Effectiveness of real-time polymerase chain reaction assay for the detection of Mycobacterium tuberculosis in pathological samples: a systematic review and meta-analysis.

BACKGROUND: Rapid and accurate diagnosis of tuberculosis (TB) is key to manage the disease and to control and prevent its transmission. Many established diagnostic methods suffer from low sensitivity or delay of timely results and are inadequate for rapid detection of Mycobacterium tuberculosis (MTB) in pulmonary and extra-pulmonary clinical samples. This study examined whether a real-time polymerase chain reaction (RT-PCR) assay, with a turn-a-round time of 2 h, would prove effective for routine detection of MTB by clinical microbiology laboratories. METHODS: A systematic literature search was performed for publications in any language on the detection of MTB in pathological samples by RT-PCR assay. The following sources were used MEDLINE via PubMed, EMBASE, BIOSIS Citation Index, Web of Science, SCOPUS, ISI Web of Knowledge and Cochrane Infectious Diseases Group Specialised Register, grey literature, World Health Organization and Centres for Disease Control and Prevention websites. Forty-six studies met set inclusion criteria. Generated pooled summary estimates (95% CIs) were calculated for overall accuracy and bivariate meta-regression model was used for meta-analysis. RESULTS: Summary estimates for pulmonary TB (31 studies) were as follows: sensitivity 0.82 (95% CI 0.81-0.83), specificity 0.99 (95% CI 0.99-0.99), positive likelihood ratio 43.00 (28.23-64.81), negative likelihood ratio 0.16 (0.12-0.20), diagnostic odds ratio 324.26 (95% CI 189.08-556.09) and area under curve 0.99. Summary estimates for extra-pulmonary TB (25 studies) were as follows: sensitivity 0.70 (95% CI 0.67-0.72), specificity 0.99 (95% CI 0.99-0.99), positive likelihood ratio 29.82 (17.86-49.78), negative likelihood ratio 0.33 (0.26-0.42), diagnostic odds ratio 125.20 (95% CI 65.75-238.36) and area under curve 0.96. CONCLUSIONS: RT-PCR assay demonstrated a high degree of sensitivity for pulmonary TB and good sensitivity for extra-pulmonary TB. It indicated a high degree of specificity for ruling in TB infection from sampling regimes. This was acceptable, but may better as a rule out add-on diagnostic test. RT-PCR assays demonstrate both a high degree of sensitivity in pulmonary samples and rapidity of detection of TB which is an important factor in achieving effective global control and for patient management in terms of initiating early and appropriate anti-tubercular therapy. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42015027534 .

Conformation and Dynamics of Human Urotensin II and Urotensin Related Peptide in Aqueous Solution.

Conformation and dynamics of the vasoconstrictive peptides human urotensin II (UII) and urotensin related peptide (URP) have been investigated by both unrestrained and enhanced-sampling molecular-dynamics (MD) simulations and NMR spectroscopy. These peptides are natural ligands of the G-protein coupled urotensin II receptor (UTR) and have been linked to mammalian pathophysiology. UII and URP cannot be characterized by a single structure but exist as an equilibrium of two main classes of ring conformations, open and folded, with rapidly interchanging subtypes. The open states are characterized by turns of various types centered at K8Y9 or F6W7 predominantly with no or only sparsely populated transannular hydrogen bonds. The folded conformations show multiple turns stabilized by highly populated transannular hydrogen bonds comprising centers F6W7K8 or W7K8Y9. Some of these conformations have not been characterized previously. The equilibrium populations that are experimentally difficult to access were estimated by replica-exchange MD simulations and validated by comparison of experimental NMR data with chemical shifts calculated with density-functional theory. UII exhibits approximately 72% open:28% folded conformations in aqueous solution. URP shows very similar ring conformations as UII but differs in an open:folded equilibrium shifted further toward open conformations (86:14) possibly arising from the absence of folded N-terminal tail-ring interaction. The results suggest that the different biological effects of UII and URP are not caused by differences in ring conformations but rather by different interactions with UTR.

Can Simulations and Modeling Decipher NMR Data for Conformational Equilibria? Arginine-Vasopressin.

Arginine vasopressin (AVP) has been suggested by molecular-dynamics (MD) simulations to exist as a mixture of conformations in solution. The (1)H and (13)C NMR chemical shifts of AVP in solution have been calculated for this conformational ensemble of ring conformations (identified from a 23 µs molecular-dynamics simulation). The relative free energies of these conformations were calculated using classical metadynamics simulations in explicit water. Chemical shifts for representative conformations were calculated using density-functional theory. Comparison with experiment and analysis of the results suggests that the (1)H chemical shifts are most useful for assigning equilibrium concentrations of the conformations in this case. (13)C chemical shifts distinguish less clearly between conformations, and the distances calculated from the nuclear Overhauser effect do not allow the conformations to be assigned clearly. The (1)H chemical shifts can be reproduced with a standard error of less than 0.24 ppm (<2.2 ppm for (13)C). The combined experimental and theoretical results suggest that AVP exists in an equilibrium of approximately 70% saddlelike and 30% clinched open conformations. Both newly introduced statistical metrics designed to judge the significance of the results and Smith and Goodman's DP4 probabilities are presented.

A Three-Site Mechanism for Agonist/Antagonist Selective Binding to Vasopressin Receptors.

Molecular-dynamics simulations with metadynamics enhanced sampling reveal three distinct binding sites for arginine vasopressin (AVP) within its V2 -receptor (V2 R). Two of these, the vestibule and intermediate sites, block (antagonize) the receptor, and the third is the orthosteric activation (agonist) site. The contacts found for the orthosteric site satisfy all the requirements deduced from mutagenesis experiments. Metadynamics simulations for V2 R and its V1a R-analog give an excellent correlation with experimental binding free energies by assuming that the most stable binding site in the simulations corresponds to the experimental binding free energy in each case. The resulting three-site mechanism separates agonists from antagonists and explains subtype selectivity.

In Silico Adoption of an Orphan Nuclear Receptor NR4A1.

A 4.1 µs molecular dynamics simulation of the NR4A1 (hNur77) apo-protein has been undertaken and a previously undetected druggable pocket has become apparent that is located remotely from the 'traditional' nuclear receptor ligand-binding site. A NR4A1/bis-indole ligand complex at this novel site has been found to be stable over 1 µs of simulation and to result in an interesting conformational transmission to a remote loop that has the capacity to communicate with a NBRE within a RXR-α/NR4A1 heterodimer. Several features of the simulations undertaken indicate how NR4A1 can be affected by alternate-site modulators.

Conformation and dynamics of 8-Arg-vasopressin in solution.

Arginine-vasopressin was subjected to a long (11 µs) molecular dynamics simulation in aqueous solution. Analysis of the results by DASH and principal components analyses revealed four main ring conformations that move essentially independently of the faster-moving tail region. Two of these conformations (labeled "saddle") feature well-defined ß-turns in the ring and conserved transannular hydrogen bonds, whereas the other two ("open") feature neither. The conformations have been identified and defined and are all of sufficient stability to be considered candidates for biological conformations in their cognate receptors.

Aromatase: the enzyme and its inhibition.

The genetic control, mechanism of action and inhibition of aromatase for potential therapeutic benefits in the arena of hormone dependent illness continues to be of considerable interest. The work presented here concentrates on recent advances in the mechanistic aspects of the aromatase enzyme and the nature of the chemical entities that lead to its inhibition.

DASH: a novel analysis method for molecular dynamics simulation data. Analysis of ligands of PPAR-gamma.

A novel molecular dynamics (MD) analysis algorithm, DASH, is introduced in this paper. DASH has been developed to utilize the sequential nature of MD simulation data. By adjusting a set of parameters, the sensitivity of DASH can be controlled, allowing molecular motions of varying magnitudes to be detected or ignored as desired, with no knowledge of the number of conformations required being prerequisite. MD simulations of three synthetic ligands of the orphan nuclear receptor PPARgamma were generated in vacuo using Tripos's SYBYL and used as the training set for DASH. Two X-ray crystal structures of PPARgamma complexed with Rosiglitazone were compared to gain knowledge of the pharmacophoric conformation; this showed that the conformation of the ligand is significantly different between the two structures, indicating that there is no distinct conformation in which rosiglitazone binds to PPARgamma but multiple binding modes. An investigation into simulation length was carried out. A simulation of 5 ns was found to give highly variable results, whereas a simulation of 25 ns gave a representative window of motion for molecules of this size. DASH was compared with Ward's hierarchical cluster analysis method. The results show that DASH analysis is as good as Ward analysis in some areas (e.g. conformation identification) and is superior in others (e.g. speed and input size).

QSAR studies of the pyrethroid insecticides. Part 3. A putative pharmacophore derived using methodology based on molecular dynamics and hierarchical cluster analysis.

Previous studies of the conformational behaviour of a group of synthetic pyrethroid insecticides have been extended to a more structurally diverse set. This includes compounds with different backbones and differing stereochemistry, with both Types I and II biological activity. These compounds also encompass a large range of biological activities. A parameterisation of the CHARMM force field for these compounds has been performed and the extra parameters are reported. Conformational sampling, using molecular dynamics (MD), has been performed for each of the 41 active structures. The accessible conformations of each have been characterised by the values of the common torsion angles using hierarchichal cluster analysis (HCA). A further CA, based on the centroids derived from the conformational sampling, identified a conformation common to at least 39 of the 41 structures. The critical torsion angles of this conformation lie at the centre of the molecule about the ester linkage and are defining an extended conformation, which differs from the minimum energy conformation of deltamethrin used previously. This may represent a putative pharmacophore for kill. The methods used here improve significantly on those used previously. The CHARMM force field was parameterised for the compounds and an improved method of conformational sampling, based on centroid clustering, has also been used.

An investigation of the ionophoric characteristics of destruxin A.

Destruxin A, a cyclohexadepsipeptide related to the enniatins and beauvericin, exhibits ionophoric properties. Calcium ion mobilization across liposomal membrane barriers, for example, has been demonstrated using the calcium ion-sensitive dyes Arsenazo III and Fura-2. Initial molecular mechanics/molecular dynamics calculations indicate the potential for destruxin A to form a coordination complex with calcium in which the divalent cation is bound at the center of a sandwich formed by two molecules of destruxin A. This novel calcium ion binding may help explain the diverse biological effects exhibited by the destruxins.