The miniature CRISPR-Cas12m effector binds DNA to block transcription.

CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window.

Inhibition of cytochrome P450 epoxygenase promotes endothelium-to-mesenchymal transition and exacerbates doxorubicin-induced cardiovascular toxicity.

BACKGROUND: Doxorubicin (DOX) is a potent chemotherapy widely used in treating various neoplastic diseases. However, the clinical use of DOX is limited due to its potential toxic effect on the cardiovascular system. Thus, identifying the pathway involved in this toxicity may help minimize chemotherapy risk and improve cancer patients' quality of life. Recent studies suggest that Endothelial-to-Mesenchymal transition (EndMT) and endothelial toxicity contribute to the pathogenesis of DOX-induced cardiovascular toxicity. However, the molecular mechanism is yet unknown. Given that arachidonic acid and associated cytochrome P450 (CYP) epoxygenase have been involved in endothelial and cardiovascular function, we aimed to examine the effect of suppressing CYP epoxygenases on DOX-induced EndMT and cardiovascular toxicity in vitro and in vivo. METHODS AND RESULTS: To test this, human endothelial cells were treated with DOX, with or without CYP epoxygenase inhibitor, MSPPOH. We also investigated the effect of MSPPOH on the cardiovascular system in our zebrafish model of DOX-induced cardiotoxicity. Our results showed that MSPPOH exacerbated DOX-induced EndMT, inflammation, oxidative stress, and apoptosis in our endothelial cells. Furthermore, we also show that MSPPOH increased cardiac edema, lowered vascular blood flow velocity, and worsened the expression of EndMT and cardiac injury markers in our zebrafish model of DOX-induced cardiotoxicity. CONCLUSION: Our data indicate that a selective CYP epoxygenase inhibitor, MSPPOH, induces EndMT and endothelial toxicity to contribute to DOX-induced cardiovascular toxicity.

Unlocking the secrets: exploring the influence of the aryl hydrocarbon receptor and microbiome on cancer development.

Gut microbiota regulates various aspects of human physiology by producing metabolites, metabolizing enzymes, and toxins. Many studies have linked microbiota with human health and altered microbiome configurations with the occurrence of several diseases, including cancer. Accumulating evidence suggests that the microbiome can influence the initiation and progression of several cancers. Moreover, some microbiotas of the gut and oral cavity have been reported to infect tumors, initiate metastasis, and promote the spread of cancer to distant organs, thereby influencing the clinical outcome of cancer patients. The gut microbiome has recently been reported to interact with environmental factors such as diet and exposure to environmental toxicants. Exposure to environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) induces a shift in the gut microbiome metabolic pathways, favoring a proinflammatory microenvironment. In addition, other studies have also correlated cancer incidence with exposure to PAHs. PAHs are known to induce organ carcinogenesis through activating a ligand-activated transcriptional factor termed the aryl hydrocarbon receptor (AhR), which metabolizes PAHs to highly reactive carcinogenic intermediates. However, the crosstalk between AhR and the microbiome in mediating carcinogenesis is poorly reviewed. This review aims to discuss the role of exposure to environmental pollutants and activation of AhR on microbiome-associated cancer progression and explore the underlying molecular mechanisms involved in cancer development.

6-Formylindolo(3,2-b)carbazole Dampens Inflammation and Reduces Endotoxin-Induced Kidney Injury via Nrf2 Activation.

Patients with sepsis are at a high risk of morbidity and mortality due to multiple organ injuries caused by pathological inflammation. Although sepsis is accompanied by multiple organ injuries, acute renal injury is a significant contributor to sepsis morbidity and mortality. Thus, dampening inflammation-induced renal injury may limit severe consequences of sepsis. As several studies have suggested that 6-formylindolo(3,2-b)carbazole (FICZ) is beneficial for treating various inflammatory diseases, we aimed to examine the potential protective effect of FICZ on the acute endotoxin-induced sepsis model of kidney injury. To test this, male C57Bl/6N mice were injected with FICZ (0.2 mg/kg) or vehicle 1 h prior to an injection of either lipopolysaccharides (LPS) (10 mg/kg), to induce sepsis, or phosphate-buffered saline for 24 h. Thereafter, gene expression of kidney injury and pro-inflammatory markers, circulating cytokines and chemokines, and kidney morphology were assessed. Our results show that FICZ reduced LPS-induced acute injury in kidneys from LPS-injected mice. Furthermore, we found that FICZ dampens both renal and systemic inflammation in our sepsis model. Mechanistically, our data indicated that FICZ significantly upregulates NAD(P)H quinone oxidoreductase 1 and heme oxygenase 1 via aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) in the kidneys to lessen inflammation and improve septic acute kidney injury. Overall, the data of our study show that FICZ possesses a beneficial reno-protective effect against sepsis-induced renal injury via dual activation of AhR/Nrf2.

Insight into the physiological and pathological roles of the aryl hydrocarbon receptor pathway in glucose homeostasis, insulin resistance, and diabetes development.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor that mediates the toxicities of several environmental pollutants. Decades of research have been carried out to understand the role of AhR as a novel mechanism for disease development. Its involvement in the pathogenesis of cancer, cardiovascular diseases, rheumatoid arthritis, and systemic lupus erythematosus have long been known. One of the current hot research topics is investigating the role of AhR activation by environmental pollutants on glucose homeostasis and insulin secretion, and hence the pathogenesis of diabetes mellitus. To date, epidemiological studies have suggested that persistent exposure to environmental contaminants such as dioxins, with subsequent AhR activation increases the risk of specific comorbidities such as obesity and diabetes. The importance of AhR signaling in various molecular pathways highlights that the role of this receptor is far beyond just xenobiotic metabolism. The present review aims at providing significant insight into the physiological and pathological role of AhR and its regulated enzymes, such as cytochrome P450 1A1 (CYP1A1) and CYP1B1 in both types of diabetes. It also provides a comprehensive summary of the current findings of recent research studies investigating the role of the AhR/CYP1A1 pathway in insulin secretion and glucose hemostasis in the pancreas, liver, and adipose tissues. This review further highlights the molecular mechanisms involved, such as gluconeogenesis, hypoxia-inducible factor (HIF), oxidative stress, and inflammation.

Characterization of Genomic Variants Associated with Resistance to Bedaquiline and Delamanid in Naive Mycobacterium tuberculosis Clinical Strains.

The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants' role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.

TB drug susceptibility testing in high fluoroquinolone resistance settings.

BACKGROUND: The insurgence of resistance to key drugs of the BPaLM (bedaquiline + pretomanid + moxifloxacin) regimen is a major concern. In settings with widespread resistance to fluoroquinolones (FQs), like Pakistan, new technologies, such as Xpert® MTB/XDR, may ensure drug resistance upfront screening. This study aims to assess MTB/XDR's performance in detecting FQs and isoniazid resistance, proposing a renewed diagnostic algorithm for drug-resistant TB (DR-TB). METHODS: This cross-sectional prospective study, approved by the local ethical committee, collected samples from people newly and previously diagnosed with TB over 6 months. Xpert® MTB/RIF Ultra, MTB/XDR, Genotype® MTBDRplus, Genotype® MTBDRsl, culture, and phenotypic drug susceptibility testing (pDST) for relevant drugs (including bedaquiline and levofloxacin) were performed. Next-generation sequencing (NGS) resolved discordances between MTB/XDR and pDST results. RESULTS: The analysis showed that MTB/XDR has 91.5% and 88.2% sensitivity and 99.5% and 97.7% specificity in detecting respectively isoniazid (INH) and resistance to FQs, demonstrating that MTB/XDR meets the WHO targets for INH resistance detection at the peripheral level. NGS effectively resolved discordances between MTB/XDR and pDST results. CONCLUSIONS: The obtained results allowed designing the proposed diagnostic algorithm for rapid identification of DR-TB, ensuring rapid and equitable access to drug susceptibility testing for TB, ultimately improving TB care and control.

CONTEXTE: La recrudescence de la résistance aux médicaments clés du régime BPaLM (bédaquiline + prétomanide + moxifloxacine) est une préoccupation majeure. Dans les contextes où la résistance aux fluoroquinolones (FQ) est répandue, comme le Pakistan, de nouvelles technologies, telles que Xpert® MTB/XDR, peuvent assurer un dépistage initial de la résistance aux médicaments. Cette étude vise à évaluer la performance de MTB/XDR dans la détection des FQ et de la résistance à l'isoniazide, en proposant un algorithme de diagnostic renouvelé pour la TB pharmacorésistante (DR-TB, pour l'anglais «drug-resistant TB ¼ ). MÉTHODES: Cette étude prospective transversale, approuvée par le comité d'éthique local, a recueilli des échantillons de personnes nouvellement diagnostiquées et précédemment diagnostiquées avec la TB pendant 6 mois. Xpert® MTB/RIF Ultra, MTB/XDR, le GenoType® MTBDRplus, le GenoType® MTBDRsl, la culture et des tests phénotypiques de sensibilité aux médicaments (pDST, pour l'anglais «phenotypic drug susceptibility testing ¼ ) pour les médicaments pertinents (y compris la bédaquiline et la lévofloxacine) ont été effectués. Le séquençage de nouvelle génération (NGS, pour l'anglais «next-generation sequencing ¼ ) a résolu les discordances entre les résultats MTB/XDR et pDST. RÉSULTATS: L'analyse a montré que le MTB/XDR a une sensibilité de 91,5% et 88,2% et une spécificité de 99,5% et 97,7% dans la détection respectivement de l'isoniazide et de la résistance aux FQ, démontrant que le MTB/XDR répond aux objectifs de l'OMS pour la détection de la résistance à l'isoniazide au niveau périphérique. NGS a efficacement résolu les discordances entre les résultats MTB/XDR et pDST. CONCLUSIONS: Les résultats obtenus ont permis de concevoir l'algorithme de diagnostic proposé pour l'identification rapide de la DR-TB, garantissant un accès rapide et équitable aux tests de sensibilité aux médicaments pour la TB, améliorant ainsi la prise en charge et le contrôle de la TB.

2022 TB programme review in Pakistan: strengthening governance, with better patient diagnosis and treatment.

BACKGROUND: In Pakistan, 84% of healthcare is provided by the private sector. We conducted an epidemiological and programme review for TB to document progress and guide further efforts. METHODS: Surveillance and data systems were assessed before analysing epidemiological data. We reviewed the programme at federal, provincial and peripheral levels and compiled national data along with WHO estimates to describe the evolution of epidemiological and programme indicators. RESULTS: In 2021, of the estimated number of TB cases, 55% of overall cases and 18% of drug-resistant cases were diagnosed and treated respectively. The contribution of the private sector in case detection increased from 30% in 2017 to 40% by 2021. For newly diagnosed pulmonary TB cases, the overall proportion of confirmed cases was 52%. In 2021, testing for rifampicin resistance among confirmed cases was 66% for new and 84% for previously treated patients. The treatment success rate exceeded 90% for drug susceptible TB. The main challenges identified were a funding gap (60% in 2021-2023), fragmented electronic systems for data collection and suboptimal coordination among provinces. CONCLUSIONS: The main challenges prevent further progress in controlling TB. By addressing these, Pakistan could improve coverage of interventions, including diagnosis and treatment. Bacteriological confirmation using recommended diagnostics also requires further optimisation.

CONTEXTE: Au Pakistan, le secteur privé assure 84% des services de santé. Une étude épidémiologique et programmatique a été réalisée sur la TB afin de recueillir des informations sur les avancées réalisées et de guider les actions à venir. MÉTHODES: Les systèmes de surveillance et de données ont été évalués préalablement à l'analyse des données épidémiologiques. Nous avons examiné le programme aux niveaux fédéral, provincial et local et compilé les données nationales ainsi que les estimations de l'OMS afin de décrire l'évolution des indicateurs épidémiologiques et du programme. RÉSULTATS: En 2021, environ 55% de l'ensemble cas de TB et 18% des cas résistants aux médicaments ont été diagnostiqués et traités respectivement. La contribution du secteur privé dans la détection des cas est passée de 30% en 2017 à 40% en 2021. La proportion totale de cas confirmés pour les nouveaux diagnostics de TB pulmonaire s'élevait à 52%. En 2021, les tests de résistance à la rifampicine parmi les cas confirmés s'élevaient à 66% pour les nouveaux patients et de 84% pour les patients déjà traités. Le taux de réussite du traitement a dépassé 90% pour la TB sensible aux médicaments. Les défis majeurs comprennent un manque de financement (60% pour la période 2021­2023), des systèmes électroniques de collecte de données fragmentés et une coordination insuffisante entre les provinces. CONCLUSIONS: Les défis majeurs entravent les avancées dans la lutte contre la TB. En les mettant en évidence, le Pakistan pourrait améliorer la portée des interventions, y compris le diagnostic et le traitement. Il est également essentiel d'optimiser la confirmation bactériologique en utilisant les diagnostics recommandés.

Glycyrrhizic Acid Nanoparticles Subside the Activity of Methicillin-Resistant Staphylococcus aureus by Suppressing PBP2a.

Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) are classified as high-risk infections that can lead to death, particularly among older individuals. Nowadays, plant nanoparticles such as glycyrrhizic acid are recognized as efficient bactericides against a wide range of bacterial strains. Recently, scientists have shown interest in plant extract nanoparticles, derived from natural sources, which can be synthesized into nanomaterials. Interestingly, glycyrrhizic acid is rich in antioxidants as well as antibacterial agents, and it exhibits no adverse effects on normal cells. In this study, glycyrrhizic acid nanoparticles (GA-NPs) were synthesized using the hydrothermal method and characterized through physicochemical techniques such as UV-visible spectrometry, DLS, zeta potential, and TEM. The antimicrobial activity of GA-NPs was investigated through various methods, including MIC assays, anti-biofilm activity assays, ATPase activity assays, and kill-time assays. The expression levels of mecA, mecR1, blaR1, and blaZ genes were measured by quantitative RT-qPCR. Additionally, the presence of the penicillin-binding protein 2a (PBP2a) protein of S. aureus and MRSA was evaluated by a Western blot assay. The results emphasized the fabrication of GA nanoparticles in spherical shapes with a diameter in the range of 40-50 nm. The data show that GA nanoparticles exhibit great bactericidal effectiveness against S. aureus and MRSA. The treatment with GA-NPs remarkably reduces the expression levels of the mecA, mecR1, blaR1, and blaZ genes. PBP2a expression in MRSA was significantly reduced after treatment with GA-NPs. Overall, this study demonstrates that glycyrrhizic acid nanoparticles have potent antibacterial activity, particularly against MRSA. This research elucidates the inhibition mechanism of glycyrrhizic acid, which involves the suppressing of PBP2a expression. This work emphasizes the importance of utilizing plant nanoparticles as effective antimicrobial agents against a broad spectrum of bacteria.

Enhancement of Vitamin C's Protective Effect against Thimerosal-Induced Neurotoxicity in the Cerebral Cortex of Wistar Albino Rats: An In Vivo and Computational Study.

Vitamin C was examined to ameliorate the neurotoxicity of thimerosal (THIM) in an animal model (Wistar albino rats). In our work, oxidative and antioxidative biomarkers such as SOD, LPO, and GSH were investigated at various doses of THIM with or without concurrent vitamin C administration. Furthermore, the adverse effects of THIM on hepatic tissue and cerebral cortex morphology were examined in the absence or presence of associated vitamin C administration. Also, we studied the effect of vitamin C on the metallothionein isoforms (MT-1, MT-2, and MT-3) in silico and in vivo using the RT-PCR assay. The results showed that the antioxidant biomarker was reduced as the THIM dose was raised and vice versa. THIM-associated vitamin C reduced the adverse effects of the THIM dose. The computation studies demonstrated that vitamin C has a lower ΔG of -4.9 kcal/mol compared to -4.1 kcal/mol for THIM to bind to the MT-2 protein, which demonstrated that vitamin C has a greater ability to bind with MT-2 than THIM. This is due to multiple hydrogen bonds that exist between vitamin C and MT-2 residues Lys31, Gln23, Cys24, and Cys29, and the sodium ion represents key stabilizing interactions. Hydrogen bonds involve electrostatic interactions between hydrogen atom donors (e.g., hydroxyl groups) and acceptors (e.g., carbonyl oxygens). The distances between heavy atoms are typically 2.5-3.5 Å. H-bonds provide directed, high-affinity interactions to anchor the ligand to the binding site. The five H-bonds formed by vitamin C allow it to form a stable complex with MT, while THIM can form two H-bonds with Gln23 and Cys24. This provides less stabilization in the binding pocket, contributing to the lower affinity compared to vitamin C. The histopathological morphologies in hepatic tissue displayed an expansion in the portal tract and the hepatocytes surrounding the portal tract, including apoptosis, binucleation, and karyomegaly. The histopathological morphologies in the brain tissue revealed a significant decrease in the number of Purkinje cells due to THIM toxicity. Interestingly, THIM toxicity was associated with hemorrhage and astrogliosis. Both intracellular and vasogenic edema appeared as the concentrations of THIM rose. Finally, vitamin C ameliorated the adverse effect on the cerebral cortex in Wistar albino rats.

Mutation of TBCE causes hypoparathyroidism-retardation-dysmorphism and autosomal recessive Kenny-Caffey syndrome.

The syndrome of congenital hypoparathyroidism, mental retardation, facial dysmorphism and extreme growth failure (HRD or Sanjad-Sakati syndrome; OMIM 241410) is an autosomal recessive disorder reported almost exclusively in Middle Eastern populations. A similar syndrome with the additional features of osteosclerosis and recurrent bacterial infections has been classified as autosomal recessive Kenny-Caffey syndrome (AR-KCS; OMIM 244460). Both traits have previously been mapped to chromosome 1q43-44 (refs 5,6) and, despite the observed clinical variability, share an ancestral haplotype, suggesting a common founder mutation. We describe refinement of the critical region to an interval of roughly 230 kb and identification of deletion and truncation mutations of TBCE in affected individuals. The gene TBCE encodes one of several chaperone proteins required for the proper folding of alpha-tubulin subunits and the formation of alpha-beta-tubulin heterodimers. Analysis of diseased fibroblasts and lymphoblastoid cells showed lower microtubule density at the microtubule-organizing center (MTOC) and perturbed microtubule polarity in diseased cells. Immunofluorescence and ultrastructural studies showed disturbances in subcellular organelles that require microtubules for membrane trafficking, such as the Golgi and late endosomal compartments. These findings demonstrate that HRD and AR-KCS are chaperone diseases caused by a genetic defect in the tubulin assembly pathway, and establish a potential connection between tubulin physiology and the development of the parathyroid.

Care-seeking pathways and diagnostic delays in extrapulmonary TB patients.

SETTING: This study was conducted at a private tertiary hospital engaged with the TB control programme in the city of Lahore, Pakistan. OBJECTIVE: To assess the healthcare-seeking pathways, different delays and factors associated with delays among the patients who presented in the outpatient department with tuberculous lymphadenitis and pleuritis, the most common manifestations of extrapulmonary TB. DESIGN: This cross-sectional study was conducted prospectively from April 2016 to August 2017. RESULTS: The median age of the 339 patients analysed was 22 years (IQR 17-30); tuberculous lymphadenitis was predominant in females (63%), while pleuritis affected more males (64%). Overall, 62% reported seeking care from healthcare providers before diagnosis, of whom 62% sought care from private facilities, 32% visited facilities >2 times and 8% visited traditional healers. Diagnostic delay was associated with tuberculous lymphadenitis, age 15-44 years, poor socio-economic status and poor TB knowledge. CONCLUSION: There was considerable delay in the management of extrapulmonary TB patients, and the health-system delay was the major contributor, leading to increased patient suffering. Efforts towards minimising health-system delay need to be prioritised for patient screening and diagnosis, with a feasible algorithm that is workable in resource-limited settings.

CONTEXTE: Cette étude a été menée dans un hôpital tertiaire privé participant au programme de lutte contre la TB dans la ville de Lahore, au Pakistan. OBJECTIF: Évaluer les parcours de recherche de soins, les différents délais et les facteurs associés aux délais parmi les patients qui se sont présentés au service des consultations externes avec une lymphadénite et une pleurite tuberculeuses, les manifestations les plus courantes de la TB extrapulmonaire. MÉTHODE: Cette étude transversale a été menée prospectivement d'avril 2016 à août 2017. RÉSULTATS: L'âge médian des 339 patients analysés était de 22 ans (IQR 17­30) ; la lymphadénite tuberculeuse prédominait chez les femmes (63%), tandis que la pleurite touchait davantage les hommes (64%). Dans l'ensemble, 62% des patients ont déclaré avoir eu recours à des prestataires de soins de santé avant le diagnostic, dont 62% dans des établissements privés, 32% ont visité des établissements >2 fois et 8% ont consulté des guérisseurs traditionnels. Le retard de diagnostic était associé à la lymphadénite tuberculeuse, à l'âge de 15 à 44 ans, à un statut socio-économique défavorable et à un manque de connaissances sur la TB. CONCLUSION: La prise en charge des patients atteints de TB extrapulmonaire accuse un retard considérable, dont le système de santé est le principal responsable, ce qui accroît les souffrances des patients. Les efforts visant à minimiser le retard du système de santé doivent être priorisés pour le dépistage et le diagnostic des patients, avec un algorithme réalisable dans des environnements aux ressources limitées.

Experimental and theoretical investigations of the effect of bis-phenylurea-based aliphatic amine derivative as an efficient green corrosion inhibitor for carbon steel in HCl solution.

A novel bis-phenylurea-based aliphatic amine (BPUA) was prepared via a facile synthetic route, and evaluated as a potential green organic corrosion inhibitor for carbon steel in 1.0 M HCl solutions. NMR spectroscopy experiments confirmed the preparation of the targeted structure. The corrosion inhibitory behavior of the prospective green compound was explored experimentally by electrochemical methods and theoretically by DFT-based quantum chemical calculations. Obtained results revealed an outstanding performance of BPUA, with efficiency of 95.1% at the inhibitor concentration of 50 mg L-1 at 25 °C. The novel compound has improved the steel resistivity and noticeably reduced the corrosion rate from 33 to 1.7 mils per year. Furthermore, the adsorption study elucidates that the mechanism of the corrosion inhibition activity obeys Langmuir isotherm with mixed physisorption/chemisorption modes for BPUA derivatives on the steel surface. Calculated Gibb's free energy of the adsorption process ranges from -35 to -37 kJ mol-1. The SEM morphology analysis validates the electrochemical measurements and substantiates the corrosion-inhibiting properties of BPUA. Additionally, the eco-toxicity assessment on human epithelial MCF-10A cells proved the environmental friendliness of the BPUA derivatives. Density functional theory (DFT) calculations correlated the inhibitor's chemical structure with the corresponding inhibitory behavior. Quantum descriptors disclosed the potentiality of BPUA adsorption onto the surface through the heteroatom-based functional groups and aromatic rings.

Ultrastructural analysis of zinc oxide nanospheres enhances anti-tumor efficacy against Hepatoma.

Zinc oxide nanomaterial is a potential material in the field of cancer therapy. In this study, zinc oxide nanospheres (ZnO-NS) were synthesized by Sol-gel method using yeast extract as a non-toxic bio-template and investigated their physicochemical properties through various techniques such as FTIR, XR, DLS, and TEM. Furthermore, free zinc ions released from the zinc oxide nanosphere suspended medium were evaluated by using the ICP-AS technique. Therefore, the cytotoxicity of ZnO nanospheres and released Zn ions on both HuH7 and Vero cells was studied using the MTT assay. The data demonstrated that the effectiveness of ZnO nanospheres on HuH7 was better than free Zn ions. Similarly, ZnO-Ns were significantly more toxic to HuH7 cell lines than Vero cells in a concentration-dependent manner. The cell cycle of ZnO-Ns against Huh7 and Vero cell lines was arrested at G2/M. Also, the apoptosis assay using Annexin-V/PI showed that apoptosis of HuH7 and Vero cell lines by ZnO nanospheres was concentration and time-dependent. Caspase 3 assay results showed that the apoptosis mechanism may be intrinsic and extrinsic pathways. The mechanism of apoptosis was determined by applying the RT-PCR technique. The results revealed significantly up-regulated Bax, P53, and Cytochrome C, while the Bcl2 results displayed significant down-regulation and the western blot data confirmed the RT-PCR data. There is oxidative stress of the ZnO nanospheres and free Zn+2 ions. Results indicated that the ZnO nanospheres and free Zn+2 ions induced oxidative stress through increasing reactive oxygen species (ROS) and lipid peroxidation. The morphology of the HuH7 cell line after exposure to ZnO nanospheres at different time intervals revealed the presence of the chromatin condensation of the nuclear periphery fragmentation. Interestingly, the appearance of canonical ultrastructure features of apoptotic morphology of Huh7, Furthermore, many vacuoles existed in the cytoplasm, the majority of which were lipid droplets, which were like foamy cells. Also, there are vesicles intact with membranes that are recognized as swollen mitochondria.

Diverse role of endocannabinoid system in mammalian male reproduction.

Endocannabinoid system (ECS) is known for its modulatory role in numerous physiological processes in the body. Endocannabinoids (eCBs) are endogenous lipid molecules which function both centrally and peripherally. The ECS is best studied in the central nervous system (CNS), immune system as well as in the metabolic system. The role of ECS in male reproductive system is emerging and the presence of a complete enzymatic machinery to synthesize and metabolize eCBs has been demonstrated in male reproductive tract. Endocannabinoid concentrations and alterations in their levels have been reported to affect the functioning of spermatozoa. A dysfunctional ECS has also been linked to the development of prostate cancer, the leading cause of cancer related mortality among male population. This review is an attempt to provide an insight into the significant role of endocannabinoids in male reproduction and further summarize recent findings that demonstrate the manner in which the endocannabinoid system impacts male sexual behavior and fertility.

Vaginal birth after two caesarean sections (VBAC-2)-a systematic review with meta-analysis of success rate and adverse outcomes of VBAC-2 versus VBAC-1 and repeat (third) caesarean sections.

BACKGROUND: Trial of vaginal birth after Caesarean (VBAC) is considered acceptable after one caesarean section (CS), however, women wishing to have trial after two CS are generally not allowed or counselled appropriately of efficacy and complications. OBJECTIVE: To perform a systematic review of literature on success rate of vaginal birth after two caesarean sections (VBAC-2) and associated adverse maternal and fetal outcomes; and compare with commonly accepted VBAC-1 and the alternative option of repeat third CS (RCS). SEARCH STRATEGY: We searched MEDLINE, EMBASE, CINAHL, Cochrane Library, Current Controlled Trials, HMIC Database, Grey Literature Databases (SIGLE, Biomed Central), using search terms Caesarean section, caesarian, C*rean, C*rian, and MeSH headings 'Vaginal birth after caesarean section', combined with second search string two, twice, second, multiple. SELECTION CRITERIA: No randomised studies were available, case series or cohort studies were assessed for quality (STROBE), 20/23 available studies included. DATA COLLECTION AND ANALYSIS: Two independent reviewers selected studies and abstracted and tabulated data and pooled estimates were obtained on success rate, uterine rupture and other adverse maternal and fetal outcomes. Meta-analyses were performed using RevMan-5 to compare VBAC-1 versus VBAC-2 and VBAC-2 versus RCS. MAIN RESULTS: VBAC-2 success rate was 71.1%, uterine rupture rate 1.36%, hysterectomy rate 0.55%, blood transfusion 2.01%, neonatal unit admission rate 7.78% and perinatal asphyxial injury/death 0.09%. VBAC-2 versus VBAC-1 success rates were 4064/5666 (71.1%) versus 38 814/50 685 (76.5%) (P < 0.001); associated uterine rupture rate 1.59% versus 0.72% (P < 0.001) and hysterectomy rates were 0.56% versus 0.19% (P = 0.001) respectively. Comparing VBAC-2 versus RCS, the hysterectomy rates were 0.40% versus 0.63% (P = 0.63), transfusion 1.68% versus 1.67% (P = 0.86) and febrile morbidity 6.03% versus 6.39%, respectively (P = 0.27). Maternal morbidity of VBAC-2 was comparable to RCS. Neonatal morbidity data were too limited to draw valid conclusions, however, no significant differences were indicated in VBAC-2, VBAC-1 and RCS groups in NNU admission rates and asphyxial injury/neonatal death rates (Mantel-Haenszel). CONCLUSIONS: Women requesting for a trial of vaginal delivery after two caesarean sections should be counselled appropriately considering available data of success rate 71.1%, uterine rupture rate 1.36% and of a comparative maternal morbidity with repeat CS option.

Implementing universal rifampicin testing for TB in Pakistan: results and implications for the TB response.

SETTING: A large specialised chest hospital in Pakistan.OBJECTIVE: To study outcomes of comprehensive Xpert® MTB/RIF and universal rifampicin testing.DESIGN: A hospital-based, 3-year retrospective record review of registered TB patients.RESULTS: During the study, 11 744 TB patients were registered when the number of annual notifications remained stagnant. Xpert use increased dramatically in adults with pulmonary TB (PTB) from less than 1% tested with Xpert alone in 2016, to 81.6% by 2018. Significant increases were seen in bacteriologically confirmed PTB in adults from 64% to 77.3% (P < 0.001). No remarkable changes were seen in testing and confirmation of PTB in children or extrapulmonary TB. Significantly lower (P < 0.001) rifampicin resistance (RR) was observed among those with smear-negative compared to those with smear-positive results among new (1.0% vs. 5.1%) and previously treated PTB cases (2.0% vs. 14.4%). Most importantly, a significant decline in RR was observed among previously treated individuals, from 15.4% (95%CI 12.2-19.0) to 8.6% (95%CI 6.6-11.0) during the study. A decrease in RR was also documented in newly diagnosed PTB patients, but this was not statistically significant.CONCLUSION: Universal rifampicin testing is feasible in adult PTB patients; a decline in RR was seen among previously treated individuals with PTB.

Primary drug resistance in extra-pulmonary tuberculosis: a hospital-based prospective study from Pakistan.

SETTING: Tertiary level specialised tuberculosis (TB) hospital.OBJECTIVE: To determine the prevalence of antimicrobial resistance in new extra-pulmonary tuberculosis (EPTB) cases.DESIGN: Prospective cross-sectional study. Presumptive EPTB patients with enlarged lymph nodes or pleural effusion having no history of TB treatment were enrolled. Specimens were tested for smear, Xpert® MTB/RIF and culture. Indirect drug susceptibility testing (DST) was performed using MGIT 960 and line-probe assays (LPA).RESULTS: Among 671 cases, 255 were bacteriologically confirmed and 185 DSTs were performed. Multidrug resistance (MDR-TB) was reported in 2.2% (95%CI 0.6-5.4), any resistance to rifampicin (RMP) in 2.7% (95%CI 0.9-6.2), isoniazid (INH) in 7.6% (95%CI 4.1-12.4), ethambutol in 1.1% (95%CI 0.1-3.9), pyrazinamide in 2.2% (95%CI 0.9-5.5) and fluoroquinolones (FQ) in 6.0% (95%CI 3.0-10.4). The sensitivity and specificity of LPA-DST was 100% and >98.8% respectively for RMP, INH and FQ. Among 82 cases with RMP of the results available for all three methods used, five were reported to be resistant on Xpert but all five were susceptible on MGIT 960 and four on MTBDRplus.CONCLUSION: Prevalence of RMP resistance in new EPTB cases is 2.7% (95%CI 0.9-6.2). Caution is warranted for RMP resistance detected using Xpert in EPTB samples with a very low bacterial load.

Sputum smear microscopy in the Xpert® MTB/RIF era.

A balanced perspective is advocated for the assessment and application of the most recent and the oldest diagnostic methods for pulmonary tuberculosis (TB)-the molecular Xpert® MTB/RIF assay and microscopy for acid-fast bacilli. We discuss their respective merits and shortcomings and identify threats that may hamper their use in TB control. Neither test on its own provides all the information needed for diagnosis and treatment monitoring. Considering all aspects important for both individual patient care and disease control, neither seems 'better' than the other. The required advancement of microscopy had already been hampered before the introduction of the GeneXpert technology by unsuccessful and probably misguided attempts to decentralise culture-based diagnosis and drug susceptibility testing. It seems evident that systematic replacement of microscopy by Xpert is not a viable option for the foreseeable future. Instead, the two methods should complement each other to arrive at a comprehensive, accessible and continuous service for a maximum number of patients. This will intrinsically prioritise targeting the most potent transmitters with the worst prognosis, simultaneously offering optimised prospects for efficient TB control. New microscopy and Xpert applications are expected to ultimately make control programmes independent of culture-based methods in diagnosis, treatment monitoring and outcome assessment.

Synthesis of a novel hexaazatriphenylene derivative for the selective detection of copper ions in aqueous solution.

A hexaazatriphenylene (HAT) derivative, naphtho[2,3-h]naphtho[2',3':7,8]quinoxalino[2,3-a]naphtho[2',3':7,8]quinoxalino[2,3-c]phenazine-5,10,15,20,25,30-hexaone (NQH) was synthesized, characterized, and found to have novel properties in being selective toward the detection of copper (Cu2+) ions. The capability of NQH to be employed as a colorimetric, chemo-fluorescence and electrochemical sensor for the detection of Cu2+ was demonstrated by performing UV-Vis absorbance, fluorescence intensity, and cyclic voltammetry (CV) measurements. The interaction between NQH and Cu2+ was initially observed with an obvious color change from yellow to brown upon the addition of Cu2+ ions to NQH. The interaction was also confirmed by UV-Vis absorbance, fluorescence intensity, and mass spectroscopy (MS/MS) measurements. UV absorbance, fluorescence and CV of NQH toward Cu2+ showed good linearity with a detection limit of 3.32 µM, 2.20 µM and 0.78 µM, respectively, which are lower than the toxicity levels of copper in drinking water (20-30 µM) set by the U.S. Environmental Protection Agency (EPA) and World Health Organization (WHO). A 1 : 2 stoichiometry complexation between NQH and Cu2+ was confirmed by Job's plot and MS/MS. In addition, the selectivity and sensitivity of the NQH compound towards Cu2+ ions were further confirmed by performing CV on a screen printed flexible and planar electrochemical sensor.