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BACKGROUND: Tuberculosis (TB) as a foremost infectious disease adds massive burden to morbidity and mortality rate, despite of well-structured TB control programs around the globe. Inappropriate health care management system and poor implementation on standard in relevance to TB, remain some reasons causative to TB prevalence and its rising antimicrobial resistance. Health Care Workers (HCWs) laboring as a part of TB control system, are the vital warriors in achieving the goals of TB End Strategy by 2035. Their performance is influenced by their knowledge, attitude, and practices (KAP) toward this infectious disease. This study aimed to signify the role of KAP score of health care Workers in the better control and prevention of TB in the Islamabad Capital Territory (ICT), Pakistan. METHODS: A cross-sectional study on Knowledge, Attitude and Practice study of Tuberculosis (TB) among health care Workers, was done in ICT, which is the capital of Pakistan. The KAP of TB was collected for the 306 Health Care Workers from all the Islamabad TB referring health facilities which refer the TB patients for testing to the National Reference Laboratory, Islamabad Pakistan. Eligible health care workers were requested to respond on KAP questionnaire after informed consent. KAP questionnaire comprised of knowledge, attitude, and practices section including demographic information. All the data was analyzed using IBM SPSS statistics 21. One Way Analysis of Variance (ANOVA) was applied to calculate KAP mean score against different variables. On the significant data sets of ANOVA output, Tukey's Multiple Comparison Test was applied for pairwise comparison. Pearson correlation coefficient was utilized to explore the association between two qualitative variables. The non-parametric tests were applied to evaluate difference of KAP score in relation to demographic covariates individually. RESULTS: From June to July 2023, we conducted TB KAP study among Health Care Workers of ICT, Pakistan. The average age was 33 years (range 26-30 years). Majority of the recruited subjects were not being trained for dealing with TB infection. The results demonstrated that Health Care Workers working were lacking their knowledge about mode of TB transmission, best diagnostic technique, and contraction of TB infections. The mean knowledge, attitude and practices mean scores were 15.05 (SD = 3.96), 83.68 (SD = 15.74) and 6.31 (SD = 2.21), respectively. Mean knowledge score of Health Care Workers were significantly related to their educational level and occupation while no significant association was declared with working experience as TB staff. Pearson coefficient of attitude score with knowledge of Health Care Workers was of weak level (0.28). Practice mean score was correlated to knowledge mean score at a moderate level (r = 0.40). On the other hand, practice score was r = 0.29 with attitude mean score had shown weak level correlation. A number of demographic factors were strongly linked to each of the mean score of knowledge, attitude, and practices. CONCLUSION: These findings highlighted the significant involvement of education, profession, and professional trainings in the better knowledge, attitude, and practices of the TB related health care Workers. For a better management system of infectious diseases like TB, a well-trained and professionally competent staff of Health Care Workers is important so as to achieve the goal of TB-End strategy by 2035 from Pakistan, which is the 5th highest burden country for TB.

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Glioblastoma multiforme (GBM) IDH-wildtype is the most prevalent brain malignancy in adults. However, molecular mechanisms, which leads to GBM have not been completely elucidated. Granulocyte colony-stimulating factor (GCSF), Granulocyte colony-stimulating factor receptor GCSFR, and Signal transducers and activators of transcription 3 (STAT3) have been involved in the occurrence and development of various cancers, but their role in GBM is little known. Herein, we have investigated the gene and protein expression of GCSF, GCSFR, and STAT3 in 21 tissue biopsy samples and also in tumor associated normal tissue (TANT) samples derived from glioblastoma patients, which revealed significantly differential expression of these genes. To validate our findings, we performed a comprehensive integrated analysis of transcriptomic and proteomic profiling of respective genes by retrieving GBM RNA-sequence data from Genome Atlas Databases. GO and KEGG analysis revealed enrichment in disease-related pathways, such as JAK/STAT pathway activation, which were associated with GBM progression. We further performed computational docking analysis of potential drug candidate Nisin against GCSF, and the results were validated in vitro through cytotoxic activity assay using a human glioblastoma cell line SF-767 in a dose-dependent manner. Our comprehensive analysis reveals that GCSF augments glioma progression, and its blockade with anticancer bacteriocin peptide Nisin can potentially inhibit the growth and metastasis of GBM.

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BACKGROUND: Glioblastoma (GBM) harbors significant genetic heterogeneity, high infiltrative capacity, and patterns of relapse following many therapies. The expression of nuclear factor kappa-B (NF-κB p65 (RelA)) and signaling pathways is constitutively activated in GBM through inflammatory stimulation such as tumor necrosis factor-alpha (TNFα), cell invasion, motility, abnormal physiological stimuli, and inducible chemoresistance. However, the underlying anti-tumor and anti-proliferative mechanisms of NF-κB p65 (RelA) and TNFα are still poorly defined. This study aimed to investigate the expression profiling of NF-κB p65 (RelA) and TNFα as well as the effectiveness of celecoxib along with temozolomide (TMZ) in reducing the growth of the human GBM cell line SF-767. METHODS: genome-wide expression profiling, enrichment analysis, immune infiltration, quantitative expression, and the Microculture Tetrazolium Test (MTT) proliferation assay were performed to appraise the effects of celecoxib and TMZ. RESULTS: demonstrated the upregulation of NF-κB p65 (RelA) and TNFα and celecoxib reduced the viability of the human glioblastoma cell line SF-767, cell proliferation, and NF-κB p65 (RelA) and TNFα expression in a dose-dependent manner. Overall, these findings demonstrate for the first time how celecoxib therapy could mitigate the invasive characteristics of the human GBM cell line SF-767 by inhibiting the NF-κB mediated stimulation of the inflammatory cascade. CONCLUSION: based on current findings, we propose that celecoxib as a drug candidate in combination with temozolomide might dampen the transcriptional and enzymatic activities associated with the aggressiveness of GBM and reduce the expression of GBM-associated NF-κB p65 (RelA) and TNFα inflammatory genes expression.

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Osteoarthritis occurs when protective cartilage of bones worn out. Similarlty, cartilage damage occurs mainly in the pannus cartilage in rheumatoid arthritis. It is a potentially debilitating condition, affecting women two to three times more often than men. The cause and prognosis of rheumatoid and osteoarthritis are still poorly known. However, advances in the study of disease pathogenesis have encouraged the creation of new therapeutics with improved outcomes. The purpose of this study is to investigate the differentially expressed genes potentially involved in dysregulated rheumatoid arthritis (RA) and their association to other types of arthritis, including osteoarthritis (OA). Complete RNAs were isolated for RNA expression profiling using next-generation sequencing from human primary cultured normal and RA chondrocytes. From RNA sequencing results 250 differentially expressed genes were identified using bioinformatics analysis, of which 32 were found to be significantly playing role in RA pathogenesis and its associated diseases. Molecular ontologies of the identified genes showed they are connected to Innate immune response, Protein phosphorylation, Transcription initiation from RNA polymerase II promoter, Immune response, Neoplasms of bones, as well as osteorthritis, and Rheumatoid arthritis. Among the identified genes, TRAF1, TRAF2, BAMP, STX11, MEOX2, AES, REL, FHL3, PNMA1, SGTA, LZTS2, SIAH2, PNMA1, and TFCP2 were found to be highly enriched in the protein-protein interaction network. The significant cross talks were found in Hypertrophic cardiomyopathy, Small cell lung cancer, Proteasome, p53 signaling pathway, Arrhythmogenic right ventricular cardiomyopathy, Small cell lung cancer, SNARE interactions in vesicular transport, RIG-I-like receptor signaling pathway, and Hypertrophic cardiomyopathy pathways. The results offer new opportunities for target gene control in RA and OA cartilage destruction.

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Exosomes are 20-150nm cell secreting nano-bodies that helps in the transportation of various biomolecules, including micro ribonucleic acid (miRNA) in the human body during both normal and diseased conditions. The current review was planned to summarise the role of miRNA carried by circulatory exosomes in cancer. miRNA is responsible for contribution in cancer, regulation of gene expression, interfering in biological pathways, gene silencing or amplification, and also has a role in cancer resistance. (miRNA) plays a dynamic role in this process by regulating the genes related to drug resistance, cell proliferation, cell cycle and apoptosis through a tissue-specific fashion. Owing to its significances, micro ribonucleic acid has been reported to be the key regulator of cancer, metastasis and also a factor in cancer resistance, and is a better source of possible potential diagnostic biomarkers. Though many studies have explored the biological roles of RNAs in cancer, many facts are needed to be investigated for clinical applications.

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Severe acute respiratory syndrome virus 2 (SARS-CoV-2) belongs to the single-stranded positive-sense RNA family. The virus contains a large genome that encodes four structural proteins, small envelope (E), matrix (M), nucleocapsid phosphoprotein (N), spike (S), and 16 nonstructural proteins (nsp1-16) that together, ensure replication of the virus in the host cell. Among these proteins, the interactions of N and Nsp3 are essential that links the viral genome for processing. The N proteins reside at CoV RNA synthesis sites known as the replication-transcription complexes (RTCs). The N-terminal of N has RNA-binding domain (N-NTD), capturing the RNA genome while the C-terminal domain (N-CTD) anchors the viral Nsp3, a component of RTCs. Although the structural information has been recently released, the residues involved in contacts between N-CTD with Nsp3 are still unknown. To find the residues involved in interactions between two proteins, three-dimensional structures of both proteins were retrieved and docked using HADDOCK. Residues at N-CTD were detected in interaction with L499, R500, K501, V502, P503, T504, D505, N506, Y507, I508, T509, K529, K530K532, S533 of Nsp3 and N-NTD to synthesize SARS-CoV-2 RNA. The interaction between Nsp3 and CTD of N protein may be a potential drug target. The current study provides information for better understanding the interaction between N protein and Nsp3 that could be a possible target for future inhibitors.

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BACKGROUND: Search for new drug targets is becoming imperative these days, given that marketed chemotherapeutic drugs have lost their efficacy against harmful agents because of adaptability to climatic changes and co-evolving vectors to new hosts. In the wake of such a challenge, the prominence of biochemical studies is increasing by way of exploring selective enzymes and investigating their structural and functional properties through biochemical kinetic parameter Km for the application of IC50 using designed drugs. Recently, discovered Adenine Aminohydrolase (EC 3.5.4.2) in Leishmania has been found to be absent in mammalian purine salvage pathway and thus considered as a promising drug target against infectious agents. OBJECTIVES: The objective of this study is to isolate and characterize AAH by learning its kinetic mode of action using preferred substrate Adenine and additives estimated through expected product formation Hypoxanthine. Bioassays designed to measure exact Enzyme kinetic parameter Km value through establishing hyperbolic curve of an enzyme reaction with the use of exact values of cellular quantities for IC50 application under experimental conditions devised by presteady state approach for SSA validity. METHODS: Following saturation kinetic, the plot of hyperbolic equilibrium curve developed using initial rates of product formation as a function of (Si) through forward shift under circumstance dG0 the system allows product and reactant favored reactions in relation to (Ef) ≈ [E0 = KM] until complete saturation and estimates Km and Vmax of enzyme system under applied conditions. M-M equation used to assess experimental initial rate data for estimation of Km on excel using Solver and nonlinear least square coefficient correlation "R2" using logarithmic equation for nonlinear curve assessment. RESULTS: UV/Vis spectrophotometer selectively analyzed reacting components confirming Enzyme characteristic reaction constant Km equal toi15. 0 ± 2 µ mol acquired from the Hyperbolic curve developed through the use of exact (Si) ranges at selected parameter Km and Vmax. The curve assessed by Michaelis Menten equation provides Km value=14.99 µmol and non-linear least square coefficient correlation "R2" value equal to 0.9895, along with that optimized lysis buffer formulation. In the docked complexes, the interactive amino acids identified were MSE441, ALA 364, GLN363, MSE518, VAL362, GLY517, ASP538, ALA445, TYR521, and TYR444. 2D interactions revealed hydrophobic and alkyl interactions at the noncompetitive binding site of the enzyme and therefore recommended as potential inhibitors against 3ICS protein. CONCLUSION: This study encourages biochemical analysis of the novel enzymes with the use of presteady state rationale in association with the computational tools as an effective way of designing drugs in a short time against selective enzymes to meet the current challenge efficiently.

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Exosomes are the extracellular vesicles secreted normally by most of the cells, containing important bioactive molecules including lipids, carbohydrates, protein, DNA and RNA resulting in cell to cell communication and many other biological activities. In this review we have focused on different insight onto exosomes to cover its basic mechanism, biogenesis, biomolecules it carries and how they are altering secondary sites. In cancerous cells these tiny bodies are reported to be secreted aberrantly and through paracrine signalling contributes in metastasis. Each type of cancer cells exosomes is unique with types of load inside, thus behave with an individual pattern to transfer cancer load from origin to other sites. Because of its involvement in cancer metastasis and its role as biomarkers in early stage disease identification and also as suitable particles for drug delivery system, Exosomes research has been focal field since last two decades. Currently exosomes are the hot area of research and because of their biologically important structure and composition some studies have also been conducted to use them as early stage biomarkers in different diseases and also by a modification these could also be a biocompatible source in drug delivery. The current researches data, results and advancement in exosome studies are quit promising and are positive indication in resolving many clinical complexities in future but still further investigations are needed to evaluate the clinical importance and applications of exosomes in detail.

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Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently was declared a pandemic by world health organization (WHO) Due to sudden outbreaks, currently, no completely effective vaccine or drug is clinically approved. Several therapeutic strategies can be envisaged to prevent further mortality and morbidity. Based on the past contribution of traditional Chinese medicines (TCM) and immune-based therapies as a treatment option in crucial pathogen outbreaks, we aimed to summarize potential therapeutic strategies that could be helpful to stop further spread of SARS-CoV-2 by effecting its structural components or modulation of immune responses. Several TCM with or without modification could be effective against the structural protein, enzymes, and nucleic acid should be tested from available libraries or to identify their immune-stimulatory activities to enhance several antiviral biological agents for effective elimination of SARS-CoV-2 from the host. TCM is not only effective in the direct inhibition of virus attachment and internalization in a cell but can also prevent their replication and can also help to boost up host immune response. Immune-modulatory effects of TCMs may lead to new medications and can guide us for the scientific validity of drug development. Besides, we also summarized the effective therapies in clinical for controlling inflammation. This review will be not only helpful for the current situation of COVID-19, but can also play a major role in such epidemics in the future.

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A central approach for better understanding the forces involved in maintaining protein structures is to investigate the protein folding and thermodynamic properties. The effect of the folding process is often disturbed in mutated states. To explore the dynamic properties behind mutations, molecular dynamic (MD) simulations have been widely performed, especially in unveiling the mechanism of drug failure behind mutation. When comparing wild type (WT) and mutants (MTs), the structural changes along with solvation free energy (SFE), and Gibbs free energy (GFE) are calculated after the MD simulation, to measure the effect of mutations on protein structure. Pyrazinamide (PZA) is one of the first-line drugs, effective against latent Mycobacterium tuberculosis isolates, affecting the global TB control program 2030. Resistance to this drug emerges due to mutations in pncA and rpsA genes, encoding pyrazinamidase (PZase) and ribosomal protein S1 (RpsA) respectively. The question of how the GFE may be a measure of PZase and RpsA stabilities, has been addressed in the current review. The GFE and SFE of MTs have been compared with WT, which were already found to be PZA-resistant. WT structures attained a more stable state in comparison with MTs. The physiological effect of a mutation in PZase and RpsA may be due to the difference in energies. This difference between WT and MTs, depicted through GFE plots, might be useful in predicting the stability and PZA-resistance behind mutation. This study provides useful information for better management of drug resistance, to control the global TB problem.

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Lipases with high tolerance to temperature play a significant role in industry from food manufacturing to waste management systems. Thus, there is a need to investigate these enzymes from different geographical areas to look out for a more thermo-stable one. Characterization of lipases through experimental approaches is time consuming process and sometimes the results are ambiguous due to errors. However, integration of computational technologies is quite useful for prediction of optimized conditions. Such technologies can be applied as synthetic biology, which has many major applications in engineered biological approaches for accurate prediction of effects of different physical and chemical parameters on the system. In this study, cloning and expression of a lipase gene from Bacillus amyloliquefaciens, isolated from a novel geographical region of Pakistan, in Escherichia coli DH5α cells followed by sequencing was carried out. To isolate thermostable lipase producing strains, all the samples were kept at 50 °C. Genomic DNA was isolated and signal peptide (1-32 residues) sequence was chopped (ΔSPLipase). The ΔSPLipase was amplified and expressed in Linearized p15TV-L vector. The purified lipase appeared as single band of approximately 26 kDa. Suitable conditions of factors required for maximum lipase activity such as temperature, pH, substrate, organic solvent, detergents and metal ions were predicted through synthetic biology approach and further confirmed in wet lab. The predicted suitable factors for enzyme were almost similar to those determined experimentally. The optimum enzyme activity was recorded at pH 8 and 50 °C temperature. Interestingly, the activity of enzyme was found on a number of solvents, metal ions, detergents, and surfactants. The predicted optimum values and their experimental confirmations highlights the importance of integrated synthetic biology approaches in wet lab experiments. The characterized lipase of B. amyloliquefaciens at molecular level from Pakistani strains displayed good activity on a range of factors that implies this strain to be used for application in industrial level production.

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Pyrazinamide (PZA) is an important component of first-line anti-tuberculosis (anti-TB) drugs. The anti-TB agent is activated into an active form, pyrazinoic acid (POA), by Mycobacterium tuberculosis (MTB) pncA gene encoding pyrazinamidase (PZase). The major cause of PZA-resistance has been associated with mutations in the pncA gene. We have detected several novel mutations including V131F, Q141P, R154T, A170P, and V180F (GeneBank Accession No. MH461111) in the pncA gene of PZA-resistant isolates during PZA drug susceptibility testing followed by pncA gene sequencing. Here, we investigated molecular mechanism of PZA-resistance by comparing the results of experimental and molecular dynamics. The mutants (MTs) and wild type (WT) PZase structures in apo and complex with PZA were subjected to molecular dynamic simulations (MD) at the 40 ns. Multiple factors, including root mean square deviations (RMSD), binding pocket, total energy, dynamic cross correlation, and root mean square fluctuations (RMSF) of MTs and WT were compared. The MTs attained a high deviation and fluctuation compared to WT. Binding pocket volumes of the MTs, were found, lower than the WT, and the docking scores were high than WT while shape complementarity scores were lower than that of the WT. Residual motion in MTs are seemed to be dominant in anti-correlated motion. Mutations at locations, V131F, Q141P, R154T, A170P, and V180F, might be involved in the structural changes, possibly affecting the catalytic property of PZase to convert PZA into POA. Our study provides useful information that will enhance the understanding for better management of TB. AbbreviationsDSTdrug susceptibility testingΔelecelectrostatic energyLJLowenstein-Jensen mediumMGITmycobacterium growth indicator tubesMTsmutantsMDmolecular dynamic simulationsMTBMycobacterium tuberculosisNALC-NaOHN-acetyl-l-cysteine-sodium hydroxideNIHNational Institutes of HealthNPTamount of substance (N), pressure (P) temperature (T)NVTmoles (N), volume (V) temperature (T)PZasepyrazinamidaseΔpspolar solvation energyPTRLProvincial Tuberculosis Reference LaboratoryRMSDroot mean square deviationsRMSFroot mean square fluctuationsΔSASAsolvent accessible surface area energyTBtuberculosisGTotaltotal binding free energyΔvdWVan der Waals energyWTwild typeCommunicated by Ramaswamy H. Sarma.

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Pyrazinamide (PZA) is the only drug for the elimination of latent Mycobacterium tuberculosis (MTB) isolates. However, due to the increased number of PZA-resistance, the chances of the success of global TB elimination seems to be more prolonged. Recently, marine natural products (MNPs) as an anti-TB agent have received much attention, where some compounds extracted from marine sponge, Haliclona sp. exhibited strong activity under aerobic and hypoxic conditions. In this study, we screened articles from 1994 to 2019 related to marine natural products (MNPs) active against latent MTB isolates. The literature was also mined for the major regulators to map them in the form of a pathway under the dormant stage. Five compounds were found to be more suitable that may be applied as an alternative to PZA for the better management of resistance under latent stage. However, the mechanism of actions behind these compounds is largely unknown. Here, we also applied synthetic biology to analyze the major regulatory pathway under latent TB that might be used for the screening of selective inhibitors among marine natural products (MNPs). We identified key regulators of MTB under latent TB through extensive literature mining and mapped them in the form of regulatory pathway, where SigH is negatively regulated by RshA. PknB, RshA, SigH, and RNA polymerase (RNA-pol) are the major regulators involved in MTB survival under latent stage. Further studies are needed to screen MNPs active against the main regulators of dormant MTB isolates. To reduce the PZA resistance burden, understanding the regulatory pathways may help in selective targets of MNPs from marine natural sources.

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Resistance to key first-line drugs is a major hurdle to achieve the global end tuberculosis (TB) targets. A prodrug, pyrazinamide (PZA) is the only drug, effective in latent TB, recommended in drug resistance and susceptible Mycobacterium tuberculosis (MTB) isolates. The prodrug conversion into active form, pyrazinoic acid (POA), required the activity of pncA gene encoded pyrazinamidase (PZase). Although pncA mutations have been commonly associated with PZA resistance but a small number of resistance cases have been associated with mutationss in RpsA protein. Here in this study a total of 69 PZA resistance isolates have been sequenced for pncA mutations. However, samples that were found PZA resistant but pncA wild type (pncAWT), have been sequenced for rpsA and panD genes mutation. We repeated a drug susceptibility testing according to the WHO guidelines on 18 pncAWT MTB isolates. The rpsA and panD genes were sequenced. Out of total 69 PZA resistant isolates, 51 harbored 36 mutations in pncA gene (GeneBank Accession No. MH46111) while, fifteen different mutations including seven novel, were detected in the fourth S1 domain of RpsA known as C-terminal (MtRpsACTD) end. We did not detect any mutations in panD gene. Among the rpsA mutations, we investigated the molecular mechanism of resistance behind mutations, D342N, D343N, A344P, and I351F, present in the MtRpsACTD through molecular dynamic simulations (MD). WT showed a good drug binding affinity as compared to mutants (MTs), D342N, D343N, A344P, and I351F. Binding pocket volume, stability, and fluctuations have been altered whereas the total energy, protein folding, and geometric shape analysis further explored a significant variation between WT and MTs. In conclusion, mutations in MtRpsACTD might be involved to alter the RpsA activity, resulting in drug resistance. Such molecular mechanism behind resistance may provide a better insight into the resistance mechanism to achieve the global TB control targets.

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Background: The global burden of tuberculosis (TB) and antibiotic resistance is attracting the attention of researchers to develop some novel and rapid diagnostic tools. Although, the conventional methods like culture are considered as the gold standard, they are time consuming in diagnostic procedure, during which there are more chances in the transmission of disease. Further, the Xpert MTB/RIF assay offers a fast diagnostic facility within 2 h, but due to low sensitivity in some sample types may lead to more serious state of the disease. The role of computer technologies is now increasing in the diagnostic procedures. Here, in the current study we have applied the artificial neural network (ANN) that predicted the TB disease based on the TB suspect data. Methods: We developed an approach for prediction of TB, based on an ANN. The data was collected from the TB suspects, guardians or care takers along with samples, referred by TB units and health centers. All the samples were processed and cultured. Data was trained on 12,636 records of TB patients, collected during the years 2016 and 2017 from the provincial TB reference laboratory, Khyber Pakhtunkhwa, Pakistan. The training and test set of the suspect data were kept as 70 and 30%, respectively, followed by validation and normalization. The ANN takes the TB suspect's information such as gender, age, HIV-status, previous TB history, sample type, and signs and symptoms for TB prediction. Results: Based on TB patient data, ANN accurately predicted the Mycobacterium tuberculosis (MTB) positive or negative with an overall accuracy of >94%. Further, the accuracy of the test and validation were found to be >93%. This increased accuracy of ANN in the detection of TB suspected patients might be useful for early management of disease to adopt some control measures in further transmission and reduce the drug resistance burden. Conclusion: ANNs algorithms may play an effective role in the early diagnosis of TB disease that might be applied as a supportive tool. Modern computer technologies should be trained in diagnostics for rapid disease management. Delays in TB diagnosis and initiation treatment may allow the emergence of new cases by transmission, causing high drug resistance in countries with a high TB burden.

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Pyrazinamide (PZA) is an essential first line antitubercular drug, which plays a crucial role in tuberculosis treatment. The PZA, which is considered as a pro-drug needs an enzyme of mycobacterial pyrazinamidase (PZase) for its conversion into an active form pyrazinoic acid. Further, this active form of PZA inhibits the ribosomal proteins S1, which facilitates the transfer-mRNA complex formation throughout the translation. The spontaneous mutations in RpsA have been found to be associated with PZA drug resistance. However, the drug resistance mechanism is still unclear. Furthermore, there is no such information available about the structural dynamics of RpsA protein because of mutations that confer Pyrazinoic acid resistance. Moreover, a total of 18 clinical PZA-resistant isolates were investigated and found to be pncAWT, which allowed exploration of the resistance mechanism of RpsA in the mutated state. Samples were repeated for the drug susceptibility testing followed by RpsA gene sequencing. A total of 11 clinical isolates harbored a total of 15 mutations. Almost half of the total strains (7/15) were observed to be in the conserved region of RpsA and known as Mycobacterium tuberculosis C-terminal domain. In the current study, (2/7) mutation T370P (mutant 1) and W403G (mutant 2) were explored to ensure the RpsA resistance mechanism through essential dynamics simulation. The essential dynamics study results revealed that the distal loop mutations drastically altered the conformation of RpsA both in the absence (-) and presence (+) of pyrazinoic acid drug for two reasons: (1) dramatic alteration or reduction in the binding pattern of pyrazinoic acid with active site residues observed and (2) a clear image of the opening and closing switching mechanism was seen upon the distal site mutation on nearby 310-helixes beside the pyrazinoic acid binding site. This switch was found to consistently remain closed only in wild type systems, while it was open in the mutant systems. We called such distance impact an "allosteric effect." The overall mechanistic investigations will provide useful information behind drug resistance for better understanding to manage tuberculosis.

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BACKGROUND: Pyrazinamide (PZA) is an important component of first-line drugs because of its distinctive capability to kill subpopulations of persistent Mycobacterium tuberculosis (MTB). The prodrug (PZA) is converted to its active form, pyrazinoic acid (POA) by MTB pncA-encoded pyrazinamidase (PZase). Mutation in pncA is the most common and primary cause of PZA resistance. The aim of the present study was to explore the molecular characterization of PZA resistance in a Pashtun-dominated region of Khyber Pakhtunkhwa, Pakistan. METHODS: We performed drug susceptibility testing (DST) on 753 culture-positive isolates collected from the Provincial Tuberculosis Control Program Khyber Pakhtunkhwa using the BACTEC MGIT 960 PZA method. In addition, the pncA gene was sequenced in PZA-resistant isolates, and PZA susceptibility testing results were used to determine the sensitivity and specificity of pncA gene mutations. RESULTS: A total of 69 isolates were PZA resistant (14.8%). Mutations were investigated in 69 resistant, 26 susceptible and one H37Rv isolates by sequencing. Thirty-six different mutations were identified in PZA-resistant isolates, with fifteen mutations, including 194_203delCCTCGTCGTG and 317_318delTC, that have not been reported in TBDRM and GMTV Databases and previous studies. Mutations Lys96Thr and Ser179Gly were found in the maximum number of isolates (n = 4 each). We did not detect mutations in sensitive isolates, except for the synonymous mutation 195C > T (Ser65Ser). The sensitivity and specificity of the pncA sequencing method were 79.31% (95% CI, 69.29 to 87.25%) and 86.67% (95% CI, 69.28 to 96.24%). CONCLUSION: Mutations in the pncA gene in circulating isolates of geographically distinct regions, especially in high-burden countries, should be investigated for better control and management of drug-resistant TB. Molecular methods for the investigation of PZA resistance are better than DST.

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In an effort to discover the mechanism of resistance offered by Mycobacterium tuberculosis (Mtb) toward the pyrazinamide (PZA) drug, an extensive molecular dynamics strategy was employed. PZA is a first-line prodrug that effectively cuts therapy time by 33% (from 9 to 6 months). Pyrazinamidase enzyme (PZase), encoded by the pncA gene, is responsible for the activation of prodrug PZA into pyrazinoic acid (POA). POA is toxic and potently inhibits the growth of latent Mtb even at low pH values. PZA resistance is caused by three genes pncA, rpsA, and panD. Among them, the pncA gene contributes 72-99% to the resistance. Hence, the present study focused on the novel mutations N11K, P69T, and D126N in the pncA gene. In the present study, the possible mechanism of these three mutations was studied through molecular dynamics simulation and docking techniques. Our in-depth analysis and results are in strong agreement with our experimental observation. The binding pocket analysis showed that mutations decrease the volume of the active site and hinder the correct orientation of PZA drug in the active site. Moreover, the Patchdock score was found to be low as compared to WT showing the disturbance of shape complementarity between PZase and PZA drug. These mutations were found to disturb the position of the Fe2+ ion. Among the mutations, D126N allosterically disturbed the position of the Fe2+ ion. MMGBSA analyses showed that these mutations decrease the binding affinity toward the PZA drug. In conclusion, mutations N11K, P69T, and D126N result in weak binding affinity with PZA and also cause significant structural deformations that lead to PZA resistance. This study provides useful information that mutations in other than active parts may also cause protein folding and ligand displacement effects, altering the biological functions.

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Pyrazinamide (PZA) is an important component of first-line antituberculosis drugs activated by Mycobacterium tuberculosis pyrazinamidase (PZase) into its active form pyrazinoic acid. Mutations in the pncA gene have been recognized as the major cause of PZA resistance. We detected some novel mutations, Leucine19Arginine (L19R), Arginine140Histidine (R140H), and Glutamic acid144 Lysine (E144K), in the pncA gene of PZA-resistant isolates in our wet lab PZA drug susceptibility testing and sequencing. As the molecular mechanism of resistance of these variants has not been reported earlier, we have performed multiple analyses to unveil different mechanisms of resistance because of PZase mutations L19R, R140H, and E144K. The mutants and native PZase structures were subjected to comprehensive computational molecular dynamics (MD) simulations at 100 nanoseconds in apo and drug-bound form. Mutants and native PZase binding pocket were compared to observe the consequence of mutations on the binding pocket size. Hydrogen bonding, Gibbs free energy, and natural ligand Fe +2 effect were also analyzed between native and mutants. A significant variation between native and mutant PZase structure activity was observed. The native PZase protein docking score was found to be the maximum, showing strong binding affinity in comparison with mutants. MD simulations explored the effect of the variants on the biological function of PZase. Hydrogen bonding, metal ion Fe +2 deviation, and fluctuation also seemed to be affected because of the mutations L19R, R140H, and E144K. The variants L19R, R140H, and E144K play a significant role in PZA resistance, altering the overall activity of native PZase, including metal ion Fe +2 displacement and free energy. This study offers valuable evidence for better management of drug-resistant tuberculosis.

RESUMEN

Pyrazinamide (PZA) is an important component of first-line anti-tuberculosis drugs which is converted into active form, pyrazinoic acid (POA), by Mycobacterium tuberculosis (MTB) pncA gene encoded, pyrazinamidase (PZase). Mutations in pncA are detected in >70% of PZA resistant isolates but, noticeably, not in all. In this study, we selected 18 PZA-resistant but wild type pncA (pncAWT) MTB isolates. Drug susceptibility testing (DST) of all the isolates were repeated at the critical concentration of PZA drug. All these PZA-resistance but pncAWT isolates were subjected to RpsA sequencing. Fifteen different mutations were identified in eleven isolates, where seven were present in a conserved region including, Ser324Phe, Glu325Lys, Gly341Arg. As the molecular mechanism of resistance behind these variants has not been reported earlier, we have performed multiple analysis to unveil the mechanisms of resistance behind mutations S324F, E325K, and G341R. The mutant and wild type RpsA structures were subjected to comprehensive computational molecular dynamic simulations at 50 ns. Root mean square deviation (RMSD), Root mean square fluctuation (RMSF), and Gibbs free energy of mutants were analyzed in comparison with wild type. Docking score of wild type-RpsA has been found to be maximum, showing a strong binding affinity in comparison with mutants. Pocket volume, RMSD and RMSF have also been found to be altered, whereas total energy, folding effect (radius of gyration) and shape complimentarily analysis showed that variants S324F, E325K, and G341R have been playing a significant role behind PZA-resistance. The study offers valuable information for better management of drug resistance tuberculosis.