An incoming nucleotide imposes an anti to syn conformational change on the templating purine in the human DNA polymerase-iota active site

Substrate-induced conformational change of the protein is the linchpin of enzymatic reactions. Replicative DNA polymerases, for example, convert from an open to a closed conformation in response to dNTP binding. Human DNA polymerase-iota (hPoliota), a member of the Y family of DNA polymerases, differs strikingly from other polymerases in its much higher proficiency and fidelity for nucleotide incorporation opposite template purines than opposite template pyrimidines. We present here a crystallographic analysis of hPoliota binary complexes, which together with the ternary complexes show that, contrary to replicative DNA polymerases, the DNA, and not the polymerase, undergoes the primary substrate-induced conformational change. The incoming dNTP "pushes" templates A and G from the anti to the syn conformation dictated by a rigid hPoliota active site. Together, the structures posit a mechanism for template selection wherein dNTP binding induces a conformational switch in template purines for productive Hoogsteen base pairing.     

A Mycobacterium tuberculosis Sigma Factor Network Responds to Cell-Envelope Damage by the Promising Anti-Mycobacterial Thioridazine

Background

Novel therapeutics are urgently needed to control tuberculosis (TB). Thioridazine (THZ) is a candidate for the therapy of multidrug and extensively drug-resistant TB.

Methodology/Principal Findings

We studied the impact of THZ on Mycobacterium tuberculosis (Mtb) by analyzing gene expression profiles after treatment at the minimal inhibitory (1x MIC) or highly inhibitory (4x MIC) concentrations between 1–6 hours. THZ modulated the expression of genes encoding membrane proteins, efflux pumps, oxido-reductases and enzymes involved in fatty acid metabolism and aerobic respiration. The Rv3160c-Rv3161c operon, a multi-drug transporter and the Rv3614c/3615c/3616c regulon, were highly induced in response to THZ. A significantly high number of Mtb genes co-expressed with σ^B (the σ^B regulon) was turned on by THZ treatment. σ^B has recently been shown to protect Mtb from envelope-damage. We hypothesized that THZ damages the Mtb cell-envelope, turning on the expression of the σ^B regulon. Consistent with this hypothesis, we present electron-microscopy data which shows that THZ modulates cell-envelope integrity. Moreover, the Mtb mutants in σ^H and σ^E, two alternate stress response sigma factors that induce the expression of σ^B, exhibited higher sensitivity to THZ, indicating that the presence and expression of σ^B allows Mtb to resist the impact of THZ. Conditional induction of σ^B levels increased the survival of Mtb in the presence of THZ.

Conclusions/Significance

THZ targets different pathways and can thus be used as a multi-target inhibitor itself as well as provide strategies for multi-target drug development for combination chemotherapy. Our results show that the Mtb sigma factor network comprising of σ^H, σ^E and σ^B plays a crucial role in protecting the pathogen against cell-envelope damage.     

Downregulation of ADIPOQ and PPARγ2 Gene Expression in Subcutaneous Adipose Tissue of Obese Adolescents With Hepatic Steatosis

Hepatic steatosis is associated with hypoadiponectinemia. The mechanism(s) resulting in lower serum adiponectin levels in obese adolescents with fatty liver is unknown. In two groups of equally obese adolescents, but discordant for hepatic fat content, we measured adiponectin, leptin, peroxisome proliferator–activated receptor γ 2 (PPARγ2) and tumor necrosis factor‐α (TNFα) gene expression in the abdominal subcutaneous adipose tissue (SAT). Twenty six adolescents with similar degrees of obesity underwent a subcutaneous periumbilical adipose tissue biopsy, in addition to metabolic (oral glucose tolerance test, and hyperinsulinemic—euglycemic clamp), and imaging studies (magnetic resonance imaging (MRI), DEXA). Using quantitative real‐time‐PCR; adiponectin, PPARγ2, TNFα, and leptin mRNA were measured. Based on a hepatic fat content (hepatic fat fraction, HFF) >5.5%, measured by fast MRI, the subjects were divided into low and high HFF group. In addition to the hypoadiponectinemia in the high HFF group, we found that the expression of adiponectin as well as PPARγ2 in the SAT was significantly decreased in this group. No differences were noted for TNFα and leptin plasma or mRNA levels between the groups. An inverse relationship was observed between adiponectin or PPARγ2 expression and hepatic fat content, whereas, adiponectin expression was positively related to PPARγ2 expression. Independent of overall obesity, a reduced expression of adiponectin and PPARγ2 in the abdominal SAT is associated with high liver fat content, as well as with insulin resistance in obese adolescents.     

A dominant dwarf shrub increases diversity of herbaceous plant communities in a Trans-Himalayan rangeland

Plant communities are structured by both competition and facilitation. The interplay between the two interactions can vary depending on environmental factors, nature of stress, and plant traits. However, whether positive or negative interactions dominate in regions of high biotic and abiotic stress remains unclear. We studied herbaceous plant communities associated with a dwarf shrub Caragana versicolor in semi-arid, high altitude Trans-Himalayan rangelands of Spiti, India. We surveyed 120 pairs of plots (within and outside shrub canopies) across four watersheds differing in altitude, aspect, and dominant herbivores. Herbaceous communities within shrub canopies had 25% higher species richness, but similar abundance when compared to communities outside the canopy, with the shrub edge having higher diversity than the centre of the canopy. Grasses and erect forbs showed positive associations with the shrub, while prostrate plants occurred at much lower abundance within the canopy. Rare species showed stronger positive associations with Caragana than abundant species. Experimental removal of herbaceous vegetation from within shrub canopies led to 42% increase in flowering in Caragana, indicating a cost to the host shrubs. Our study indicates a robust pattern of a dwarf shrub facilitating local community diversity across this alpine landscape, increasing diversity at the plot level, facilitating rare species, and yet incurring a cost to hosts from the presence of herbaceous plants. Given these large influences of this shrub on the vegetation of these high altitude rangelands, we suggest that the shrub microhabitat be explicitly considered in any analyses of ecosystem health in such rangelands.     

Microdissection approaches in tuberculosis research

Mycobacterium tuberculosis, a globally significant pathogen, results in active or latent tuberculosis. The granuloma is the characteristic lesion that offers insight into host–pathogen interactions in these distinct states. Microdissection provides a way to isolate and consequently investigate specific tissue sections. We review various techniques available and in use.     

TRPM7 regulates gastrulation during vertebrate embryogenesis

During gastrulation, cells in the dorsal marginal zone polarize, elongate, align and intercalate to establish the physical body axis of the developing embryo. Here we demonstrate that the bifunctional channel-kinase TRPM7 is specifically required for vertebrate gastrulation. TRPM7 is temporally expressed maternally and throughout development, and is spatially enriched in tissues undergoing convergent extension during gastrulation. Functional studies reveal that TRPM7's ion channel, but not its kinase domain, specifically affects cell polarity and convergent extension movements during gastrulation, independent of mesodermal specification. During gastrulation, the non-canonical Wnt pathway via Dishevelled (Dvl) orchestrates the activities of the GTPases Rho and Rac to control convergent extension movements. We find that TRPM7 functions synergistically with non-canonical Wnt signaling to regulate Rac activity. The phenotype caused by depletion of the Ca²⁺- and Mg²⁺-permeant TRPM7 is suppressed by expression of a dominant negative form of Rac, as well as by Mg²⁺ supplementation or by expression of the Mg²⁺ transporter SLC41A2. Together, these studies demonstrate an essential role for the ion channel TRPM7 and Mg²⁺ in Rac-dependent polarized cell movements during vertebrate gastrulation.