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.     

Changes in species composition,diversity and biomass of herbaceous plant traits due to N amendment in a dry tropical environment of India

Aim European and North American studies have suggested that nitrogen (N) depositions reduce plant diversity and increase primary productivity due to changes in plant traits. To predict the vegetation response to future global change, experimental validations from other regions are widely needed. We assessed the effects of N treatment by urea fertilization on the diversity and biomass of the herbaceous plant traits (HPTs) in a dry tropical environment of India.Methods Diversity and biomass of different HPTs were determined on the basis of data collected in year 2010, from 135, 1 m × 1 m plots distributed over 15 locations. The plots were treated with urea fertilizer in different doses (Control, 60kgNha^-1 yr^-1 and 120kg N ha^-1 yr^-1) since 1st January 2007. The plots were ordinated and data were subjected to appropriate statistical analyses.Important findings Correspondence analysis (CA) suggested uniqueness of species composition due to N amendment. Species number and biomass of the trait categories varied due to N fertilization and traits. All studied trait categories (except N-fixers) yielded maximum mean species number at moderate level of N fertilization. Different levels of N fertilization exhibited different species diversity–primary productivity (D-P) relationships. Further, study showed reduction in plant diversity due to increase in biomass at high rates of N addition.Conclusions Tall, erect, non N-fixers, annuals, grasses HPTs were favoured by N enrichment. N dose above 60kg enhanced the biomass of fast growing, erect, annuals, non N-fixers, nitrophilic HPTs. The changes in traits with N addition, especially the increase in annuals and grasses and decrease in typically N-rich N-fixers, have implications for sustainable cattle production.     

Extraction of rapamycin (sirolimus) from Streptomyces rapamycinicus using ultrasound

The study was designed to investigate the use of ultrasound-assisted extraction (UAE) of rapamycin (sirolimus) from bacterial strain of Streptomyces rapamycinicus NRRL 5491. To achieve the maximum extraction yield, various parameters were optimized which include S. rapamycinicus (10?g) of biomass in toluene (50?mL), temperature (20°C), acoustic intensity (35.67?W/cm²), and duty cycle (40%) for 4?min extraction time with probe tip length of 0.5?cm dipped into extraction solvent from the surface. The maximum extraction yield 60.15?±?0.01?mg/L was attained under the mentioned optimum parameters. The use of ultrasound for the extraction of rapamycin shows about twofold increase in the yield as compared to the conventional solid–liquid extraction (29.7?±?0.2?mg/L). The study provides the effective UAE technique to produce potential value-added products.     

Isolation of indigenous Staphylococcus sciuri from chromium-contaminated paddy field and its application for reduction of Cr(VI) in rice plants cultivated in pots

Accumulation of Cr(VI) in rice seeds cultivated in Cr-contaminated soil of the Sundarbans (India) is an environmental problem. Cr(VI) concentration in this soil was 6.2 ± 0.3 mg/kg, whereas total chromium was 32.04 ± 1.60 mg/kg. A Cr(VI)-removing bacterium isolated from Cr-contaminated paddy field soil of Sundarbans was identified as Staphylococcus sciuri. Enrichment culture of S. sciuri was applied to pot cultivation of rice in Cr-contaminated soil. After 8 weeks, 71 ± 3% Cr(VI) (final concentration 2.15 ± 0.01 mg/kg) and 65 ± 2% total Cr removal (end concentration 11.3 ± 0.5 mg/kg) were attained in bacterium-treated soils. Growth parameters indicated healthy development of plants cultivated in bacterium-treated soils that was not observed in control plants. Total Cr removal attained in rice seeds of plants cultivated in bacterium-treated soils compared with control rice seeds was 78 ± 4%. Total Cr concentration in test seeds was 0.72 ± 0.05 mg/kg (World Health Organization [WHO] permissible limit: 1.30 mg/kg), whereas the same in control seeds was 3.27 ± 0.16 mg/kg. Cr(VI) reduction achieved in rice seeds cultivated in bacterium-treated soil compared with control rice seeds was 95 ± 5%. Cr(VI) concentration in rice seeds cultivated in treated soil was 0.050 ± 0.003 mg/kg, whereas the same in untreated control was 0.93 ± 0.05 mg/kg. Successful paddy field soil bioremediation by any Staphylococcus species was demonstrated for the first time.     

Intelligent image analysis (IIA) using artificial neural network (ANN) for non-invasive estimation of chlorophyll content in micropropagated plants of potato

An artificial neural network (ANN) was used to analyze photometric features extracted from the digitized images of leaves from in vitro-regenerated potato plants for non-invasive estimation of chlorophyll content. A MATLAB®-based, feed-forward, backpropagation-type network was developed for an input layer (three input elements), with one hidden layer (one node) and one output layer representing the predicted chlorophyll content. A significant influence of training function during optimization of ANN modeling was observed. Among the 11 training functions tested, “trainlm” was found to be the best on the basis of comparative analysis of root-mean-square error (RMSE) at zero epoch. A significant correlation between the model-predicted and Soil-Plant Analysis Development (SPAD) meter-measured relative chlorophyll contents was obtained when the mean brightness ratio (rgb) parameters were used. Compared to a red (R), green (G), and blue (B) color space model, the rgb model exhibited better performance with a significant correlation (R ² = 0.85). Incorporation of photometric features, such as luminosity (L), blue (B)/L, and green (G)/L, with rgb failed to improve the performance of the network. The developed Intelligent image analysis (IIA) system was able to estimate in real time the chlorophyll content of in vitro-regenerated leaves for assessment of plant nutrient status during micropropagation.     

Role of pH on dimeric interactions for DENV envelope protein: an insight from molecular dynamics study

The entry of dengue viruses is mediated by pH triggering in the host cells. Here we have studied the DENV E protein stability and binding of its units at low and normal pH using MD and MM-PB/SA method for the first time. To investigate the role of pH in dissociation of dimeric protein, we have performed a concise study of hydrogen bonding and other interactions between units of dimer at low and normal pH. The Generalized Born calculation connotes that dimeric unit was relatively less stable and less proned for dimerisation at low pH. Our results provide a theoretical verification for previous assumptions of pH triggering mechanism of dengue envelope protein. During the pH alteration, we found a large decrement in salt bridges which were observed at normal pH. We also compared the flexibility of each unit and found that they exhibit different fluctuations during molecular dynamics simulations.     

Minireview: structural insights into early folding events using continuous-flow time-resolved small-angle X-ray scattering

Small-angle X-ray scattering (SAXS) is a powerful method for obtaining quantitative structural information on the size and shape of proteins, and it is increasingly used in kinetic studies of folding and association reactions. In this minireview, we discuss recent developments in using SAXS to obtain structural information on the unfolded ensemble and early folding intermediates of proteins using continuous-flow mixing devices. Interfacing of these micromachined devices to SAXS beamlines has allowed access to the microsecond time regime. The experimental constraints in implementation of turbulence and laminar flow-based mixers with SAXS detection and a comparison of the two approaches are presented. Current improvements and future prospects of microsecond time-resolved SAXS and the synergy with ab initio structure prediction and molecular dynamics simulations are discussed.