Mycobacterium tuberculosis Response Regulators, DevR and NarL, Interact in Vivo and Co-regulate Gene Expression during Aerobic Nitrate Metabolism

Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M. tuberculosis NarL/NarS as a functional two-component system and identified His(241) and Asp(61) as conserved phosphorylation sites in NarS and NarL, respectively. Transcriptional profiling between M. tuberculosis H37Rv and Delta narL mutant strain during exponential growth in broth cultures with or without nitrate defined an similar to 30-gene NarL regulon that exhibited significant overlap with DevR-regulated genes, thereby implicating a role for the DevR response regulator in the regulation of nitrate metabolism. Notably, expression analysis of a subset of genes common to NarL and DevR regulons in M. tuberculosis Delta devR, Delta devS Delta dosT, and Delta narL mutant strains revealed that in response to nitrite produced during aerobic nitrate metabolism, the DevRS/DosT regulatory system plays a primary role that is augmented by NarL. Specifically, NarL itself was unable to bind to the narK2, acg, and Rv3130c promoters in phosphorylated or unphosphorylated form; however, its interaction with DevR similar to P resulted in cooperative binding, thereby enabling co-regulation of these genes. These findings support the role of physiologically derived nitrite as a metabolic signal in mycobacteria. We propose NarL-DevR binding, possibly as a heterodimer, as a novel mechanism for co-regulation of gene expression by the DevRS/DosT and NarL/NarS regulatory systems.

Phase stability of silver particles embedded calcium phosphate bioceramics

In this paper, we report the compositional variation-dependent phase stability of hydroxyapatite (Ca-10(PO4)(6)(OH)(2)) on doping with silver. The transformation of hydroxyapatite to (beta/alpha) tricalcium phosphate phases during sintering has been explored using Raman spectroscopy and X-ray diffraction techniques. The optical absorption spectroscopy analysis reveals the presence of Ag+ ions at low doping levels. As the doping increases, abundance of Ag particles is enhanced.

Calcium binding properties of calcium dependent protein kinase 1 (CaCDPK1) from Cicer arietinum

Calcium plays a crucial role as a secondary messenger in all aspects of plant growth, development and survival. Calcium dependent protein kinases (CDPKs) are the major calcium decoders, which couple the changes in calcium level to an appropriate physiological response. The mechanism by which calcium regulates CDPK protein is not well understood. In this study, we investigated the interactions of Ca2+ ions with the CDPK1 isoform of Cicer arietinum (CaCDPK1) using a combination of biophysical tools. CaCDPK1 has four different EF hands as predicted by protein sequence analysis. The fluorescence emission spectrum of CaCDPK1 showed quenching with a 5 nm red shift upon addition of calcium, indicating conformational changes in the tertiary structure. The plot of changes in intensity against calcium concentrations showed a biphasic curve with binding constants of 1.29 mu M and 120 mu M indicating two kinds of binding sites. Isothermal calorimetric (ITC) titration with CaCl2 also showed a biphasic curve with two binding constants of 0.027 mu M and 1.7 mu M. Circular dichroism (CD) spectra showed two prominent peaks at 208 and 222 nm indicating that CaCDPK1 is a alpha-helical rich protein. Calcium binding further increased the alpha-helical content of CaCDPK1 from 75 to 81%. Addition of calcium to CaCDPK1 also increased fluorescence of 8-anilinonaphthalene-1-sulfonic acid (ANS) indicating exposure of hydrophobic surfaces. Thus, on the whole this study provides evidence for calcium induced conformational changes, exposure of hydrophobic surfaces and heterogeneity of EF hands in CaCDPK1. (C) 2015 Elsevier GmbH. All rights reserved.

Linking carbon metabolism to carotenoid production in mycobacteria using Raman spectroscopy

Bacteria can utilize multiple sources of carbon for growth, and for pathogenic bacteria like Mycobacterium tuberculosis, this ability is crucial for survival within the host. In addition, phenotypic changes are seen in mycobacteria grown under different carbon sources. In this study, we use Raman spectroscopy to analyze the biochemical components present in M. smegmatis cells when grown in three differently metabolized carbon sources. Our results show that carotenoid biosynthesis is enhanced when M. smegmatis is grown in glucose compared to glycerol and acetate. We demonstrate that this difference is most likely due to transcriptional upregulation of the carotenoid biosynthesis operon (crt) mediated by higher levels of the stress-responsive sigma factor SigF. Moreover, we find that increased SigF and carotenoid levels correlate with greater resistance of glucose-grown cells to oxidative stress. Thus, we demonstrate the use of Raman spectroscopy in unraveling unknown aspects of mycobacterial physiology and describe a novel effect of carbon source variation on mycobacteria.

Twinning induced enhancement of fracture toughness in ultrafine grained Hydroxyapatite-Calcium Titanate composites

Despite being highly bioactive and biocompatible, the limitations of monolithic hydroxyapatite (HA) include extremely low fracture toughness, poor electrical conductivity. While addressing these issues, the present study demonstrates how CaTiO3 (CT) addition to HA can be utilized to obtain a combination of long crack fracture toughness (1.7 MPa m(1/2) SEVNB technique) and flexural strength of 98-155 MPa (3-point bending) and a moderate tensile strength (diametral compression) of 17-36 MPa. The enhancement in fracture resistance in spark plasma sintered HA-CT composites has been explained in reference to the observed twin morphology. TEM reveals the presence of twins in CT grains due to 1800 rotation about 101]. The measured properties along with our earlier reports on biocompatibility and electrical properties make HA-CT suitable for bone tissue engineering applications. When compared with other competing HA-based biocomposites, HA-CT composites are found to have a better combination of properties useful for medium load bearing implant applications. (C) 2015 Elsevier Ltd. All rights reserved.

First-in-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating M-tuberculosis

Development of effective therapies to eradicate persistent, slowly replicating M. tuberculosis (Mtb) represents a significant challenge to controlling the global TB epidemic. To develop such therapies, it is imperative to translate information from metabolome and proteome adaptations of persistent Mtb into the drug discovery screening platforms. To this end, reductive sulfur metabolism is genetically and pharmacologically implicated in survival, pathogenesis, and redox homeostasis of persistent Mtb. Therefore, inhibitors of this pathway are expected to serve as powerful tools in its preclinical and clinical validation as a therapeutic target for eradicating persisters. Here, we establish a first functional HTS platform for identification of APS reductase (APSR) inhibitors, a critical enzyme in the assimilation of sulfate for the biosynthesis of cysteine and other essential sulfur-containing molecules. Our HTS campaign involving 38?350 compounds led to the discovery of three distinct structural classes of APSR inhibitors. A class of bioactive compounds with known pharmacology displayed potent bactericidal activity in wild-type Mtb as well as MDR and XDR clinical isolates. Top compounds showed markedly diminished potency in a conditional Delta APSR mutant, which could be restored by complementation with Mtb APSR. Furthermore, ITC studies on representative compounds provided evidence for direct engagement of the APSR target. Finally, potent APSR inhibitors significantly decreased the cellular levels of key reduced sulfur-containing metabolites and also induced an oxidative shift in mycothiol redox potential of live Mtb, thus providing functional validation of our screening data. In summary, we have identified first-in-class inhibitors of APSR that can serve as molecular probes in unraveling the links between Mtb persistence, antibiotic tolerance, and sulfate assimilation, in addition to their potential therapeutic value.

Regional research priorities in brain and nervous system disorders

The characteristics of neurological, psychiatric, developmental and substance-use disorders in low-and middle-income countries are unique and the burden that they have will be different from country to country. Many of the differences are explained by the wide variation in population demographics and size, poverty, conflict, culture, land area and quality, and genetics. Neurological, psychiatric, developmental and substance-use disorders that result from, or are worsened by, a lack of adequate nutrition and infectious disease still afflict much of sub-Saharan Africa, although disorders related to increasing longevity, such as stroke, are on the rise. In the Middle East and North Africa, major depressive disorders and post-traumatic stress disorder are a primary concern because of the conflict-ridden environment. Consanguinity is a serious concern that leads to the high prevalence of recessive disorders in the Middle East and North Africa and possibly other regions. The burden of these disorders in Latin American and Asian countries largely surrounds stroke and vascular disease, dementia and lifestyle factors that are influenced by genetics. Although much knowledge has been gained over the past 10 years, the epidemiology of the conditions in low-and middle-income countries still needs more research. Prevention and treatments could be better informed with more longitudinal studies of risk factors. Challenges and opportunities for ameliorating nervous-system disorders can benefit from both local and regional research collaborations. The lack of resources and infrastructure for health-care and related research, both in terms of personnel and equipment, along with the stigma associated with the physical or behavioural manifestations of some disorders have hampered progress in understanding the disease burden and improving brain health. Individual countries, and regions within countries, have specific needs in terms of research priorities.

Regulation of Acetate Metabolism and Acetyl Co-a Synthetase 1 (ACS1) Expression by Methanol Expression Regulator 1 (Mxr1p) in the Methylotrophic Yeast Pichia pastoris

Methanol expression regulator 1 (Mxr1p) is a zinc finger protein that regulates the expression of genes encoding enzymes of the methanol utilization pathway in the methylotrophic yeast Pichia pastoris by binding to Mxr1p response elements (MXREs) present in their promoters. Here we demonstrate that Mxr1p is a key regulator of acetate metabolism as well. Mxr1p is cytosolic in cells cultured in minimal medium containing a yeast nitrogen base, ammonium sulfate, and acetate (YNBA) but localizes to the nucleus of cells cultured in YNBA supplemented with glutamate or casamino acids as well as nutrient-rich medium containing yeast extract, peptone, and acetate (YPA). Deletion of Mxr1 retards the growth of P. pastoris cultured in YNBA supplemented with casamino acids as well as YPA. Mxr1p is a key regulator of ACS1 encoding acetyl-CoA synthetase in cells cultured in YPA. A truncated Mxr1p comprising 400 N-terminal amino acids activates ACS1 expression and enhances growth, indicating a crucial role for the N-terminal activation domain during acetate metabolism. The serine 215 residue, which is known to regulate the expression of Mxr1p-activated genes in a carbon source-dependent manner, has no role in the Mxr1p-mediated activation of ACS1 expression. The ACS1 promoter contains an Mxr1p response unit (MxRU) comprising two MXREs separated by a 30-bp spacer. Mutations that abrogate MxRU function in vivo abolish Mxr1p binding to MxRU in vitro. Mxr1p-dependent activation of ACS1 expression is most efficient in cells cultured in YPA. The fact that MXREs are conserved in genes outside of the methanol utilization pathway suggests that Mxr1p may be a key regulator of multiple metabolic pathways in P. pastoris.

Dual role of arginine metabolism in establishing pathogenesis

Arginine is an integral part of host defense when invading pathogens are encountered. The arginine metabolite nitric oxide (NO) confers antimicrobial properties, whereas the metabolite ornithine is utilized for polyamine synthesis. Polyamines are crucial to tissue repair and anti-inflammatory responses. iNOS/arginase balance can determine Th1/Th2 response. Furthermore, the host arginine pool and its metabolites are utilized as energy sources by various pathogens. Apart from its role as an immune modulator, recent studies have also highlighted the therapeutic effects of arginine. This article sheds light upon the roles of arginine metabolism during pathological conditions and its therapeutic potential.

Structural analysis of dihydrofolate reductases enables rationalization of antifolate binding affinities and suggests repurposing possibilities

Antifolates are competitive inhibitors of dihydrofolate reductase ( DHFR), a conserved enzyme that is central to metabolism and widely targeted in pathogenic diseases, cancer and autoimmune disorders. Although most clinically used antifolates are known to be target specific, some display a fair degree of cross-reactivity with DHFRs from other species. A method that enables identification of determinants of affinity and specificity in target DHFRs from different species and provides guidelines for the design of antifolates is currently lacking. To address this, we first captured the potential druggable space of a DHFR in a substructure called the `supersite' and classified supersites of DHFRs from 56 species into 16 `site-types' based on pairwise structural similarity. Analysis of supersites across these site-types revealed that DHFRs exhibit varying extents of dissimilarity at structurally equivalent positions in and around the binding site. We were able to explain the pattern of affinities towards chemically diverse antifolates exhibited by DHFRs of different site-types based on these structural differences. We then generated an antifolate-DHFR network by mapping known high-affinity antifolates to their respective supersites and used this to identify antifolates that can be repurposed based on similarity between supersites or antifolates. Thus, we identified 177 human-specific and 458 pathogen-specific antifolates, a large number of which are supported by available experimental data. Thus, in the light of the clinical importance of DHFR, we present a novel approach to identifying differences in the druggable space of DHFRs that can be utilized for rational design of antifolates.

Maintaining local displacive disorders in Na0.5Bi0.5TiO3 piezoceramics by K0.5Bi0.5TiO3 substitution

A comprehensive global and local structural characterization using X-ray and neutron diffraction, and EXAFS have been performed to investigate the effect of electrical poling on the structure of (1-x)Na0.5Bi0.5TiO3-(x)K0.5Bi0.5TiO3 compositions. While the annealed samples showed definite indication of presence of local displacive disorders in all the compositions studied (0

Integration of holographic sensors into microfluidics for the real-time pH sensing of L. casei metabolism

We describe developments in the integration of analyte specific holographic sensors into PDMS-based microfluidic devices for the purpose of continuous, low-impact monitoring of extra-cellular change in micro-bioreactors. Holographic sensors respond to analyte concentration via volume change, which makes their reduction in size and integration into spatially confined fluidics difficult. Through design and process modification many of these constraints have been addressed, and a microfluidics-based device capable of real-time monitoring of the pH change caused by Lactobacillus casei fermentation is presented as a general proof-of-concept for a wide array of possible devices.

Regulation of Energy Metabolism by the Extracytoplasmic Function (ECF) σ Factors of Arcobacter butzleri

11 p.