Promoter characterization of relZ-bifunctional (pp)pGpp synthetase in mycobacteria.

The second messenger guanosine 3',5'-bis(diphosphate)/guanosine tetraphosphate (ppGpp) and guanosine 3'-diphosphate 5'-triphosphate/guanosine pentaphosphate (pppGpp) ((p)ppGpp) has been shown to be crucial for the survival of mycobacteria under hostile conditions. Unexpectedly, deletion of primary (p)ppGpp synthetase-Rel did not completely diminish (p)ppGpp levels leading to the discovery of novel bifunctional enzyme-RelZ, which displayed guanosine 5'-monophosphate,3'-diphosphate (pGpp), ppGpp, and pppGpp ((pp)pGpp) synthesis and RNAseHII activity. What conditions does it express itself under, and does it work in concert with Rel? The regulation of its transcription and whether the Rel enzyme plays a role in such regulation remain unclear. In this article, we have studied relZ promoter and compared its activity with rel promoter in different growth conditions. We observed that the promoter activity of relZ was constitutive; it is weaker than rel promoter, lies within 200 bp upstream of translation-start site, and it increased under carbon starvation. Furthermore, the promoter activity of relZ was compromised in the rel-knockout strain in the stationary phase. Our study unveils the dynamic regulation of relZ promoter activity by SigA and SigB sigma factors in different growth phases in mycobacteria. Importantly, elucidating the regulatory network of RelZ would enable the development of the targeted interventions for treating mycobacterial infections.

Oral rhabdomyosarcoma of mandibular region: A case report.

Rhabdomyosarcoma is a malignant neoplasm of mesenchymal cells, showing varying degrees of striated muscle cell differentiation. The most common sites of occurrence are the head and neck (40%), genitourinary tract (25%), and extremities (20%). Rhabdomyosarcoma is anatomically divided into two categories including parameningeal and nonparameningeal. It predominantly occurs in children while rarely found in adults, and involvement of the oral cavity accounts for only 10%-20% of all head and neck cases. The present case is of oral rhabdomyosarcoma of a 27-year-old woman, involving the mandibular region and demonstrates its clinical, radiological, histological, and immunohistochemical findings.

Tale of Twin Bifunctional Second Messenger (p)ppGpp Synthetases and Their Function in Mycobacteria.

M. tuberculosis, an etiological agent of tuberculosis, requires a long treatment regimen due to its ability to respond to stress and persist inside the host. The second messenger (p)ppGpp-mediated stress response plays a critical role in such long-term survival, persistence, and antibiotic tolerance which may also lead to the emergence of multiple drug resistance. In mycobacteria, (pp)pGpp molecules are synthesized predominantly by two bifunctional enzymes-long RSH-Rel and short SAS-RelZ. The long RSH-Rel is a major (p)ppGpp synthetase and hydrolase. How it switches its activity from synthesis to hydrolysis remains unclear. RelMtb mutant has been reported to be defective in biofilm formation, cell wall function, and persister cell formation. The survival of such mutants has also been observed to be compromised in infection models. In M. smegmatis, short SAS-RelZ has RNase HII activity in addition to (pp)Gpp synthesis activity. The RNase HII function of RelZ has been implicated in resolving replication-transcription conflicts by degrading R-loops. However, the mechanism and regulatory aspects of such a regulation remain elusive. In this article, we have discussed (p)ppGpp metabolism and its role in managing the stress response network of mycobacteria, which is responsible for long-term survival inside the host, making it an important therapeutic target.

Comparative effectiveness of low-level laser therapy and transcutaneous electrical nerve stimulation in symptomatic Temporomandibular Disorders: A Randomised Control Trial.

BACKGROUND: Myofascial pain is one of the most common facial region pains, presenting itself with various signs and symptoms like tenderness of masticatory muscles and difficulty in mouth opening. Considering its multifactorial aetiology various treatment modalities are present. OBJECTIVE: The aim of this study is to compare the effectiveness of transcutaneous electrical nerve stimulation (TENS)and low-level laser therapy (LLLT) for the patients of temporomandibular disorders (TMDS). METHODS: The study was conducted with 20 patients diagnosed with TMDS. Group A received LLLT at 660 nm, 6 J/point, two sessions/week for 4 weeks and group B received TENS at 2-250 Hz, two sessions/week for 4 weeks. RESULTS: Pain Score decreased and mouth opening increased in both groups with time, however, the difference between the two groups was not statistically significant. Right and left lateral excursions showed improvement at different intervals in both groups. However, the LLLT group showed significant improvement. CONCLUSION: Clinical trial showed improvement in visual analogue scale (VAS), maximum mouth opening (MMO) and lateral excursion in the different time intervals in both the groups with LLLT showed more improvement with lateral excursions movements.

An Unusual Presentation of Superior Mesenteric Venous Occlusion in Mild COVID-19.

SARS-CoV-2, an etiological agent of COVID-19, has been reported to inflict remarkably diverse manifestations in different subjects across the globe. Though patients with COVID-19 predominantly have fever, respiratory and constitutional symptoms, atypical presentations are becoming increasingly evident. COVID-19 may predispose to both venous and arterial thromboembolism due to excessive inflammation, hypoxia, immobilization, and diffuse intravascular coagulation in moderate to severe symptomatic cases. In this case report, we are reporting thromboembolic complications of COVID-19 in a mild symptomatic subject incidentally diagnosed with mesenteric venous occlusion with no abdominal symptoms. Early recognition of the abdominal symptoms, diagnosis, initiation of anticoagulants, and timely surgical intervention may improvise the outcome in a patient with COVID-19 infection-induced mesenteric thrombosis. Superior mesenteric artery and venous thrombosis may lead to subsequent ischemia necessitating emergency laparotomy. Thus, the usage of low-dose anticoagulants in all the patients of COVID-19 irrespective of the categorization into mild, moderate, and severe COVID-19 disease should be considered.

Vitamin C and its therapeutic potential in the management of COVID19.

COVID19 has emerged as one of the worst pandemics in the history of mankind. Several vaccines have been approved by different government agencies worldwide, but data on their efficacy and safety are limited, and distribution remains a massive challenge. As per WHO, personal immunity is vital for protection against COVID19. Earlier, Vitamin C-mediated pathways have been shown to play critical role in boosting immunity attributed to its antioxidant properties. Recently, the involvement of such pathways in protection against COVID19 has been suggested. The controlled doses of Vitamin C administered through intravenous (IV) injections are being studied for determining its role in the prognosis of COVID19. In this article, we have discussed the potential role of Vitamin C in the management in COVID19 patients and presented recent clinical trials data. Additionally, we have elaborated the possibility of administering Vitamin C through inhalers in order to achieve local high concentration and the challenges of such approach.

In vivo assessment of a single adenine mutation in 5'UTR of Endothelin-1 gene in paediatric cases with severe pulmonary hypertension: an observational study.

OBJECTIVE: Endothelin-1 plays an important role in the pathogenesis of severe pulmonary hypertension. The + 139 'A', adenine insertion variant in 5'UTR of edn1 gene has been reported to be associated with increased expression of Endothelin-1 in vitro. The aim of present study was to explore the association of this variant with the circulating levels of Endothelin-1 in vivo using archived DNA and plasma samples from 38 paediatric congenital heart disease (cyanotic and acyanotic) patients with severe pulmonary hypertension. RESULTS: The plasma Endothelin-1 levels were highly varied ranging from 1.63 to75.16 pg/ml. The + 139 'A' insertion variant in 5'UTR of edn1 was seen in 8 out of 38 cases with only one acyanotic sample demonstrating homozygosity of inserted 'A' allele at + 139 site (4A/4A genotype). The plasma Endothelin-1 levels in children with homozygous variant 3A/3A genotype were comparable in cyanotic and acyanotic groups. Lone 4A/4A acyanotic sample had ET-1 levels similar to the median value of ET-1 associated with 3A/3A genotype and was absent in cyanotic group presumably due to deleterious higher ET-1 levels. The discussed observations, limited by the small sample size, are suggestive of homozygous adenine insertion variant posing a risk in cyanotic babies with Severe Pulmonary Hypertension.

Use of the International League Against Epilepsy (ILAE) 1989, 2010, and 2017 Classification of Epilepsy in children in a low-resource setting: A hospital-based cross-sectional study.

OBJECTIVES: This cross-sectional study was designed to test the applicability of the 1989, 2010, and 2017 International League Against Epilepsy (ILAE) classification of epilepsy in children from a resource-limited setting in India. METHODS: Classification of seizure types and syndromes was done through parental interviews and review of medical records in children with epilepsy aged one month to 18 years. Available investigations including EEG, MRI, and metabolic/genetic tests were used in classifying patients as per the 1989, 2010, and 2017 ILAE (level II-epilepsy type) classification. We compared the proportion of children remaining unclassified by each scheme. RESULTS: Seven hundred and twenty-six children (436 males, mean age 6.4 ± 4.6 years) were enrolled. Using the 1989 ILAE classification, we were able to classify 95.7%, and 82.6% children by the 2010 scheme. The 2017 ILAE classification could classify all 726 children at level I (seizure type), 664 (91.0%) children at level II (epilepsy type), and an electroclinical syndrome could be identified in 409 (56.1%) of the children. An etiology could be identified in 75%, perinatal brain injury being the most frequent. West syndrome was the most common electroclinical syndrome, identified in 22.7% patients. The 1989 ILAE classification system was superior to the 2010 system (P = .01) in epilepsy classification. There was no difference between the 1989 and 2017 schemes (P = .31) or the 2010 and 2017 schemes (P = .10). SIGNIFICANCE: The 2017 ILAE classification, being multidimensional, allowed classification of children who could not undergo extensive evaluation due to economic constraints and also provided room for overlapping etiologies.

A novel function of Mycobacterium tuberculosis chaperonin paralog GroEL1 in copper homeostasis.

Among the two GroEL paralogs in Mycobacterium tuberculosis, GroEL1 and GroEL2, GroEL1 has a characteristic histidine-rich C terminus. Since histidine richness is likely to be involved in metal binding, we attempted to decipher the role of GroEL1 in chelating metals and the consequence on M. tuberculosis physiology. Isothermal titration calorimetry showed that GroEL1 binds copper and other metals. Mycobacterial viability assay, redox balance, and DNA protection assay concluded that GroEL1 protects from copper stress in vitro. Solution X-ray scattering and constrained modeling of GroEL1 -/+ copper ions showed reorientation of the apical domain as seen in functional assembly. We conclude that the duplication of chaperonin genes in M. tuberculosis might have led to their evolutionary divergence and consequent functional divergence of chaperonins.

Development of a highly effective low-cost vaporized hydrogen peroxide-based method for disinfection of personal protective equipment for their selective reuse during pandemics.

BACKGROUND: Personal Protective Equipment (PPE) is required to safely work with biological agents of bacterial (i.e. Mycobacterium tuberculosis) or viral origin (Ebola and SARS). COVID-19 pandemic especially has created unforeseen public health challenges including a global shortage of PPE needed for the safety of health care workers (HCWs). Although sufficient stocks of PPE are currently available, their critical shortage may develop soon due to increase in demand and depletion of existing supply lines. To empower our HCWs and ensure their continued protection, proactive measures are urgently required to develop procedures to safely decontaminate the PPEs to allow their "selective reuse" during contingency situations. METHODS: Herein, we have successfully developed a decontamination method based on vaporized hydrogen peroxide (VHP). We have used a range of concentration of hydrogen peroxide to disinfect PPE (coveralls, face-shields, and N-95 masks). To ensure a proper disinfection, we have evaluated three biological indicators namely Escherichia coli, Mycobacterium smegmatis and spores of Bacillus stearothermophilus, considered as the gold standard for disinfection processes. We next evaluated the impact of repeated VHP treatment on physical features, permeability, and fabric integrity of coveralls and N-95 masks. Next, we performed Scanning Electron Microscopy (SEM) to evaluate microscopic changes in fiber thickness of N-95 masks, melt blown layer or coverall body suits. Considering the fact that any disinfection procedure should be able to meet local requirements, our study included various regionally procured N-95 masks and coveralls available at our institute All India Institute of Medical Sciences (AIIMS), New Delhi, India. Lastly, the practical utility of VHP method developed herein was ascertained by operationalizing a dedicated research facility disinfecting used PPE during COVID-19. RESULTS: Our prototype studies show that a single VHP cycle (7-8% Hydrogen peroxide) could disinfect PPE and PPE housing room of about 1200 cubic feet (length10 ft × breadth 10 ft × height 12 ft) in less than 10 min, as noted by a complete loss of B. stearothermophilus spore revival. The results are consistent and reproducible as tested in over 10 cycles in our settings. Further, repeated VHP treatment did not result in any physical tear, deformity or other appreciable change in the coverall and N-95 masks. Our permeation tests evaluating droplet penetration did not reveal any change in permeability post-VHP treatments. Also, SEM analysis indeed revealed no significant change in fiber thickness or damage to fibers of coveralls or melt blown layer of N-95 masks essential for filtration. There was no change in user comfort and experience following VHP treatment of PPE. Based on results of these studies, and parameters developed and optimized, an institutional research facility to disinfect COVID-19 PPE is successfully established and operationalized with more than 80% recovery rate for used PPE post-disinfection. CONCLUSIONS: Our study, therefore, successfully establishes the utility of VHP to effectively disinfect PPE for a possible reuse as per the requirements. VHP treatment did not damage coveralls, cause physical deformity and also did not alter fabric architecture of melt blown layer. We observed that disinfection process was successful consistently and therefore believe that the VHP-based decontamination model will have a universal applicability and utility. This process can be easily and economically scaled up and can be instrumental in easing global PPE shortages in any biosafety facility or in health care settings during pandemic situation such as COVID-19.

Air quality forecasting using artificial neural networks with real time dynamic error correction in highly polluted regions.

Air pollution is an important issue, especially in megacities across the world. There are emission sources within and also in the regions around these cities, which cause fluctuations in air quality based on prevailing meteorological conditions. Short term air quality forecasting is used not to just possibly mitigate forthcoming high air pollution episodes, but also to plan for reduced exposures of residents. In this study, a model using Artificial Neural Networks (ANN) has been developed to forecast pollutant concentration of PM10, PM2.5, NO2, and O3 for the current day and subsequent 4 days in a highly polluted region (32 different locations in Delhi). The model has been trained using meteorological parameters and hourly pollution concentration data for the year 2018 and then used for generating air quality forecasts in real-time. It has also been equipped with Real Time Correction (RTC), to improve the quality of the forecasts by dynamically adjusting the forecasts based on the model performance during the past few days. The model without RTC performs decently, but with RTC the errors are further reduced in forecasted values. The utility of the model has been demonstrated in real-time and model validations were performed for the whole year of 2018 and also independently for 2019. The model shows very good performance for all the pollutants on several evaluation metrics. Coefficient of correlations for various pollutants varies from 0.79-0.88 to 0.49-0.68 between the Day0 to Day4 forecasts. Lowest deterioration of performance was observed for ozone over the four days of forecasts. Use of RTC further improves the model performance for all pollutants.

Amine functionalized and Fe3O4 incorporated activated carbon for bisphenol-A separation.

Activated carbon (AC) is the most commonly used adsorbent for water purification, although the dispersive nature of AC in aqueous solution poses a serious problem. To overcome this limitation, AC was magnetized with iron oxide using iron salts as precursor. Further to enhance its effectiveness, it was impregnated with Aliquat 336. Different characterization techniques (Fourier transform infrared spectroscopy (FT-IR), field emission scanning electron microscopy (FE-SEM), along with energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD)) were used to analyze the adsorbent. Furthermore, the value of the pH at which the overall charge on the surface of the adsorbent is neutral was found by pH drift method. The modified form of the activated carbon was used to treat the aqueous solution of bisphenol-A in the batch as well as in the continuous mode of operation. In batch mode, the data were validated using equilibrium and kinetic models, and in continuous mode, data were fitted with the Thomas, Adams-Bohart, and bed depth service time (BDST) fixed bed adsorption models. Also, the changes in Gibb's free energy, enthalpy, and entropy were estimated from the temperature study. The design of an adsorption column is proposed to treat 10,000 L/day of an industrial effluent containing BPA.

A study on the prevalence of depression and the severity of depression in patients of chronic obstructive pulmonary disease in a semi-urban Indian population.

Chronic obstructive pulmonary disease (COPD) is one of the few respiratory diseases which is associated with a number of comorbidities. Psychiatric disease like depression is a very important comorbidity of COPD because it decreases the feeling of wellbeing in the patient and also interferes with the compliance with medication thereby increasing the risk of hospitalization in the COPD patient. A cross-sectional study was done for two years in the department of pulmonary medicine at Era's Lucknow medical college and hospital, Lucknow. A total of 150 patients were enrolled for the study after a clinico-radiological screening for the diagnosis confirmed on spirometry. After the confirmation of the diagnosis of COPD in these patients, they were screened for depression using the PHQ-9 scale in our department of pulmonary medicine. The confirmation of the diagnosis of depression was done according to the ICD-10 guidelines for depression and the severity of depression was graded using HAM-D scale in the department of psychiatry at our institute. The most common age group enrolled in the study was 51-60 years of age (40.67%). One hundred and fifteen patients (76.7%) of the enrolled patients were smokers while the remaining 35 patients (23.3%) were non-smokers. Depression was found to be present in 46 out of the total 150 patients in the study. Thus, the prevalence of depression in our study was 30.67%. Depression was seen in COPD groups B,C and D. Out of the 46 patients of COPD with depression,18 had mild depression (39.13%), 26 had moderate depression (56.52%) and 2 had severe depression (4.35%). Hence, depression of all grades (i.e., mild, moderate and severe depression) is seen in COPD groups B, C and D.

Multifaceted remodeling by vitamin C boosts sensitivity of Mycobacterium tuberculosis subpopulations to combination treatment by anti-tubercular drugs.

Bacterial dormancy is a major impediment to the eradication of tuberculosis (TB), because currently used drugs primarily target actively replicating bacteria. Therefore, decoding of the critical survival pathways in dormant tubercle bacilli is a research priority to formulate new approaches for killing these bacteria. Employing a network-based gene expression analysis approach, we demonstrate that redox active vitamin C (vit C) triggers a multifaceted and robust adaptation response in Mycobacterium tuberculosis (Mtb) involving ~ 67% of the genome. Vit C-adapted bacteria display well-described features of dormancy, including growth stasis and progression to a viable but non-culturable (VBNC) state, loss of acid-fastness and reduction in length, dissipation of reductive stress through triglyceride (TAG) accumulation, protective response to oxidative stress, and tolerance to first line TB drugs. VBNC bacteria are reactivatable upon removal of vit C and they recover drug susceptibility properties. Vit C synergizes with pyrazinamide, a unique TB drug with sterilizing activity, to kill dormant and replicating bacteria, negating any tolerance to rifampicin and isoniazid in combination treatment in both in-vitro and intracellular infection models. Finally, the vit C multi-stress redox models described here also offer a unique opportunity for concurrent screening of compounds/combinations active against heterogeneous subpopulations of Mtb. These findings suggest a novel strategy of vit C adjunctive therapy by modulating bacterial physiology for enhanced efficacy of combination chemotherapy with existing drugs, and also possible synergies to guide new therapeutic combinations towards accelerating TB treatment.

Genome-wide expression profiling establishes novel modulatory roles of vitamin C in THP-1 human monocytic cell line.

BACKGROUND: Vitamin C (vit C) is an essential dietary nutrient, which is a potent antioxidant, a free radical scavenger and functions as a cofactor in many enzymatic reactions. Vit C is also considered to enhance the immune effector function of macrophages, which are regarded to be the first line of defence in response to any pathogen. The THP-1 cell line is widely used for studying macrophage functions and for analyzing host cell-pathogen interactions. RESULTS: We performed a genome-wide temporal gene expression and functional enrichment analysis of THP-1 cells treated with 100 µM of vit C, a physiologically relevant concentration of the vitamin. Modulatory effects of vitamin C on THP-1 cells were revealed by differential expression of genes starting from 8 h onwards. The number of differentially expressed genes peaked at the earliest time-point i.e. 8 h followed by temporal decline till 96 h. Further, functional enrichment analysis based on statistically stringent criteria revealed a gamut of functional responses, namely, 'Regulation of gene expression', 'Signal transduction', 'Cell cycle', 'Immune system process', 'cAMP metabolic process', 'Cholesterol transport' and 'Ion homeostasis'. A comparative analysis of vit C-mediated modulation of gene expression data in THP-1cells and human skin fibroblasts disclosed an overlap in certain functional processes such as 'Regulation of transcription', 'Cell cycle' and 'Extracellular matrix organization', and THP-1 specific responses, namely, 'Regulation of gene expression' and 'Ion homeostasis'. It was noteworthy that vit C modulated the 'Immune system' process throughout the time-course. CONCLUSIONS: This study reveals the genome-wide effects of physiological levels of vit C on THP-1 gene expression. The multitude of effects impacted by vit C in macrophages highlights its role in maintaining homeostasis of several cellular functions. This study provides a rational basis for the use of the Vitamin C- THP-1 cell model, to study biochemical and cellular responses to stresses, including infection with M. tuberculosis and other intracellular pathogens.

The pleiotropic transcriptional response of Mycobacterium tuberculosis to vitamin C is robust and overlaps with the bacterial response to multiple intracellular stresses.

Mycobacterium tuberculosis (Mtb) owes its success as a pathogen in large measure to its ability to exist in a persistent state of 'dormancy' resulting in a lifelong latent tuberculosis (TB) infection. An understanding of bacterial adaptation during dormancy will help in devising approaches to counter latent TB infection. In vitro models have provided valuable insights into bacterial adaptation; however, they have limitations because they do not disclose the bacterial response to the intracellular environment wherein the bacteria are simultaneously exposed to multiple stresses. We describe the pleiotropic response of Mtb in the vitamin C (vit C) model of dormancy developed in our laboratory. Vit C mediates a rapid regulation of genes representing ~14 % of the genome in Mtb cultures. The upregulated genes were better represented in lipid, intermediary metabolism and regulatory protein categories. The downregulated genes mainly related to virulence, detoxification, information pathways and cell wall processes. A comparison of this response to that in other models indicates that vit C generates a multiple-stress environment for axenic Mtb cultures that resembles a macrophage-like environment. The bacterial response to vit C resembles responses to gaseous stresses such as hypoxia and nitric oxide, oxidative and nitrosative stresses, nutrient starvation and, notably, the activated macrophage environment itself. These responses demonstrate that the influence of vit C on Mtb gene expression extends well beyond the DevR dormancy regulon. A detailed characterization of the response to vit C is expected to disclose useful strategies to counter the adaptive mechanisms essential to Mtb dormancy.