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1.  A Novel F420-dependent Anti-oxidant Mechanism Protects Mycobacterium tuberculosis against Oxidative Stress and Bactericidal Agents 
Molecular microbiology  2012;87(4):744-755.
Summary
Mycobacterium tuberculosis (Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F420-dependent anti-oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F420-deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/NAD+ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F420− mutants are hypersensitive to mycobactericidal agents such as Isoniazid, Moxifloxacin and Clofazimine that elevate oxidative stress. The Mtb deazaflavin-dependent nitroreductase (Ddn) and its two homologues Rv1261c and Rv1558 encode for an F420H2 dependent quinone reductase (Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyze an F420H2 specific obligate two-electron reduction of endogenous quinones, thereby competing with the one-electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F420 biosynthesis pathway or Fqr-class proteins as a mechanism to potentiate the action of bactericidal agents.
doi:10.1111/mmi.12127
PMCID: PMC3567243  PMID: 23240649
F420; Mtb; Mycobacterium; TB; oxidative stress; quinone reductase
2.  Disaccharide Analysis of Glycosaminoglycans Using Hydrophilic Interaction Chromatography and Mass Spectrometry 
Analytical chemistry  2013;85(2):1138-1145.
Heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) participate in many important biological processes. Quantitative disaccharide analysis of HS and CS/DS is essential for the characterization of GAGs and enables modeling of the GAG domain structure. Methods involving enzymatic digestion and chemical depolymerization have been developed to determine the type and location of sulfation/acetylation modifications as well as uronic acid epimerization. Enzymatic digestion generates disaccharides with Δ-4,5-unsaturation at the non-reducing end. Chemical depolymerization with nitrous acid retains the uronic acid epimerization. This work shows the use of hydrophilic interaction liquid chromatography (HILIC)-MS for quantification of both enzyme-derived and nitrous acid depolymerization products for structural analysis of HS and CS/DS. This method enables biomedical researchers to determine complete disaccharide profiles on GAG samples using a single LC-MS platform.
doi:10.1021/ac3030448
PMCID: PMC3557806  PMID: 23234263
4.  Exploring the Mode of Action of Bioactive Compounds by Microfluidic Transcriptional Profiling in Mycobacteria 
PLoS ONE  2013;8(7):e69191.
Most candidate anti-bacterials are identified on the basis of their whole cell anti-bacterial activity. A critical bottleneck in the early discovery of novel anti-bacterials is tracking the structure activity relationship (SAR) of the novel compounds synthesized during the hit to lead and lead optimization stage. It is often very difficult for medicinal chemists to visualize if the novel compounds synthesized for understanding SAR of a particular scaffold have similar molecular mechanism of action (MoA) as that of the initial hit. The elucidation of the molecular MoA of bioactive inhibitors is critical. Here, a new strategy and routine assay for MoA de-convolution, using a microfluidic platform for transcriptional profiling of bacterial response to inhibitors with whole cell activity has been presented. First a reference transcriptome compendium of Mycobacterial response to various clinical and investigational drugs was built. Using feature reduction, it was demonstrated that subsets of biomarker genes representative of the whole genome are sufficient for MoA classification and deconvolution in a medium-throughput microfluidic format ultimately leading to a cost effective and rapid tool for routine antibacterial drug-discovery programs.
doi:10.1371/journal.pone.0069191
PMCID: PMC3729944  PMID: 23935951
5.  Variations of Subunit ε of the Mycobacterium tuberculosis F1Fo ATP Synthase and a Novel Model for Mechanism of Action of the Tuberculosis Drug TMC207 
The subunit ε of bacterial F1FO ATP synthases plays an important regulatory role in coupling and catalysis via conformational transitions of its C-terminal domain. Here we present the first low-resolution solution structure of ε of Mycobacterium tuberculosis (Mtε) F1FO ATP synthase and the nuclear magnetic resonance (NMR) structure of its C-terminal segment (Mtε103–120). Mtε is significantly shorter (61.6 Å) than forms of the subunit in other bacteria, reflecting a shorter C-terminal sequence, proposed to be important in coupling processes via the catalytic β subunit. The C-terminal segment displays an α-helical structure and a highly positive surface charge due to the presence of arginine residues. Using NMR spectroscopy, fluorescence spectroscopy, and mutagenesis, we demonstrate that the new tuberculosis (TB) drug candidate TMC207, proposed to bind to the proton translocating c-ring, also binds to Mtε. A model for the interaction of TMC207 with both ε and the c-ring is presented, suggesting that TMC207 forms a wedge between the two rotating subunits by interacting with the residues W15 and F50 of ε and the c-ring, respectively. T19 and R37 of ε provide the necessary polar interactions with the drug molecule. This new model of the mechanism of TMC207 provides the basis for the design of new drugs targeting the F1FO ATP synthase in M. tuberculosis.
doi:10.1128/AAC.01039-12
PMCID: PMC3535943  PMID: 23089752
6.  A peculiar liver with surgically and radiologically important variations: a case report 
Anatomy & Cell Biology  2013;46(1):82-84.
A peculiar liver was found in an adult male cadaver during a dissection class for undergraduate medical students. The quadrate lobe and fissure for the ligamentum teres were totally absent. Thus, the cystic notch on the inferior border was very broad and deep, and the fundus and body of the gall bladder popped out through this notch. The cystic duct terminated into the right hepatic duct at the porta hepatis instead of terminating into the common hepatic duct. Awareness of variations of the lobes and fissures may minimize a misdiagnosis of liver problems. The aim of the current study was to alert radiologists and surgeons about possible variations in the external appearance and anomalies of the lobes and fissures of the liver.
doi:10.5115/acb.2013.46.1.82
PMCID: PMC3615617  PMID: 23560240
Liver; Quadrate lobe; Ligamentum teres; Cystic duct
7.  Characterization of Phosphofructokinase Activity in Mycobacterium tuberculosis Reveals That a Functional Glycolytic Carbon Flow Is Necessary to Limit the Accumulation of Toxic Metabolic Intermediates under Hypoxia 
PLoS ONE  2013;8(2):e56037.
Metabolic versatility has been increasingly recognized as a major virulence mechanism that enables Mycobacterium tuberculosis to persist in many microenvironments encountered in its host. Glucose is one of the most abundant carbon sources that is exploited by many pathogenic bacteria in the human host. M. tuberculosis has an intact glycolytic pathway that is highly conserved in all clinical isolates sequenced to date suggesting that glucose may represent a non-negligible source of carbon and energy for this pathogen in vivo. Fructose-6-phosphate phosphorylation represents the key-committing step in glycolysis and is catalyzed by a phosphofructokinase (PFK) activity. Two genes, pfkA and pfkB have been annotated to encode putative PFK in M. tuberculosis. Here, we show that PFKA is the sole PFK enzyme in M. tuberculosis with no functional redundancy with PFKB. PFKA is required for growth on glucose as sole carbon source. In co-metabolism experiments, we report that disruption of the glycolytic pathway at the PFK step results in intracellular accumulation of sugar-phosphates that correlated with significant impairment of the cell viability. Concomitantly, we found that the presence of glucose is highly toxic for the long-term survival of hypoxic non-replicating mycobacteria, suggesting that accumulation of glucose-derived toxic metabolites does occur in the absence of sustained aerobic respiration. The culture medium traditionally used to study the physiology of hypoxic mycobacteria is supplemented with glucose. In this medium, M. tuberculosis can survive for only 7–10 days in a true non-replicating state before death is observed. By omitting glucose in the medium this period could be extended for up to at least 40 days without significant viability loss. Therefore, our study suggests that glycolysis leads to accumulation of glucose-derived toxic metabolites that limits long-term survival of hypoxic mycobacteria. Such toxic effect is exacerbated when the glycolytic pathway is disrupted at the PKF step.
doi:10.1371/journal.pone.0056037
PMCID: PMC3567006  PMID: 23409118
8.  InDiaMed: A Comprehensive Database of Indian Medicinal plants for Diabetes 
Bioinformation  2013;9(7):378-380.
According to International Diabetes Federation (IDF), India has 62.4 million people with diabetes and by 2030 it is predicted that the number will rise to 100 million. Studies claim that there are around 410 experimentally proven Indian medicinal plants which have anti-diabetic activity, of which the mechanism of action of 109 plants has been elucidated or reported. So, the need of the hour is to explore the claims of Indian medicinal flora and open up the facets of many Indian plants which are being examined for their beneficial role in diabetes. So, we created a database (InDiaMed) of Indian medicinal plants that captures their role in anti-diabetic activity. InDiaMed's features include chemical, pharmacological, biochemical and geographical information of the medicinal plant, scientifically relevant information of the plant, and the coherent research done on it in the field of diabetes. The database also includes the list of poly-herbal formulations which are used for treatment of diabetes in India.
Availability
http://www.indiamed.info
doi:10.6026/97320630009378
PMCID: PMC3669792  PMID: 23750084
Diabetes; Database; Medicinal Plants; Poly-herbal formulations; active constituents
9.  Cardioprotective activity of chalcones in ischemia/reperfusion-induced myocardial infarction in albino rats 
BACKGROUND:
There is a comprehensive body of experimental and clinical evidence suggesting that exogenous supplementation of natural antioxidants or augmentation of endogenous antioxidants attenuates the damage caused by myocardial infarction.
OBJECTIVE:
To evaluate the cardioprotective effects of Cl-chalcone and F-chalcone against ischemia/reperfusion (I/R)-induced myocardial infarction in rats.
METHODS:
Myocardial infarct size was measured using the staining agent 2,3,5-triphenyltetrazolium chloride. Malondialdehyde was measured in serum and heart tissue, and superoxide dismutase and catalase in heart tissue were measured spectrophotometrically.
RESULTS:
I/R resulted in significant cardiac necrosis, indicated by a rise in the end products of myocardial lipid peroxidation (malondialdehydes). A loss of antioxidative enzymes (superoxide dismutase and catalase) in heart tissue was also observed in animals subjected to in vivo myocardial I/R injury.
DISCUSSION:
The present study demonstrated that treatment with Cl-chalcone and F-chalcone significantly limited infarct size, partially but significantly attenuated the level of lipid peroxidation and moderated the loss of antioxidant reserves in rats subjected to 30 min coronary artery occlusion followed by a 4 h reperfusion in comparison with I/R groups.
CONCLUSIONS:
The results of the present study suggest that chalcones have cardioprotective activity against I/R-induced myocardial infarction in rats.
PMCID: PMC3628422  PMID: 23620697
Chalcones; Cl-chalcone; F-chalcone; Infarct size; Myocardial infarction; Reperfusion injury
10.  Evaluation on the Corrosion of the Three Ni-Cr Alloys with Different Composition 
Dental casting alloys are widely used in contact with oral tissue for many years now. With the development of new dental alloys over the past 15 years, many questions remain unanswered about their biologic safety. Concepts and current issues concerning the response to the biologic effects of dental casting alloys are presented. In this paper, samples of three commercially available nickel-chrome (Ni-cr) casting alloys (Dentaurum, Bego, Sankin) were taken to assess their corrosion behavior, using potentiodynamic polarization method (electrochemical method) with fusayama artificial saliva as an electrolyte medium to check for their biocompatibility. The parameters for corrosion rate and corrosion resistance were obtained from computer-controlled corrosion schematic instrument, namely, potentiostat through corrosion software (power CV). The results obtained were analyzed by classic Tafel analysis. Statistical analysis was done by Student's t-test and ANOVA test. It was concluded that Dentarum and Bego showed satisfactory corrosive behavior, with exception of Sankin which depicted higher corrosion rate and least resistance to corrosion. Thus, the selection of an alloy should be made on the basis of corrosion resistance and biologic data from dental manufactures.
doi:10.1155/2011/397029
PMCID: PMC3062075  PMID: 21461232
11.  2-Amino­anilinium 2-chloro­acetate 
In the crystal structure of the title compound, C6H9N2 +·ClCH2COO−, prepared by the reaction of OPDA (orthophenelynediamine) with chloro­acetic ­acid, N—H⋯O hydrogen bonds generate ladder-like chains and very weak inter­molecular C—H⋯Cl hydrogen-bonding inter­actions between the anions and cations lead to a supra­molecular network. C—H⋯O inter­actions also occur.
doi:10.1107/S1600536810024554
PMCID: PMC3007232  PMID: 21588271
13.  Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9 
Brazilian Journal of Microbiology  2009;40(2):358-366.
An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16) fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg2+, Zn2+, Cu2+, Ag2+ and Fe2+ decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/min respectively.
doi:10.1590/S1517-838220090002000028
PMCID: PMC3769737  PMID: 24031373
Pseudomonas aeruginosa SRT9; extra cellular lipases; purification; Michaelis constant
14.  Medial swivel dislocation of the talonavicular joint 
Indian Journal of Orthopaedics  2009;43(1):87-89.
Medial swivel dislocation, a variant of subtalar dislocation is uncommon. A 35 years old male presented after 6 weeks old injury to left ankle following motor cycle accident. He had pain, swelling around ankle and was unable to bear weight on left foot. Clinical examination revealed diffuse swelling and tenderness in mid foot region. His plain X rays and CT scan showed talonavicular dislocation with compression defect of the head of the talus. He was treated by open reduction and K-wire fixation. At 32 months follow up foot was painless, stable with normal range of ankle and subtalar motion.
doi:10.4103/0019-5413.45329
PMCID: PMC2739503  PMID: 19753187
Medial swivel dislocation; subtalar-subluxation; talonavicular dislocation
15.  A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy 
Nature Communications  2010;1(5):1-8.
Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine–imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.
Candidate anti-tuberculosis drugs are often identified in whole-cell screens. Here, Pethe et al. show that inappropriate carbon-source selection can lead to the identification of compounds devoid of efficacy in vivo, underlining the importance of developing predictive in vitro screens.
doi:10.1038/ncomms1060
PMCID: PMC3220188  PMID: 20975714
16.  A High-Throughput Screen To Identify Inhibitors of ATP Homeostasis in Non-replicating Mycobacterium tuberculosis 
ACS Chemical Biology  2012;7(7):1190-1197.
Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.
doi:10.1021/cb2004884
PMCID: PMC3401038  PMID: 22500615
17.  A novel F420-dependent anti-oxidant mechanism protects Mycobacterium tuberculosis against oxidative stress and bactericidal agents 
Molecular Microbiology  2012;87(4):744-755.
Mycobacterium tuberculosis (Mtb) is an aerobic bacterium that persists intracellularly in host macrophages and has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F420-dependent anti-oxidant mechanism that protects Mtb against oxidative stress. Inactivation of the fbiC gene in Mtb results in a cofactor F420-deficient mutant that is hypersensitive to oxidative stress and exhibits a reduction in NADH/NAD+ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being an important component of the pathway resulting in cell death by bactericidal agents, F420− mutants are hypersensitive to mycobactericidal agents such as isoniazid, moxifloxacin and clofazimine that elevate oxidative stress. The Mtb deazaflavin-dependent nitroreductase (Ddn) and its two homologues Rv1261c and Rv1558 encode for an F420H2-dependent quinone reductase (Fqr) function leading to dihydroquinones. We hypothesize that Fqr proteins catalyse an F420H2-specific obligate two-electron reduction of endogenous quinones, thereby competing with the one-electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones, thus protecting mycobacteria against oxidative stress and bactericidal agents. These findings open up an avenue for the inhibition of the F420 biosynthesis pathway or Fqr-class proteins as a mechanism to potentiate the action of bactericidal agents.
doi:10.1111/mmi.12127
PMCID: PMC3567243  PMID: 23240649

Results 1-17 (17)