorghum stay-green and grain yield QTL reported previously. Of the 60 genic SSRs mapped, 14 were closely linked with QTL for ten traits. A genic marker, XnhsbSFCILP67 (Sb03g028240) encoding Indole-3-acetic acid-amido synthetase GH3.5, was co-located with QTL for GLB, GLM, PGLM and GLAM on SBI-03. Genes underlying key enzymes of chlorophyll metabolism were also found in the stay-green QTL regions.

Conclusions

We validated important stay-green QTL reported in the past in sorghum and detected new QTL influencing the stay-green related traits consistently. Stg2, Stg3 and StgB were prominent in their expression. Collectively, the QTL/markers identified are likely candidates for subsequent verification for their involvement in stay-green phenotype using NILs and to develop drought tolerant sorghum varieties through marker-assisted breeding for terminal drought tolerance in sorghum.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-909) contains supplementary material, which is available to authorized users.     

Is One Sputum Specimen as Good as Two during Follow-Up Cultures for Monitoring Multi Drug Resistant Tuberculosis Patients in India?

Background

In India, the Revised National Tuberculosis Control Programme (RNTCP) has adopted the strategy of examining two specimens during follow-up culture examinations to monitor the treatment response of multi-drug resistant tuberculosis (MDR-TB) patients.

Objectives

To determine the incremental yield of the second sputum specimen during follow-up culture examinations among patients with MDR-TB and the effect on case management on changing from two to one specimen follow-up strategy.

Methods

A cross sectional record review of MDR-TB patients registered during 2008–09 under RNTCP was undertaken in three MDR-TB treatment sites of India.

Results

Of 1721 pairs of follow-up sputum culture examinations done among 220 MDR-TB patients, 451(26%) were positive with either of the two specimens; 29(1.7%) were culture positive only on the second specimen indicating the incremental yield. To detect one additional culture positive result on the second specimen, 59 specimens needed to be processed. If we had examined only one specimen, we would have missed 29 culture-positive results. By current RNTCP guidelines, however, a single specimen policy would have altered case management in only 3(0.2%) instances, where patients would have missed a one month extension of the intensive phase of MDR-TB treatment. There is no meaningful advantage in using two specimens for the monitoring of MDR-TB patients. A single specimen policy could be safely implemented with negligible clinical effect on MDR-TB patients and favourable resource implications for RNTCP.     

The Phylogeography of Y-Chromosome Haplogroup H1a1a-M82 Reveals the Likely Indian Origin of the European Romani Populations

Linguistic and genetic studies on Roma populations inhabited in Europe have unequivocally traced these populations to the Indian subcontinent. However, the exact parental population group and time of the out-of-India dispersal have remained disputed. In the absence of archaeological records and with only scanty historical documentation of the Roma, comparative linguistic studies were the first to identify their Indian origin. Recently, molecular studies on the basis of disease-causing mutations and haploid DNA markers (i.e. mtDNA and Y-chromosome) supported the linguistic view. The presence of Indian-specific Y-chromosome haplogroup H1a1a-M82 and mtDNA haplogroups M5a1, M18 and M35b among Roma has corroborated that their South Asian origins and later admixture with Near Eastern and European populations. However, previous studies have left unanswered questions about the exact parental population groups in South Asia. Here we present a detailed phylogeographical study of Y-chromosomal haplogroup H1a1a-M82 in a data set of more than 10,000 global samples to discern a more precise ancestral source of European Romani populations. The phylogeographical patterns and diversity estimates indicate an early origin of this haplogroup in the Indian subcontinent and its further expansion to other regions. Tellingly, the short tandem repeat (STR) based network of H1a1a-M82 lineages displayed the closest connection of Romani haplotypes with the traditional scheduled caste and scheduled tribe population groups of northwestern India.     

Genetic affinities of the central Indian tribal populations

Background

The central Indian state Madhya Pradesh is often called as ‘heart of India’ and has always been an important region functioning as a trinexus belt for three major language families (Indo-European, Dravidian and Austroasiatic). There are less detailed genetic studies on the populations inhabited in this region. Therefore, this study is an attempt for extensive characterization of genetic ancestries of three tribal populations, namely; Bharia, Bhil and Sahariya, inhabiting this region using haploid and diploid DNA markers.

Methodology/Principal Findings

Mitochondrial DNA analysis showed high diversity, including some of the older sublineages of M haplogroup and prominent R lineages in all the three tribes. Y-chromosomal biallelic markers revealed high frequency of Austroasiatic-specific M95-O2a haplogroup in Bharia and Sahariya, M82-H1a in Bhil and M17-R1a in Bhil and Sahariya. The results obtained by haploid as well as diploid genetic markers revealed strong genetic affinity of Bharia (a Dravidian speaking tribe) with the Austroasiatic (Munda) group. The gene flow from Austroasiatic group is further confirmed by their Y-STRs haplotype sharing analysis, where we determined their founder haplotype from the North Munda speaking tribe, while, autosomal analysis was largely in concordant with the haploid DNA results.

Conclusions/Significance

Bhil exhibited largely Indo-European specific ancestry, while Sahariya and Bharia showed admixed genetic package of Indo-European and Austroasiatic populations. Hence, in a landscape like India, linguistic label doesn''t unequivocally follow the genetic footprints.     

CD45-mediated signaling pathway is involved in Rhizoctonia bataticola lectin (RBL)-induced proliferation and Th1/Th2 cytokine secretion in human PBMC

We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate the involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.     

Effects of Dried Distillers’ Grain on Fecal Prevalence and Growth of Escherichia coli O157 in Batch Culture Fermentations from Cattle

Distillers’ grains (DG), a by-product of ethanol production, are fed to cattle. Associations between Escherichia coli O157 prevalence and feeding of DG were investigated in feedlot cattle (n = 379) given one of three diets: steam-flaked corn (SFC) and 15% corn silage with 0 or 25% dried distillers’ grains (DDG) or SFC with 5% corn silage and 25% DDG. Ten fecal samples were collected from each pen weekly for 12 weeks to isolate E. coli O157. Cattle fed 25% DDG with 5 or 15% silage had a higher (P = 0.01) prevalence of E. coli O157 than cattle fed a diet without DDG. Batch culture ruminal or fecal microbial fermentations were conducted to evaluate the effect of DDG on E. coli O157 growth. The first study utilized microbial inocula from steers fed SFC or dry-rolled corn with 0 or 25% DDG and included their diet as the substrate. Ruminal microbial fermentations from steers fed DDG had higher E. coli O157 contents than ruminal microbial fermentations from steers fed no DDG (P < 0.05) when no substrate was included. Fecal fermentations showed no DDG effect on E. coli O157 growth. In the second study with DDG as a substrate, ruminal fermentations with 0.5 g DDG had higher (P < 0.01) E. coli O157 concentrations at 24 h than ruminal fermentations with 0, 1, or 2 g DDG. In fecal fermentations, 2 g DDG resulted in a higher concentration (P < 0.05) at 24 h than 0, 0.5, or 1 g DDG. The results indicate that there is a positive association between DDG and E. coli O157 in cattle, and the findings should have important ramifications for food safety.     

A pilot study of the effects of pioglitazone and rosiglitazone on de novo lipogenesis in type 2 diabetes

Treatment of type 2 diabetes mellitus (T2DM) patients with pioglitazone results in a more favorable lipid profile, and perhaps more favorable cardiac outcomes, than treatment with rosiglitazone. Pioglitazone treatment increases VLDL-triacylglycerol clearance, but the role of de novo lipogenesis (DNL) has not been explored, and no direct comparison has been made between the thiazolidinediones (TZDs). Twelve subjects with T2DM and hypertriacylglyceridemia were randomized to either rosiglitazone or pioglitazone treatment. Stable isotope infusion studies were performed at baseline and after 20 weeks of treatment. Both treatments reduced glucose and HbA(1c) concentrations equally. Pioglitazone treatment resulted in a 40% reduction in hepatic DNL (P < 0.01) and in a 25% reduction in hepatic glucose production (P < 0.05), while rosiglitazone did not significantly change either parameter, although comparisons of changes between treatments were not significantly different. These pilot results indicate that pioglitazone reduces hepatic DNL while rosiglitazone does not. Larger follow-up studies are required to confirm differential effects of these agents definitively. The reduction in DNL may underlie altered assembly or atherogenicity of lipoprotein particles and may reflect PPARalpha or other non-PPARgamma actions on the liver by pioglitazone. These differences might help explain previously reported differences in lipid profiles and cardiovascular disease outcomes for rosiglitazone and pioglitazone.     

Effect of novel arecoline thiazolidinones as muscarinic receptor 1 agonist in Alzheimer's dementia models

The discovery of cholinergic deficit in Alzheimer's disease (AD) patient's brain has triggered research efforts, using cholinomimetic approaches for their efficacy in AD therapy. Various therapies may be of potential clinical use in AD. Among these are cholinergic agents, which include muscarinic agonists, acetylcholinesterase inhibitors, and acetylcholine releasing agents. One of the muscarinic agonists tested in AD is arecoline and its bioisosters, which are widely explored as muscarinic receptor 1 agonist (M1 receptor agonist) in AD research. In this regard, five-membered heterocyclic ring system attached arecoline basic nucleus (N-methyl tetrahydropyridines) at third position has been extensively researched on. The present research involved synthesis of arecoline thiazolidinones 5(a-j) by using dipolar addition of 3-aminopyridine and alkyl/aryl carboxaldehydes in presence of gamma ferrite as catalyst. The resulting products were methylated and reduced to get desired products. Subsequently the synthesized arecoline thiazolidinones were subjected to in vitro muscarinic receptor binding studies using male Wistar rat brain (cerebral cortex) membrane homogenate and extended this in vitro study to in vivo pharmacological evaluation of memory and learning in male Wistar rats. Four derivatives (5a-5c and 5e) showed considerable M1 receptor binding affinity (in vitro) and elicited beneficial effects in vivo memory and learning models (Rodent memory evaluation, plus and Y maze studies).