Structure-based design of small molecule inhibitors of the cagT4SS ATPase Cagα of Helicobacter pylori.

We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori. The secretion system is encoded by the cag pathogenicity island, and we chose Cagα, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cagα in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cagα subunits and mutagenesis of the binding site identified Cagα residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889, and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cagα, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect.

A mentored hands-on training model for scaling up implementation and intervention research in India: "connecting the dots".

Despite the high burden of mental disorders in low- and middle-income countries (LMICs), less than 25% of those in need have access to appropriate services, in part due to a scarcity of locally relevant, evidence-based interventions and models of care. To address this gap, researchers from India and the United States and the Indian Council of Medical Research (ICMR) collaboratively developed a "Grantathon" model to provide mentored research training to 24 new principal investigators (PIs). This included a week-long didactic training, a customized web-based data entry/analysis system and a National Coordination Unit (NCU) to support PIs and track process objectives. Outcome objectives were assessed via scholarly output including publications, awards received and subsequent grants that were leveraged. Multiple mentorship strategies including collaborative problem-solving approaches were used to foster single-centre and multicentre research. Flexible, approachable and engaged support from mentors helped PIs overcome research barriers, and the NCU addressed local policy and day-to-day challenges through informal monthly review meetings. Bi-annual formal review presentations by all PIs continued through the COVID-19 pandemic, enabling interim results reporting and scientific review, also serving to reinforce accountability. To date, more than 33 publications, 47 scientific presentations, 12 awards, two measurement tools, five intervention manuals and eight research grants have been generated in an open-access environment. The Grantathon is a successful model for building research capacity and improving mental health research in India that could be adopted for use in other LMICs.

Cloning, expression and characterization of a thermo-alkali-stable xylanase from Aspergillus oryzae LC1 in Escherichia coli BL21(DE3).

In the present investigation, cloning and overexpression of xylanase (XynF1), the main xylanase of A. oryzae LC1, was performed in prokaryotic system E. coli BL21(DE3) to produce recombinant xylanase with high titer of specific activity (1037.3 U/mg), which was 9.3-fold higher than the native strain. Further, the recombinant XynF1 of size 37 kDa was purified using Ni2+-NTA resins followed by cation exchange chromatography, which showed an 1.8-fold increase in purity with 71.4% yield. The r-XynF1 exhibited a wide range of activity at different pH (3.0-10.0) range and temperature (30-70 °C) with an optimum pH at 5.0 and temperature at 30 °C. The results from the current study, clearly demonstrate that this is an effective method to generate a recombinant enzyme with improved activity, making it useful for possible industrial applications.

Defect engineering, microstructural examination and improvement of ultrafast third harmonic generation in GaZnO nanostructures: a study of e-beam irradiation.

Electron beam induced effects on defect engineering and structural, morphological and optical properties of Ga doped ZnO (GaZnO) nanostructures for improved ultrafast nonlinear optical properties are presented. A microstructural analysis was carried out based on the Scherrer, Williamson-Hall, and size-strain models. All three models reveal a peak broadening effect upon electron beam irradiation (EBI) and the crystallite size of the films shows a decrease of 30% compared to unirradiated nanostructures. The decrease in intensity, variation in the peak position and broadening of the Raman E2H mode confirm that the EBI treatment introduces disorder into the nanostructures. The interband gap emissions observed in photoluminescence spectra are primarily due to defect-related emissions originating from intrinsic defects such as Zni, Oi, VZn, VO, VZn+, VO+ and OZn. The O1s core-level spectra show that the peak related to oxygen vacancy defects is suppressed upon EBI. Surface morphology studies reveal that the nucleation barriers of GaZnO nanostructures are reduced upon irradiation treatment resulting in a coalescence mechanism. Third harmonic generation studies show that higher electron-beam doses lead to the occurrence of enhanced THG signals due to a drastic change in the occupation of localized defect levels. Thermally induced nonlinear optical studies depict an improved χ(3) of 1.71 × 10-3 esu upon irradiation due to enhanced FCA induced TPA mechanism and non-radiative transitions which indicates the credibility of the grown films in photonic devices.

Helicobacter pylori helicase loader protein Hp0897 shows unique functions of N- and C-terminal regions.

Helicase loaders are required for the loading of helicases at the vicinity of replication origins. In Helicobacter pylori, Hp0897 has been shown to be a potential helicase loader for replicative helicase (HpDnaB) although it does not show any sequence homology with conventional DnaC like helicase loader proteins. Therefore, it is important to investigate the in vivo role of Hp0897 and structure-function analysis with respect to domain mapping of Hp0897 and HpDnaB. Although HporiC is divided into oriC1 and oriC2, the latter has been assigned as functional origin based on loading of initiator protein HpDnaA. Using chromatin immunoprecipitation (ChIP) experiment, we show preferential binding of Hp0897 at oriC2 over oriC1 like HpDnaA highlighting its helicase loader function in vivo. Furthermore, we generated series of deletion mutants for HpDnaB and Hp0897 that enabled us to map the domains of interaction between these two proteins. Interestingly, the C-terminal domain of Hp0897 (Hp0897CTD) shows stronger interaction with HpDnaB over the N-terminal region of Hp0897 (Hp0897NTD). Similar to the full-length protein, Hp0897CTD also stimulates the DNA binding activity of HpDnaB. Furthermore, overexpression of Hp0897 full-length protein in H. pylori leads to an elongated cell phenotype. While the overexpression of Hp0897CTD does not show a phenotype of cell elongation, overexpression of Hp0897NTD shows extensive cell elongation. These results highlight the possible role of Hp0897CTD in helicase loading and Hp0897NTD's unique function linked to cell division that make Hp0897 as a potential drug target against H. pylori.

Unraveling the factors and mechanism involved in persistence: Host-pathogen interactions in Helicobacter pylori.

Helicobacter pylori and humans have one of the most complex relationships in nature. How a bacterium manages to live in one of the harshest and hostile environments is a topic of unraveling mysteries. H. pylori is a prevalent species and it colonizes the human gut of more than 50% of the world population. It infects the epithelial region of antrum and persists there for a long period. Over the time of evolution, H. pylori has developed complex strategies to extend the degree of inflammation in gastric mucosa. H. pylori needs specific adaptations for initial colonization into the host environment like helical shape, flagellar movement, chemotaxis, and the production of urease enzyme that neutralizes acidic environment of the stomach. There are several factors from the bacterium as well as from the host that participate in these complex interactions. On the other hand, to establish the persistent infection, H. pylori escapes the immune system by mimicking the host antigens. This pathogen has the ability to dodge the immune system and then persist there in the form of host cell, which leads to immune tolerance. H. pylori has an ability to manipulate its own pathogen-associated molecular patterns, which leads to an inhibition in the binding with specific pattern recognition receptors of the host to avoid immune cell detection. Also, it manipulates the host metabolic homeostasis in the gastric epithelium. Besides, it has several genes, which may get involved in the acquisition of nutrition from the host to survive longer in the host. Due to the persistence of H. pylori, it causes chronic inflammation and raises the chances of gastric cancer. This review highlights the important elements, which are certainly responsible for the persistence of H. pylori in the human host.

Prediction of 30-Day Readmission for COPD Patients Using Accelerometer-Based Activity Monitoring.

Chronic obstructive pulmonary disease (COPD) claimed 3.0 million lives in 2016 and ranked 3rd among the top 10 global causes of death. Moreover, once diagnosed and discharged from the hospital, the 30-day readmission risk in COPD patients is found to be the highest among all chronic diseases. The existing diagnosis methods, such as Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2019, Body-mass index, airflow Obstruction, Dyspnea, and Exercise (BODE) index, modified Medical Research Council (mMRC), COPD assessment test (CAT), 6-minute walking distance, which are adopted currently by physicians cannot predict the potential readmission of COPD patients, especially within the 30 days after discharge from the hospital. In this paper, a statistical model was proposed to predict the readmission risk of COPD patients within 30-days by monitoring their physical activity (PA) in daily living with accelerometer-based wrist-worn wearable devices. This proposed model was based on our previously reported PA models for activity index (AI) and regularity index (RI) and it introduced a new parameter, quality of activity (QoA), which incorporates previously proposed parameters, such as AI and RI, with other activity-based indices to predict the readmission risk. Data were collected from continuous PA monitoring of 16 COPD patients after hospital discharge as test subjects and readmission prediction criteria were proposed, with a 63% sensitivity and a 37.78% positive prediction rate. Compared to other clinical assessment, diagnosis, and prevention methods, the proposed model showed significant improvement in predicting the 30-day readmission risk.

'Modulation of the enzymatic activities of replicative helicase (DnaB) by interaction with Hp0897: a possible mechanism for helicase loading in Helicobacter pylori'.

DNA replication in Helicobacter pylori is initiated from a unique site (oriC) on its chromosome where several proteins assemble to form a functional replisome. The assembly of H. pylori replication machinery is similar to that of the model gram negative bacterium Escherichia coli except for the absence of DnaC needed to recruit the hexameric DnaB helicase at the replisome assembly site. In the absence of an obvious DnaC homologue inH. pylori, the question arises as to whether HpDnaB helicase is loaded at theHp-replication origin by itself or is assisted by other unidentified protein(s). A high-throughput yeast two-hybrid study has revealed two proteins of unknown functions (Hp0897 and Hp0340) that interact with HpDnaB. Here we demonstrate that Hp0897 interacts with HpDnaB helicase in vitro as well as in vivo Furthermore, the interaction stimulates the DNA binding activity of HpDnaB and modulates its adenosine triphosphate hydrolysis and helicase activities significantly. Prior complex formation of Hp0897 and HpDnaB enhances the binding/loading of DnaB onto DNA. Hp0897, along with HpDnaB, colocalizes with replication complex at initiation but does not move with the replisome during elongation. Together, these results suggest a possible role of Hp0897 in loading of HpDnaB at oriC.

Intracellular locations of replication proteins and the origin of replication during chromosome duplication in the slowly growing human pathogen Helicobacter pylori.

We followed the position of the replication complex in the pathogenic bacterium Helicobacter pylori using antibodies raised against the single-stranded DNA binding protein (HpSSB) and the replicative helicase (HpDnaB). The position of the replication origin, oriC, was also localized in growing cells by fluorescence in situ hybridization (FISH) with fluorescence-labeled DNA sequences adjacent to the origin. The replisome assembled at oriC near one of the cell poles, and the two forks moved together toward the cell center as replication progressed in the growing cell. Termination and resolution of the forks occurred near midcell, on one side of the septal membrane. The duplicated copies of oriC did not separate until late in elongation, when the daughter chromosomes segregated into bilobed nucleoids, suggesting sister chromatid cohesion at or near the oriC region. Components of the replication machinery, viz., HpDnaB and HpDnaG (DNA primase), were found associated with the cell membrane. A model for the assembly and location of the H. pylori replication machinery during chromosomal duplication is presented.

Crystal structure and mode of helicase binding of the C-terminal domain of primase from Helicobacter pylori.

To better understand the poor conservation of the helicase binding domain of primases (DnaGs) among the eubacteria, we determined the crystal structure of the Helicobacter pylori DnaG C-terminal domain (HpDnaG-CTD) at 1.78 Å. The structure has a globular subdomain connected to a helical hairpin. Structural comparison has revealed that globular subdomains, despite the variation in number of helices, have broadly similar arrangements across the species, whereas helical hairpins show different orientations. Further, to study the helicase-primase interaction in H. pylori, a complex was modeled using the HpDnaG-CTD and HpDnaB-NTD (helicase) crystal structures using the Bacillus stearothermophilus BstDnaB-BstDnaG-CTD (helicase-primase) complex structure as a template. By using this model, a nonconserved critical residue Phe534 on helicase binding interface of DnaG-CTD was identified. Mutation guided by molecular dynamics, biophysical, and biochemical studies validated our model. We further concluded that species-specific helicase-primase interactions are influenced by electrostatic surface potentials apart from the critical hydrophobic surface residues.

Strategies adopted by gastric pathogen Helicobacter pylori for a mature biofilm formation: Antimicrobial peptides as a visionary treatment.

Enormous efforts in recent past two decades to eradicate the pathogen that has been prevalent in half of the world's population have been problematic. The biofilm formed by Helicobacter pylori provides resistance towards innate immune cells, various combinatorial antibiotics, and human antimicrobial peptides, despite the fact that these all are potent enough to eradicate it in vitro. Biofilm provides the opportunity to secrete various virulence factors that strengthen the interaction between host and pathogen helping in evading the innate immune system and ultimately leading to persistence. To our knowledge, this review is the first of its kind to explain briefly the journey of H. pylori starting with the chemotaxis, the mechanism for selecting the site for colonization, the stress faced by the pathogen, and various adaptations to evade these stress conditions by forming biofilm and the morphological changes acquired by the pathogen in mature biofilm. Furthermore, we have explained the human GI tract antimicrobial peptides and the reason behind the failure of these AMPs, and how encapsulation of Pexiganan-A(MSI-78A) in a chitosan microsphere increases the efficiency of eradication.

A Prospective Comparative Study of 3-Stitch Mesh Hernioplasty with Conventional Lichtenstein Repair.

Background: Hernioplasty, in which a mesh is used to strengthen a weakness or defect in the inguinal wall, has replaced simple tissue repair. As it is associated with low recurrence, it is considered the gold standard and is one of the most common general surgical procedures. The ideal repair should be rapid, safe and simple to do, requires minimal dissection to create sufficient space, be cost-effective and be accompanied by a brief hospital stay, reduced pain, and fewer recurrences. The aim of the present study was to compare the efficacy of 3-stitch mesh fixation with that of traditional Lichtenstein mesh fixation of inguinal hernia repair. Materials and Methods: Between July 2018 and December 2019, 59 cases of primary, uncomplicated inguinal hernias were surgically treated. Both the classical Lichtenstein technique (group A, n = 30) and the Lichtenstein technique with the three-stitch fixation method (group B, n = 29) were used on patients with inguinal hernias. Between the two groups, the mean operative times, post-surgical pain scores, average hospital stays and postoperative complications including recurrence rates were compared. Results: With a P-value of 0.001, the 3-point fixation group (group B) took 3.41 ± 0.58 min less time to fix the mesh than the Lichtenstein group (group A, 5.52 ± 0.59 min). The pain after surgery was much less for participants who had 3-point mesh fixation than for those who had conventional mesh fixation in the early (1, 3, 7 and 15 days after surgery) and late (1 month and 3 months) postoperative periods, with a P-value of 0.0001. When compared to the classical mesh fixation group, the 3-point mesh fixation group had less urinary retention, seroma and swelling. Both groups had the same number of other complications. Conclusions: The three-point hernioplasty is a simple procedure that is easier to adopt, less time-consuming, causes less trauma and has a lower risk of postoperative discomfort including chronic groin pain.

An endophytic fungus from Azadirachta indica A. Juss. that produces azadirachtin.

Azadirachtin A and its structural analogues are a well-known class of natural insecticides having antifeedant and insect growth-regulating properties. These compounds are exclusive to the neem tree, Azadirachta indica A. Juss, from where they are currently sourced. Here we report for the first time, the isolation and characterization of a novel endophytic fungus from A. indica, which produces azadirachtin A and B in rich mycological medium (Sabouraud dextrose broth), under shake-flask fermentation conditions. The fungus was identified as Eupenicillium parvum by ITS analysis (ITS1 and ITS2 regions and the intervening 5.8S rDNA region). Azadirachtin A and B were identified and quantified by LC-HRMS and LC-HRMS(2), and by comparison with the authentic reference standards. The biosynthesis of azadirachtin A and B by the cultured endophyte, which is also produced by the host neem plant, provides an exciting platform for further scientific exploration within both the ecological and biochemical contexts.

Epidermal Growth Factor Receptor Variant III Mutation, an Emerging Molecular Marker in Glioblastoma Multiforme Patients: A Single Institution Study on the Indian Population.

Background Glioblastoma is the most frequent and the most aggressive primary malignant brain tumor in adults. Standard treatment includes surgical removal of the tumor followed by concomitant chemotherapy and radiotherapy. Temozolomide, an oral alkylating agent, is currently the most commonly used chemotherapy. However, the median survival of glioblastoma multiforme (GBM) patients remains very low. Epidermal growth factor receptor variant III (EGFRvIII) is a novel marker for GBM patients of Indian origin as very few studies have been done on this molecular marker in our country. This is the first study utilizing this molecular marker among GBM patients in Rajasthan, India. This was a single institutional study that aimed to estimate the proportion of EGFRvIII mutation in GBM patients of Indian origin. Methodology This was a non-randomized, ambispective, single institutional observational study done on 35 brain tissue biopsies of histopathologically diagnosed and confirmed cases of GBM based on the World Health Organization 2007 Classification received in the pathology department of Dr. Sampurnanand Medical College, Jodhpur from 2015 to 2020 after applying inclusion and exclusion criteria. Molecular study of the EGFRvIII marker was conducted in all cases of GBM in the same institution on the RNA extracted from selected biopsy samples. Statistical analysis was performed using the SPSS version 22.0 software package (IBM Corp., Armonk, NY USA). The correlation between age and gender with EGFR-positive cases was analyzed, and EGFR positivity compared with previous studies. Results The occurrence of the EGFRvIII mutation was found to be 17.4% (6/35 cases). The mean age of presentation of a tumor with this mutation was estimated to be 54.3 years. Males were more commonly found to be affected (66.6%, 4/6 cases). Conclusions Thus, the identification of this mutation would segregate patients who may benefit from newer therapeutic approaches. In the future, personalized treatment may be advised for GBM patients depending on the presence of the EGFRvIII mutation.

Machine Learning-Based 30-Day Hospital Readmission Predictions for COPD Patients Using Physical Activity Data of Daily Living with Accelerometer-Based Device.

Chronic obstructive pulmonary disease (COPD) is a significantly concerning disease, and is ranked highest in terms of 30-day hospital readmission. Generally, physical activity (PA) of daily living reflects the health status and is proposed as a strong indicator of 30-day hospital readmission for patients with COPD. This study attempted to predict 30-day hospital readmission by analyzing continuous PA data using machine learning (ML) methods. Data were collected from 16 patients with COPD over 3877 days, and clinical information extracted from the patients' hospital records. Activity-based parameters were conceptualized and evaluated, and ML models were trained and validated to retrospectively analyze the PA data, identify the nonlinear classification characteristics of different risk factors, and predict hospital readmissions. Overall, this study predicted 30-day hospital readmission and prediction performance is summarized as two distinct approaches: prediction-based performance and event-based performance. In a prediction-based performance analysis, readmissions predicted with 70.35% accuracy; and in an event-based performance analysis, the total 30-day readmissions were predicted with a precision of 72.73%. PA data reflect the health status; thus, PA data can be used to predict hospital readmissions. Predicting readmissions will improve patient care, reduce the burden of medical costs burden, and can assist in staging suitable interventions, such as promoting PA, alternate treatment plans, or changes in lifestyle to prevent readmissions.

Gastric Perforation With Peritonitis Secondary to a Trichobezoar: A Literature Review and Report of a Rare Presentation.

Trichobezoar, a rare disorder commonly seen in psychiatric patients having a habit of plucking and eating their own hair, is a ball of hair admixed with gastro-intestinal secretions that leads to the blocking of the passage of food particles. Presentation of the disease is variable, ranging from asymptomatic to severe complications including obstruction and perforation. We report a case of a 27-year-old female patient who presented with an acute abdomen and on laparotomy, gastric perforation secondary to large gastric trichobezoar was found. The patient was treated with en bloc removal of the trichobezoar.

A comprehensive approach to discover Toxin-Antitoxin systems from human pathogen Helicobacter pylori: A poison and its antidote encapsulated in the genome.

AIM: An enormous presence and their identified role as stress managers, antibiotic resistance, persistence, and biofilm formation is the reason why the research on Toxin-Antitoxin (TA) loci is getting more and more emphasis. These set of genes consist of poison (Toxin) and its antidote (Antitoxin) expressing in an operon where the toxin inhibits the cellular process and antitoxin which can be a protein or non-coding RNA, rescues the toxin. Most recent progress in genomics and in silico studies have revealed new TA families, and types of TA on bacterial chromosome. However, there is almost no or very little is known about the TA in H. pylori. Therefore, this study aims to identify the TA genes in human pathogen using a comprehensive in silico approach. METHODOLOGY: Here, we have collected the genome-wide data of TA in H. pylori 26695 using TASmania, a new TA database. Further, entire TA dataset was validated with several other databases available for TA, operon analysis and experimental data available. KEY FINDINGS: The study revealed the presence of 80 putative TA genes in H. pylori and highlighted their similarity as well as uniqueness in comparison to other three known TA carrying human pathogens. SIGNIFICANCE: The present study indicates the presence of a large number of TA genes in H. pylori which make biofilm and goes into persistence. Hence, our innovative approach unlocks the prospect for characterizing these putative TA genes and their role as stress managers.

Acute Scrotal Abdomen: An Epitome of Negligence in Rural India.

An inguinoscrotal hernia is considered to be giant when it passes beyond the midpoint of the thigh in a standing position. It is a rare condition that can lead to complications such as obstruction and perforation. Here, we present the case of a 35-year-old male who was diagnosed with a giant inguinoscrotal hernia with transverse colon perforation peritonitis. The patient presented with acute abdomen and septic shock. On presentation, resuscitation was started and an emergency laparotomy was performed. Resection of the gangrenous bowel segment and end jejunostomy was done as damage control surgery. However, despite intensive care and efforts, the patient succumbed due to multiorgan dysfunction syndrome (MODS). This is a rare case of a giant inguinoscrotal hernia with transverse colon perforation peritonitis, leading to MODS and mortality.

Erratum to "Perceptions and practices of COVID-19 protective measures among the general public of North India" [Clin Epidemiol Glob Health 13 (2022) 100927].

[This corrects the article DOI: 10.1016/j.cegh.2021.100927.].

Perceptions and practices of COVID-19 protective measures among the general public of North India.

BACKGROUND: India has a high COVID-19 burden. The Indian government responded to the pandemic by mandating its population to adhere to certain Protective Measures (PMs). Compliance to these PMs depends on their acceptability and adaptability among the general public. AIMS: To explore the perceptions and practices of COVID-19 related PMs among the general public of North India. METHODS: Qualitative study in four administrative districts (Lucknow, Etawah, Patna and Darbhanga) of North India. Two urban and two rural districts were purposefully selected. Audio in-depth interviews (IDIs) were conducted with healthy caregivers of children (2-59 months). Data was managed using Atlast Ti and analyzed using conventional content analysis. RESULTS: From July-Sep 2020, 60 IDIs were conducted; 36.6% (22/60) were females and 26.6% (16/60) had below primary education. Respondents concurred that most people in their society flouted the recommended PMs. The reasons for poor/non-compliance with PMs were: perceived poor susceptibility to illness, perceived less severity of COVID-19 and low perceived benefits of complying with the PMs. Respondents opined that COVID-19 is less prevalent in rural areas and among the educated population. Most respondents were aware of the recommended PMs and opined that these must be followed, however subjective norms, social norms and behavioral intentions negatively impacted compliance. CONCLUSIONS: Since there was poor community perception of susceptibility to COVID-19 as well as poor perceived severity, the community did not understand the benefits of adhering to the recommended PMs. Therefore, future health communication strategies must take these into account to increase the possibility of success.