A user-friendly web portal for analyzing conformational changes in structures of Mycobacterium tuberculosis

Repair of damaged or incorrectly matched DNA is essential to the survival of all organisms. Consequently cells have devised a plentitude of pathways for repair. We have been investigating the mechanisms of mismatch repair and base excision repair. Both of these repair processes involve a large number of proteins that interact with one another as well as with DNA. Our long-term goal is to assemble complexes that are fully functional for DNA repair and to image the process of DNA repair. In addition, we wish to i) determine the stoicheometry of binding of the protein complexes to each other and to DNA, ii) monitor conformational changes due to substrate binding, iii) measure physical properties of DNA and the complexes. To accomplish this end, we have endeavored to improve techniques for solution imaging as well as those for data analysis. In this presentation I will discuss data on the stoicheometry of binding in several protein complexes and data on the physical properties of DNA.To measure the physical properties of DNA, we utilize a nanoManipulator, a modified Scanning Force Microscope with a novel, user-friendly interface that allows easy and controlled manipulation of nanometer-sized samples.

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Microscopic Observation Drug Susceptibility (MODS) Assay: A Convenient Method for Determining Antibiogram of Clinical Isolates of Mycobacterium tuberculosis in Ghana

Medical Sciences ◽

10.3390/medsci8010005 ◽

2020 ◽

Vol 8 (1) ◽

pp. 5

Author(s):

Enid Owusu ◽

Mercy Jemima Newman

Keyword(s):

Mycobacterium Tuberculosis ◽

Rural Areas ◽

Microscopic Observation ◽

Drug Susceptibility ◽

Ease Of Use ◽

Health Centers ◽

Culture Methods ◽

Susceptibility Tests ◽

User Friendly

(1) Background: Present methods for drug susceptibility tests (DST) rely on culture methods that are sophisticated and relatively faster, or a slow and cheaper option. These methods frustrate disease control; therefore, there is a need for methods that incorporate key functions of microscopy and culture, with reduced cost burden and sophistry. Thus, the purpose of this study was to identify which, among the most commonly used (in Ghana) methods, can conveniently be used at health centers located in rural areas for effective DST determination of Mycobacterium tuberculosis (MTB). (2) Methods: Mycobacterium tuberculosis isolates were tested for their susceptibility to streptomycin, isoniazid, rifampicin, ethambutol (SIRE), and pyrazinamide by microscopic observation drug susceptibility (MODS) and BACTEC MGIT 960 methods. Evaluations were based on shorter turnaround periods, rapidity, ease of use, cost, etc. A comparative analysis was statistically expressed as kappa values. (3) Results: Endpoints for drug susceptibilities by MODS averaged 13 days (7–32), whilst that for BACTEC MGIT 960 was 10 days with a further 12 days to detect resistance. Therefore, a turnaround period of 22 days was needed for DST by BACTEC MGIT 960, compared to 13 days for MODS. There were differences in correlation levels between the two methods, as determined by their kappa values. (4) Conclusion: The MODS assay was found to be less costly, more user-friendly, and still able to be conveniently used at health centers located in rural areas known to be endemic for TB, particularly in Ghana.

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Characterization of the Mycobacterium tuberculosis H37Rv alkyl hydroperoxidase AhpC points to the importance of ionic interactions in oligomerization and activity

Biochemical Journal ◽

10.1042/bj3540209 ◽

2001 ◽

Vol 354 (1) ◽

pp. 209-215 ◽

Cited By ~ 17

Author(s):

Radha CHAUHAN ◽

Shekhar C. MANDE

Keyword(s):

Mycobacterium Tuberculosis ◽

Conformational Changes ◽

Three Dimensional ◽

Enteric Bacteria ◽

Enzymic Activity ◽

Ionic Interactions ◽

Three Dimensional Model ◽

Mycobacterium Tuberculosis H37rv ◽

Resistant Strains ◽

Alkyl Hydroperoxidase

An alkyl hydroperoxidase (AhpC) has been found frequently to be overexpressed in isoniazid-resistant strains of Mycobacterium tuberculosis. These strains have an inactivated katG gene encoding a catalase peroxidase, which might render mycobacteria susceptible to the toxic peroxide radicals, thus leading to the concomitant overexpression of the AhpC. Although the overexpressed AhpC in isoniazid-resistant strains of M. tuberculosis may not directly participate in isoniazid action, AhpC might still assist M. tuberculosis in combating oxidative damage in the absence of the catalase. Here we have attempted to characterize the AhpC protein biochemically and report its functional and oligomerization properties. The alkyl hydroperoxidase of M. tuberculosis is unique in many ways compared with its well-characterized homologues from enteric bacteria. We show that AhpC is a decameric protein, composed of five identical dimers held together by ionic interactions. Dimerization of individual subunits takes place through an intersubunit disulphide linkage. The ionic interactions play a significant role in enzymic activity of the AhpC protein. The UV absorption spectrum and three-dimensional model of AhpC suggest that interesting conformational changes may take place during oxidation and reduction of the intersubunit disulphide linkage. In the absence of the partner AhpF subunit in M. tuberculosis, the mycobacterial AhpC might use small-molecule reagents, such as mycothiol, for completing its enzymic cycle.

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Epidemiology of Rifampicin Resistant Tuberculosis and Common Mutations in rpoB Gene of Mycobacterium tuberculosis: A Retrospective Study from Six Districts of Punjab (India) Using Xpert MTB/RIF Assay

Journal of Laboratory Physicians ◽

10.4103/0974-2727.180789 ◽

2016 ◽

Vol 8 (02) ◽

pp. 096-100 ◽

Cited By ~ 4

Author(s):

Ramandeep Kaur ◽

Neerja Jindal ◽

Shilpa Arora ◽

Shajla Kataria

Keyword(s):

Mycobacterium Tuberculosis ◽

Surrogate Marker ◽

Rpob Gene ◽

Multidrug Resistant ◽

Resistance Pattern ◽

Resistance Mutations ◽

Resistant Tuberculosis ◽

Mdr Tb ◽

Previously Treated Patients ◽

User Friendly

ABSTRACT Background: Xpert MTB/RIF assay has revolutionized the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (RIF), a surrogate marker for multidrug-resistant TB (MDR-TB) in <2 h. The RIF resistance pattern in Malwa region of Punjab, India, is not documented. Here, we report the epidemiology of RIF-resistant TB and mutations in rpoB gene of Mycobacterium tuberculosis (MTB). Materials and Methods: A total of 1612 specimens received between October 2013 and February 2015 were tested by Xpert MTB/RIF assay following manufacturer’s instructions. The results thus obtained were analyzed using SPSS version 20.0.0 (SPSS Inc., Chicago, IL, USA) statistical software. Result: RIF resistance was statistically higher in previously treated patients in comparison to the new patients (P = 0.006) and in patients with acid fast-Bacilli (AFB) positive smears to AFB-negative smears (P = 0.048). RIF resistance mutations in 130 specimens revealed frequency of E 73/130 (56%), B 28/130 (21.5%), D 18/130 (13.8%), A 11/130 (8.4%), and C 1/130 (0.7%) while in one specimen, mutation combination, i.e., mutations associated with more than one probe (A and B both) was present. Conclusion: Xpert MTB/RIF assay is a user-friendly screening tool for detection of MTB and RIF resistance from suspected TB/MDR cases in a shorter period of time. It could also serve as a useful technique to have simultaneous preliminary information regarding the mutation pattern of RIF resistance in MTB isolates.

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Lateral Flow Immunoassay for Naked Eye Detection of Mycobacterium tuberculosis

Journal of Sensors ◽

10.1155/2020/1365983 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Nazifah Ariffin ◽

Nor Azah Yusof ◽

Jaafar Abdullah ◽

Siti Fatimah Abd Rahman ◽

Nurul Hanun Ahmad Raston ◽

...

Keyword(s):

Developing Countries ◽

Mycobacterium Tuberculosis ◽

Point Of Care ◽

Colour Change ◽

Lateral Flow ◽

Lateral Flow Immunoassay ◽

Optical Signals ◽

Naked Eye ◽

User Friendly ◽

And Control

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. Detection and control of infectious diseases is a major problem, especially in developing countries. Lateral flow immunoassay (LFIA) has been introduced as a handheld immunoassay-based point-of-care platform for an automated detection of TB. The CFP10-ESAT6 antigen of M. tuberculosis was used as the target in early detection of TB using LFIA strip-based POC strategy. An interesting platform based on optical signals is implemented as a colour change in the detection area that is visible to the naked eye. The gold nanoparticles (AuNPs) were used as the colour probe for the detection of a target of interest. The high-resolution transmission electron microscopy (HRTEM) image and ultraviolet-visible spectrophotometer (UV-Vis) analysis confirmed that the synthesized AuNPs were appropriate for the immunoassay designed. The platform consists of AuNPs conjugated with specific antibodies (Ab) to capture the antigen of M. tuberculosis. Under the capillary effect, sandwich immunoreactions of AuNP-Ab-antigen were performed on the test pad of the immunostrip, which can be observed by the colour signal on the test line of the strip with a short assay time. Furthermore, the newly developed biosensor was utilized in CFP10-ESAT6 antigen detection in human sputum specimens with satisfactory results. The characteristic coloured bands enable visual detection (naked eye) of target analyte without instrumentation. This noninvasive diagnose system which is sputum-based detection could provide user-friendly and affordable diagnostic tests in developing countries.

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Strategic Improvements for Gross Anatomy Web-Based Teaching

Anatomy Research International ◽

10.1155/2012/146262 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

David R. Marker ◽

Krishna Juluru ◽

Chris Long ◽

Donna Magid

Keyword(s):

Web Site ◽

Gross Anatomy ◽

Web Portal ◽

High Expectations ◽

Student Opinions ◽

Radiology Teaching Files ◽

Teaching Files ◽

Radiology Teaching ◽

User Friendly ◽

The Web

Current generations of graduate students have been immersed in technology from their early school years and have high expectations regarding digital resources. To better meet the expectations of Gross Anatomy students at our institution, electronic radiology teaching files for first-year coursework were organized into a web site. The web site was custom designed to provide material that directly correlated to the Gross Anatomy dissection and lectures. Quick links provided sets of images grouped by anatomic location. Additionally, Lab and Study Companions provided specific material for the students to review prior to and after lectures and gross dissections. Student opinions of this education resource were compared to student opinions of the prior year’s digital teaching files. The new content was ranked as more user friendly (3.1 points versus 2.3 points) and more useful for learning anatomy (3.3 points versus 2.6 points). Many students reported that using the web portal was critical in helping them to better understand relationships of anatomical structures. These findings suggest that a well-organized web portal can provide a user-friendly, valuable educational resource for medical students who are studying Gross Anatomy.

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The importance of the quaternary structure to represent conformational ensembles of the major Mycobacterium tuberculosis drug target

Scientific Reports ◽

10.1038/s41598-019-50213-0 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Renata Fioravanti Tarabini ◽

Luís Fernando Saraiva Macedo Timmers ◽

Carlos Eduardo Sequeiros-Borja ◽

Osmar Norberto de Souza

Keyword(s):

Mycobacterium Tuberculosis ◽

Conformational Changes ◽

Protein Function ◽

Environmental Changes ◽

Quaternary Structure ◽

Single Point ◽

Point Mutations ◽

Conformational Ensembles ◽

Dynamics Simulations ◽

Coa Reductase

Abstract Flexibility is a feature intimately related to protein function, since conformational changes can be used to describe environmental changes, chemical modifications, protein-protein and protein-ligand interactions. In this study, we have investigated the influence of the quaternary structure of 2-trans-enoyl-ACP (CoA) reductase or InhA, from Mycobacterium tuberculosis, to its flexibility. We carried out classical molecular dynamics simulations using monomeric and tetrameric forms to elucidate the enzyme’s flexibility. Overall, we observed statistically significant differences between conformational ensembles of tertiary and quaternary structures. In addition, the enzyme’s binding site is the most affected region, reinforcing the importance of the quaternary structure to evaluate the binding affinity of small molecules, as well as the effect of single point mutations to InhA protein dynamics.

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Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

Journal of Biological Chemistry ◽

10.1074/jbc.m110.211649 ◽

2011 ◽

Vol 286 (18) ◽

pp. 16197-16207 ◽

Cited By ~ 26

Author(s):

Sebastian Reichau ◽

Wanting Jiao ◽

Scott R. Walker ◽

Richard D. Hutton ◽

Edward N. Baker ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Conformational Changes ◽

Active Site ◽

Catalytic Mechanism ◽

Condensation Reaction ◽

Shikimate Pathway ◽

Enzyme Reaction ◽

Multidrug Resistant ◽

Active Site Residues ◽

Site Water

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.

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