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Hot springs have tremendous significance due to their divulging physiochemical features. In the recent past, metagenomics has emerged as a unique methodology to explore microbiota as well as new biocatalysts possessing advantageous biochemical properties from hot springs. In the present study, metagenomics has been employed for microbial diversity exploration and identification of genes involved in various metabolic pathways among two hot springs, Manikaran and Tatapani, located in Himachal Pradesh, India. Taxonomic analysis of both metagenomes revealed the dominance of the Proteobacteria phylum. Genomic signatures of other bacterial phyla such as Chloroflexi, Actinobacteria, Bacteroidetes, Cyanobacteria, Planctomycetes, and Firmicutes were also found in significant abundance in both the metagenomes. The abundance of microorganisms belonging to genera, especially Nitrospira , Thauera , Meiothermus , Thiobacillus , Massilia , and Anaerolinea , was reported to be prevalent in the hot springs. A significant amount of metagenomic data remained taxonomically unclassified, which indeed emphasizes the scientific importance of these thermoaquatic niches. The functional potential analysis of both metagenomes revealed pathways related to carbohydrate metabolism, followed by amino acid metabolism, energy metabolism, genetic information processing, metabolism of cofactors and vitamins, membrane transporter, and signal transduction. Exploration of biomass-modifying biocatalysts enumerated the presence of glycoside hydrolases, glycosyl transferases, polysaccharide lyases, and carbohydrate esterases in the metagenomic data. Together, these findings offer an in-depth understanding of the microbial inhabitants in North-Western Himalayan hot springs and their underlying potential for various biotechnological and industrial applications., Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01248-z., Competing Interests: Conflict of interestThe authors declare that they have no conflicting interest., (© Association of Microbiologists of India 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Introduction The knee joint, an extraordinary feat of biomechanics, is prone to injuries, with the anterior cruciate ligament (ACL) often being a common victim. The intricate coordination of joint movements relies heavily on the ACL's screw-home mechanism, a crucial element for synchronizing knee movement with neighboring joints. Despite its indispensable role, the ACL is susceptible to injury, necessitating surgical intervention. While many patients experience positive outcomes following ACL reconstruction surgeries, a significant proportion face the challenge of procedure failure. The key to success lies in the healing process within the tibial and femoral bone tunnels. The post-ACL reconstruction phase introduces its own set of challenges, particularly in the context of returning to sports (RTS), underscoring the importance of reinstating neuromuscular and motor function. The trajectory of rehabilitation is influenced by factors such as graft healing, patient age, gender, pain levels, and concurrent injuries. Materials and methods This prospective observational study spanned 2.5 years, enrolling 71 patients with diagnosed ACL injuries. Arthroscopic reconstruction utilized hamstring autografts and peroneus longus autografts. A nine-month post-surgery follow-up employed the Lysholm scoring system for comprehensive evaluations. Results Over 2.5 years, 87.3% of male and 12.6% of female participants underwent arthroscopic reconstruction. Lysholm scores revealed 28.1% excellent, 45.0% good, and 26.7% fair outcomes, with no participants in the unsatisfactory range. Lysholm scores demonstrated positive outcomes, indicating the efficacy of arthroscopic reconstruction in enhancing knee function. Findings align with existing literature, emphasizing positive results from ACL reconstruction techniques and specific implants. Comparisons with related studies highlight challenges in standardized return-to-sport guidelines and underscore the need for outcome measure standardization. Conclusion The study contributes nuanced insights into ACL reconstruction outcomes, emphasizing positive functional recovery trends at the nine-month follow-up. Lysholm scores indicate favorable outcomes, supporting the procedure's effectiveness. Consideration of specific implants adds practical value. Despite limitations, this study enriches ACL reconstruction research, promoting advancements in patient care and outcomes. Ongoing research with extended follow-ups and larger cohorts will enhance understanding and refine ACL reconstruction strategies., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Soni et al.)

Introduction: Alzheimer's disease (AD) is a widespread neurodegenerative condition with complex causes and a significant global impact, particularly among the elderly. This brief introduction emphasizes AD's hallmark features and the urgent public health concern it poses, with numbers on the rise. It also highlights the potential of statins and magnesium L-threonate as a combined therapeutic approach to prevent AD and mitigate its underlying pathological features. The study's goal is to shed light on these promising interventions in a rat model induced by aluminum chloride (AlCl3)., Materials and Methods: A total of 30 aged female Wistar rats were divided into five groups (n=6/group). The vehicle control group received normal saline orally (p.o.).The model control group received AlCl3(4.2 mg/kg/day intraperitoneal (i.p.)). The standard-treated group received rivastigmine (1 mg/kg/day p.o.), and the atorvastatin-treated and atorvastatin with magnesium L-threonate-treated groups received atorvastatin (20 mg/kg/day p.o.) and atorvastatin (20 mg/kg/day) with magnesium L-threonate (604 mg/kg/day p.o. ), respectively. Cognitive functions such as radial arm maze, elevated plus maze (EPM), passive shock avoidance test, and open-field test (OFT) were performed at weekly intervals up to 28 days. After completion of the study on the 29 th day, all animals were sacrificed, and the brain was used for estimation of AchE enzyme activity, oxidative stress parameters, and histopathological analysis., Result: At the end of the fourth week, administration of atorvastatin and atorvastatin with magnesium L-threonate resulted in a decreased average time taken to reach the correct arm, reduced transfer latency (TL) in the EPM, shortened latency to reach the shock-free zone (SFZ), and an increase in rearing and counts by locomotion activity in the OFT. It also demonstrated improved anti-cholinesterase activity and suppressed oxidative stress, as indicated by a decrease in nitric oxide (NO) levels and an increase in superoxide dismutase (SOD) and catalase levels. Additionally, it led to reductions in brain changes observed in histopathological analysis., Conclusion: Atorvastatin with magnesium L-threonate provides a better beneficial protective effect against AD than atorvastatin alone. This combination can be a first choice for patients who are already taking atorvastatin in the early stages of AD., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Gangoda et al.)

Introduction Antimicrobial prophylaxis, involving short antibiotic courses preceding surgical procedures, is recommended to minimize postoperative infections. Paediatric cardiac surgeries are classified as clean procedures, though infection challenges persist due to illness severity and extended ICU stays. Antimicrobial prophylaxis varies, ranging from single doses to extended administration until catheters are removed. Typically lasting 24 to 48 hours, it has proven infection-reduction benefits. Despite these practices, uncertainties surround the optimal nature, timing, and duration of administration. This concern is amplified by escalating antimicrobial resistance driven by antibiotic overuse. Vulnerable paediatric populations bear heightened consequences of irrational antimicrobial use, contributing to global resistance trends. Yet, a defined optimal prophylaxis schedule for paediatric cardiac surgery is lacking. Importing adult guidelines may be inadequate due to paediatric research complexities and population diversity. Developing effective prophylaxis protocols is crucial for children undergoing cardiac surgery, given global antibiotic overuse and evolving drug resistance. Establishing an optimal prophylactic strategy remains a challenge, necessitating further research for evidence-based protocols to mitigate infections in this vulnerable patient cohort. Methods This study investigates antibiotic use in paediatric cardiac surgery. A retrospective analysis of 100 patients from a rural Indian hospital (2017-2018) assesses antibiotic patterns, including type, dose, duration, and adherence to prophylaxis protocols. Results In the studied cohort of paediatric cardiac surgery patients, complete compliance (100%) with antibiotic prophylaxis was observed. However, deviations were identified: 30% received antibiotics prematurely, and 30% did not align with institutional protocol criteria. Concerning antibiotic selection, 87% followed hospital policy with the recommended cefoperazone and sulbactam combination plus amikacin, while 9% received piperacillin/tazobactam + amikacin due to sepsis. Irregular use (22%) based on clinical records occurred. Furthermore, 4% received piperacillin/tazobactam + teicoplanin, with one instance of inappropriate higher antibiotic use. Regarding prophylaxis duration, only 27% adhered to the appropriate timeline, with 40% exceeding 48 hours, indicating extended use. Upon discharge, a notable proportion (45 patients) received antibiotic prescriptions. Among them, 73% were prescribed rationally, while 27% exhibited irrational antibiotic use. Conclusion The findings of this study shed a significant light on the issue of antibiotic misuse within the context of paediatric cardiac surgery. It underscores the pressing need for more stringent measures to regulate and address this concerning trend. The study underscores the pivotal importance of adhering rigorously to established protocols and guidelines for antibiotic prophylaxis. This adherence not only holds the potential to elevate the overall quality of patient care but also plays a critical role in combating the escalating challenge of antibiotic resistance. Through a concerted effort to optimize antibiotic usage, we can simultaneously enhance patient outcomes and contribute to the ongoing fight against the emergence of antibiotic-resistant strains, thus preserving the efficacy of these vital medications for future generations., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Pathan et al.)

RNA virus infections have been a leading cause of pandemics. Aided by global warming and increased connectivity, their threat is likely to increase over time. The flaviviruses are one such RNA virus family, and its prototypes such as the Japanese encephalitis virus (JEV), Dengue virus, Zika virus, West Nile virus, etc., pose a significant health burden on several endemic countries. All viruses start off their life cycle with an infected cell, wherein a series of events are set in motion as the virus and host battle for autonomy. With their remarkable capacity to hijack cellular systems and, subvert/escape defence pathways, viruses are able to establish infection and disseminate in the body, causing disease. Using this strategy, JEV replicates and spreads through several cell types such as epithelial cells, fibroblasts, monocytes and macrophages, and ultimately breaches the blood-brain barrier to infect neurons and microglia. The neurotropic nature of JEV, its high burden on the paediatric population, and its lack of any specific antivirals/treatment strategies emphasise the need for biomedical research-driven solutions. Here, we highlight the latest research developments on Japanese encephalitis virus-infected cells and discuss how these can aid in the development of future therapies., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Background: Fermented foods are the results of metabolic activities of various microorganisms. People have traditionally known how to culture desirable microorganisms, primarily lactic acid bacteria, yeasts, and filamentous molds, for the manufacture of edible foods. Yeast isolated from home-made mango pickle from Hamirpur, Himachal Pradesh, was assessed for probiotic properties and their enzymatic profiling., Results: Four yeast isolates were isolated out of which P. kudriavzevii Y33 was selected on the basis of high acid tolerance as well as broadest antimicrobial activity. The selected isolate was observed to have high acid tolerance at pH 2 and show strong antimicrobial activity against all the pathogens examined. P. kudriavzevii Y33 can also withstand high bile concentration and showed high viability index, i.e., 95% at concentration of 2% of bile. The isolate was able to demonstrate high cholesterol assimilation in medium containing ox bile and taurocholate, at 88.58 and 86.83%, respectively. The autoaggregation ability of isolate increases with increasing the time of incubation and showed 87% of autoaggregation after 24 h of incubation. P. kudriavzevii Y33 exhibited resistance towards different antibiotics, found to be positive for exopolysaccharide production and showed no hemolytic activity. The isolate was observed to produce several enzymes such as β-galactosidase, protease, amylase, phytase, and lipase., Conclusions: The results of the current study revealed that P. kudriavzevii Y33 has various beneficial qualities that suggest it could be used as probiotics. Enzymes produced by yeast isolate help in improving flavor and mineral availability in the fermented products., (© 2022. The Author(s).)

RNA virus infection triggers interferon (IFN) receptor signaling, leading to the activation of hundreds of interferon-stimulated genes (ISGs). Guanylate-binding proteins (GBPs) belong to one such IFN inducible subfamily of guanosine triphosphatases (GTPases) that have been reported to exert broad anti-microbial activity and regulate host defenses against several intracellular pathogens. Here, we investigated the role of human GBP1 (hGBP1) in Japanese encephalitis virus (JEV) infection of HeLa cells in both an IFNγ unprimed and primed environment. We observed enhanced expression of GBP1 both at transcript and protein levels upon JEV infection, and GBP1 association with the virus replication membranes. Depletion of hGBP1 through siRNA had no effect on JEV replication or virus induced cell death in the IFNγ unprimed environment. IFNγ stimulation provided robust protection against JEV infection. Knockdown of GBP1 in the primed environment upregulated expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1) and significantly reduced JEV replication. Depletion of GBP1 in an IFNγ primed environment also inhibited virus replication in human neuroblastoma SH-SH5Y cells. Our data suggests that in the presence of IFNγ, GBP1 displays a proviral role by inhibiting innate immune responses to JEV infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chhabra, Sharma and Kalia.)

Maintenance of cellular homeostasis through regulated degradation of proteins and organelles is a defining feature of autophagy. This process itself is tightly regulated in a series of well-defined biochemical reactions governed largely by the highly conserved ATG protein family. Given its crucial role in regulating protein levels under both basal and stress conditions such as starvation and infection, genetic or pharmacological perturbation of autophagy results in massive changes in the cellular proteome and impacts nearly every biological process. Therefore, studying autophagy perturbations at a global scale assumes prime importance. In recent years, quantitative mass spectrometry (MS)-based proteomics has emerged as a powerful approach to explore biological processes through global proteome quantification analysis. Tandem mass tag (TMT)-based MS proteomics is one such robust quantitative technique that can examine relative protein abundances in multiple samples (parallel multiplexing). Investigating autophagy through TMT-based MS approach can give great insights into autophagy-regulated biological processes, protein-protein interaction networks, spatiotemporal protein dynamics, and identification of new autophagy substrates. This chapter provides a detailed protocol for studying the impact of a dysfunctional autophagy pathway on the cellular proteome and pathways in a healthy vs. disease (virus infection) condition using a 16-plex TMT-based quantitative proteomics approach. We also provide a pipeline on data processing and analysis using available web-based tools., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Japanese encephalitis virus (JEV) is a flavivirus, spread by the bite of carrier Culex mosquitoes. The subsequent disease caused is Japanese encephalitis (JE), which is the leading global cause of virus-induced encephalitis. The disease is predominant in the entire Asia-Pacific region with the potential of global spread. JEV is highly neuroinvasive with symptoms ranging from mild fever to severe encephalitis and death. One-third of JE infections are fatal, and half of the survivors develop permanent neurological sequelae. Disease prognosis is determined by a series of complex and intertwined signaling events dictated both by the virus and the host. All flaviviruses, including JEV replicate in close association with ER derived membranes by channelizing the protein and lipid components of the ER. This leads to activation of acute stress responses in the infected cell-oxidative stress, ER stress, and autophagy. The host innate immune and inflammatory responses also enter the fray, the components of which are inextricably linked to the cellular stress responses. These are especially crucial in the periphery for dendritic cell maturation and establishment of adaptive immunity. The pathogenesis of JEV is a combination of direct virus induced neuronal cell death and an uncontrolled neuroinflammatory response. Here we provide a comprehensive review of the JEV life cycle and how the cellular stress responses dictate the pathobiology and resulting immune response. We also deliberate on how modulation of these stress pathways could be a potential strategy to develop therapeutic interventions, and define the persisting challenges., (Copyright © 2021. Published by Elsevier Ltd.)

Advances in proteomics have enabled a comprehensive understanding of host-pathogen interactions. Here we have characterized Japanese encephalitis virus (JEV) infection-driven changes in the mouse embryonic fibroblast (MEF) proteome. Through tandem mass tagging (TMT)-based mass spectrometry, we describe changes in 7.85 % of the identified proteome due to JEV infection. Pathway enrichment analysis showed that proteins involved in innate immune sensing, interferon responses and inflammation were the major upregulated group, along with the immunoproteasome and poly ADP-ribosylation proteins. Functional validation of several upregulated anti-viral innate immune proteins, including an active cGAS-STING axis, was performed. Through siRNA depletion, we describe a crucial role of the DNA sensor cGAS in restricting JEV replication. Further, many interferon-stimulated genes (ISGs) were observed to be induced in infected cells. We also observed activation of TLR2 and inhibition of TLR2 signalling using TLR1/2 inhibitor CU-CPT22-blocked production of inflammatory cytokines IL6 and TNF-α from virus-infected N9 microglial cells. The major proteins that were downregulated by infection were involved in cell adhesion (collagens), transport (solute carrier and ATP-binding cassette transporters), sterol and lipid biosynthesis. Several collagens were found to be transcriptionally downregulated in infected MEFs and mouse brain. Collectively, our data provide a bird's-eye view into how fibroblast protein composition is rewired following JEV infection.

Microtubule-associated protein 1 light chain 3 (MAP1LC3) is a protein with a well-defined function in autophagy, but still incompletely understood roles in several other autophagy-independent processess. Studies have shown MAP1LC3 is a host-dependency factor for the replication of several viruses. Japanese encephalitis virus (JEV), a neurotropic flavivirus, replicates on ER-derived membranes that are marked by autophagosome-negative non-lipidated MAP1LC3 (LC3-I). Depletion of LC3 exerts a profound inhibition on virus replication and egress. Here, we further characterize the role of LC3 in JEV replication, and through immunofluorescence and immunoprecipitation show that LC3-I interacts with the virus capsid protein in infected cells. This association was observed on capsid localized to both the replication complex and lipid droplets (LDs). JEV infection decreased the number of LDs per cell indicating a link between lipid metabolism and virus replication. This capsid-LC3 interaction was independent of the autophagy adaptor protein p62/Sequestosome 1 (SQSTM1). Further, no association of capsid was seen with the Gamma-aminobutyric acid receptor-associated protein family, suggesting that this interaction was specific for LC3. High-resolution protein-protein docking studies identified a putative LC3-interacting region in capsid, 56 FTAL 59, and other key residues that could mediate a direct interaction between the two proteins.

Basal autophagy is crucial for maintenance of cellular homeostasis. ATG5 is an essential protein for autophagosome formation, and its depletion has been extensively used as a tool to disrupt autophagy. Here, we characterize the impact of Atg5 deficiency on the cellular proteome of mouse embryonic fibroblasts (MEFs). Using a tandem mass tagging (TMT)-based quantitative proteomics analysis, we observe that 14% of identified proteins show dysregulated levels in atg5 -/- MEFs. These proteins were distributed across diverse biological processes, such as cell adhesion, development, differentiation, transport, metabolism, and immune responses. Several of the upregulated proteins were receptors involved in transforming growth factor β (TGF-β) signaling, JAK-STAT signaling, junction adhesion, and interferon/cytokine-receptor interactions and were validated as autophagy substrates. Nearly equal numbers of proteins, including several lysosomal proteins and enzymes, were downregulated, suggesting a complex role of autophagy/ATG5 in regulating their levels. The atg5 -/- MEFs had lower levels of key immune sensors and effectors, including Toll-like receptor 2 (TLR2), interferon regulatory factor 3 (IRF3), IRF7, MLKL, and STAT1/3/5/6, which were restored by reexpression of ATG5. While these cells could efficiently mount a type I interferon response to the double-stranded RNA (dsRNA) mimic poly(I·C), they were compromised in their inflammatory response to the bacterial pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and Pam3CSK4. Transcriptional activation and secretion of interleukin-6 (IL-6) in these cells could be recovered by ATG5 expression, supporting the role of autophagy in the TLR2-induced inflammatory response. This study provides a key resource for understanding the effect of autophagy/ATG5 deficiency on the fibroblast proteome. IMPORTANCE Autophagy performs housekeeping functions for cells and maintains a functional mode by degrading damaged proteins and organelles and providing energy under starvation conditions. The process is tightly regulated by the evolutionarily conserved Atg genes, of which Atg5 is one such crucial mediator. Here, we have done a comprehensive quantitative proteome analysis of mouse embryonic fibroblasts that lack a functional autophagy pathway ( Atg5 knockout). We observe that 14% of the identified cellular proteome is remodeled, and several proteins distributed across diverse cellular processes with functions in signaling, cell adhesion, development, and immunity show either higher or lower levels under autophagy-deficient conditions. These cells have lower levels of crucial immune proteins that are required to mount a protective inflammatory response. This study will serve as a valuable resource to determine the role of autophagy in modulating specific protein levels in cells., (Copyright © 2019 Sharma et al.)

Background: The study aimed to determine the effect of menopausal status and hormone therapy on the introitus and labia majora at the levels of histology and gene expression., Methods: Three cohorts of 10 women each (pre-menopause, post-menopause and post-menopause + hormone therapy) were selected based on the presentation of clinical atrophy and vaginal pH. Biopsies were obtained from the introitus (fourchette) and labia majora and processed for histology and gene expression analyses with microarrays. Other data collected included self-assessed symptoms, serum estradiol, testosterone, serum hormone binding globulin and the pH of the vagina and labia majora., Results: The introitus appears exquisitely sensitive to hormone status. Dramatic changes were observed in histology including a thinning of the epithelium in post-menopausal subjects with vaginal atrophy. Furthermore, there was differential expression of many genes that may contribute to tissue remodeling in the atrophic introitus. Levels of expression of genes associated with wound healing, angiogenesis, cell migration/locomotion, dermal structure, apoptosis, inflammation, epithelial cell differentiation, fatty acid, carbohydrate and steroid metabolism were significantly different in the cohort exhibiting atrophy of the introitus. While changes were also observed at the labia, that site was considerably less sensitive to hormone status. The gene expression changes observed at the introitus in this study were very similar to those reported previously in the atrophic vagina providing further evidence that these changes are associated with atrophy., Conclusions: The histological and gene expression changes occurring within the introitus after menopause may contribute to the constellation of symptoms that constitute the genitourinary syndrome of menopause., Competing Interests: MA Freedman and KB Sharma are consultants for The Procter & Gamble Company and received funding from Procter & Gamble for this study. RL Binder, MA Farage, Y Wang, C Combs, D Moore, JP Tiesman, CC Bascom, RJ Isfort and R Warren are current or former employees of Procter & Gamble., (Copyright 2019, Binder et al.)

Diphenyleneiodonium (DPI) and N-acetyl-l-cysteine (NAC), two widely used anti-oxidants, were employed to evaluate the role of oxidative stress in Japanese encephalitis virus (JEV) induced autophagy, stress responses and replication. DPI and NAC exerted opposite effects on ROS levels in JEV infected mouse neuronal cells (Neuro2a), mouse embryonic fibroblasts (MEFs) and human epithelial cells (HeLa). While NAC effectively quenched ROS, DPI enhanced ROS levels, suggesting that DPI induces oxidative stress in JEV infected cells. DPI treatment of JEV infected Neuro2a cells further blocked autophagy induction and activation of all three arms of the ER stress pathway, and, inhibited virus particle release. Autophagy induction in JEV infection has been previously shown to be linked to the activation of XBP1 and ATF6 ER stress sensors. Our data suggests that DPI mediated block of autophagy is a result of inhibition of ER stress responses and is not associated with an anti-oxidative effect. Since DPI has a wide inhibitory potential for all Flavin dependent enzymes, it is likely that the signalling pathways for ER stress and autophagy during JEV infection are modulated by DPI sensitive enzymes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Stringent regulation of antiviral signaling and cellular autophagy is critical for the host response to virus infection. However, little is known how these cellular processes are regulated in the absence of type I interferon signaling. Here, we show that ATF3 is induced following Japanese encephalitis virus (JEV) infection, and regulates cellular antiviral and autophagy pathways in the absence of type I interferons in mouse neuronal cells. We have identified new targets of ATF3 and show that it binds to the promoter regions of Stat1, Irf9, Isg15 and Atg5 thereby inhibiting cellular antiviral signaling and autophagy. Consistent with these observations, ATF3-depleted cells showed enhanced antiviral responses and induction of robust autophagy. Furthermore, we show that JEV replication was significantly reduced in ATF3-depleted cells. Our findings identify ATF3 as a negative regulator of antiviral signaling and cellular autophagy in mammalian cells, and demonstrate its important role in JEV life cycle.

Endoplasmic reticulum (ER) stress and autophagy are key cellular responses to RNA virus infection. Recent studies have shown that Japanese encephalitis virus (JEV)-induced autophagy negatively influences virus replication in mouse neuronal cells and embryonic fibroblasts, and delays virus-induced cell death. Here, we evaluated the role of ER stress pathways in inducing autophagy during JEV infection. We observed that JEV infection of neuronal cells led to activation of all three sensors of ER stress mediated by eIF2α/PERK, IRE1/XBP1 and ATF6. The kinetics of autophagy induction as monitored by levels of SQSTM1 and LC3-II paralleled activation of ER stress. Inhibition of the eIF2α/PERK pathway by siRNA-mediated depletion of proteins and by the PERK inhibitor had no effect on autophagy and JEV replication. However, depletion of XBP1 and ATF6, alone or in combination, prevented autophagy induction and significantly enhanced JEV-induced cell death. JEV-infected cells depleted of XBP1 or ATF6 showed reduced transcription of ER chaperones, ERAD components and autophagy genes, resulting in reduced protein levels of the crucial autophagy effectors ATG3 and BECLIN-1. Conversely, pharmacological induction of ER stress in JEV-infected cells further enhanced autophagy and reduced virus titres. Our study thus demonstrates that a crucial link exists between the ER stress pathways and autophagy in virus-infected cells, and that these processes are highly regulated during virus infection.

Rationale: Flexible bronchoscopy is performed by clinicians representing multiple medical specialties in a variety of settings. Given the increasing importance of competency-based assessment in postgraduate training, it is important that this skill be assessed within a competency-based framework using a valid measurement tool., Objectives: The purpose of this study was to design and validate a practical, competency-based bronchoscopy assessment tool that could be applied to trainees in a clinical setting., Methods: Focus groups of expert physicians were formed in Ottawa, Canada representing adult medical specialties routinely engaged in preparing trainees to perform flexible bronchoscopy (respiratory medicine, critical care, thoracic surgery and anesthesia). The focus groups were charged with identifying themes and items relevant to the assessment of competency in bronchoscopy. By an iterative process, a bronchoscopy assessment tool was developed, the Ontario Bronchoscopy Assessment Tool (OBAT). The tool was evaluated by first using it to assess learners in a pilot study, refining it based on the results, and then testing the OBAT again in a validation study., Measurements and Main Results: The initial tool consisted of 19 items, organized into the following groups: preprocedure planning, sedation and monitoring, technical skill, diagnostic skill, and post-procedure planning. The tool demonstrated high reliability (0.91) and discriminated junior from senior trainees. Based on the results of the pilot, the tool was simplified to a 12-item scale with three subscales: preprocedure planning, technical skills, and post-procedure planning. In the validation study, the assessment tool remained highly reliable (0.92) and discriminated junior from senior trainees with an estimated eight assessments per trainee., Conclusions: The OBAT demonstrates promise as a reliable tool to assess trainee competence for bronchoscopy in clinical settings.

Pulmonary arteriovenous malformations (AVMs) are commonly supplied by the pulmonary arterial system and rarely by the systemic bronchial circulation. The authors outline the case of a young woman with pulmonary AVMs as part of hereditary hemorrhagic telangiectasia with the uncommon presentation of massive hemoptysis. Management of her recurrent, life-threatening pulmonary hemorrhage was complicated by pulmonary AVMs that were supplied by both the pulmonary and systemic bronchial arterial circulatory systems. Transcatheter embolotherapy of the higher pressure bronchial systemic circuit was necessary for acute hemostasis.

Objective: To examine the prognostic significance of elevated urinary beta human chorionic gonadotrophin (beta-hCG) in patients with bladder cancer., Patients and Methods: Total beta-hCG was measured in the urine of 142 patients referred for cystoscopic examination. Patients were followed for a minimum of 17 months and grouped according to stage of disease. Because the water output by individual patients varied, urinary creatinine levels were measured as an indicator of the concentration of the urine sample. Patient outcomes were correlated with urinary total beta-hCG levels both corrected and uncorrected for creatinine concentration. After correcting for urinary creatinine levels, 40 patients were excluded because the sample was too dilute (undetectable beta-hCG and a creatinine level of < 4 mmol/L). A further four patients were excluded as they had concurrent malignancies not in the bladder and one patient was lost to follow up., Results: None of the 52 patients with benign conditions, nine of the 27 with Ta-T1, and nine of the 25 with T2-T4 bladder disease had urinary total beta-hCG levels > 3.74 IU/mmol/L creatinine. There was no significant association between urinary total beta-hCG concentrations and the rates of recurrence or progression for Ta-T1 disease at 17 months of follow-up. For patients with T2-T4 disease there was a significant association with widespread metastasis (P < 0.01) and mortality (P < 0.01) at 17 months of follow-up. These associations persisted when urinary total beta-hCG levels were not corrected for urinary creatinine concentration (metastasis, P < 0.01; mortality, P = 0.07; Kaplan-Meier survival time analysis, uncorrected for creatinine P = 0.027, corrected for creatinine P < 0.001). This association could not be accounted for by differences in age, histopathology or treatment., Conclusion: Although sample concentration was a serious confounding factor, after correcting for dilution using the creatinine content, the elevated urinary levels of total beta-hCG indicated those T2-T4 lesions which were likely to metastasize and those patients likely to die early. If this test is to be used clinically, concentrated samples, i.e. early-morning urine, and a more sensitive beta-hCG assay are required. Nevertheless, for T2-T4 bladder tumours, an elevated pre-treatment level of urinary beta-hCG is a marker of poor prognosis and may prove useful in deciding appropriate therapy.

A total of 190 Salmonella typhimurium strains encountered in different parts of India were characterized on the basis of plasmid profile, phage type and antimicrobial resistance pattern. Recent trends in the epidemiology of R-plasmids were also studied. The majority of S. typhimurium strains (90.5%) were untypable by phage typing. Only 18 strains (9.5%) were phage typable. The phage untypable strains isolated from northern (57) central (65), and southern (50) regions of India could be subgrouped into 24, 12 and 16 different plasmid profiles respectively. Heterogeneity was the prominent feature although most of the plasmid profiles were related among strains isolated from particular place. A great diversity among small plasmids (2.7-8.3 kb) made subgrouping of majority strains (71%) with R-pattern ApCmKmSmSuTcTp possible. Conjugation studies and plasmid profile analysis of transconjugants revealed all the strains to harbour non conjugative non-auto transmissible plasmids with the exception of 7.2 and 2.7 kb plasmids which were not mobilizable.

First trimester maternal serum levels of pregnancy-associated plasma protein A(PAPP-P) are reduced in women with a Down's syndrome pregnancy. We have examined the concentration of this molecule in the amniotic (AF) and extra-embryonic coelomic (EECF) fluids surrounding the developing fetus. Maternal serum levels of PAPP-A were elevated in all samples and steadily rose from a median of 480 mIU/1 at 8 weeks to a median of 6375 mIU/1 at 14 weeks gestation. Levels of PAPP-A were low in EECF and undetectable in the AF until 14 weeks gestation. This pattern of distribution is in contrast to that of most other trophoblast-associated antigens. This may reflect PAPP-A physiology and its specific production by the syncytiotrophoblast.

Adhesive and invasive properties were compared with plasmid profile in S. Typhimurium strains of phage type 99(10) and 36(10). All strains of phage type 99 were multiple drug resistant (ApCmKmSmSuTcTp) and harboured small plasmids of 2.4-5.2 MDal. Six strains of the phage type 36 had ApCmTc R-pattern and two with only ampicillin resistance, carried plasmids of molecular size 2.6-5.2 MDal; two strains were sensitive to all antibiotics and devoid of plasmids. None of the strains were found to harbour high molecular weight plasmids. All plasmid positive strains of phage types 99 and 36 could be divided into two groups of three plasmid patterns each, which were phage type specific. All plasmid positive and negative strains adhered and invaded HeLa cells to different degrees. No correlation could be established between plasmid profile and adhesion invasion characteristics. High molecular weight plasmids therefore are unlikely to be essential for adhesion and invasion.

Plasmid profile analysis and antibiotic resistance pattern determination were carried out for 117 phage untypable S. Typhimurium strains. Majority of the strains (82%) were resistant to all the seven antibiotics tested, R-pattern being ApCmKmSmSuTcTp, rest (12%) showed heterogenous R-patterns. Plasmid DNA analysis revealed phage untypable strains to harbour large (58.8-114.3 MDal), intermediate size (36 MDal, 42 MDal) and small (1.8-5.2 MDal) plasmids with varying molecular weights. All the phage untypable strains could be subgrouped by plasmid profile analysis into 23 plasmid patterns. Plasmid profile analysis could discriminate large number of phage untypable strains on the basis of their plasmid pattern.

Fresh cardiac valvular tissues and atrial appendages removed from 106 Indian patients with rheumatic heart disease at the time of corrective cardiac surgery were examined to determine the characteristics of valvular interstitial lymphocytic infiltrates using conventional histologic staining along with indirect immunofluorescent techniques. Precise identification of the phenotypic profiles of inflammatory mononuclear cells was attempted using anti-IgG, anti-Ia, and monoclonal mouse hybridoma reagents identifying T cells (OKT3) as well as T cell subsets (OKT4 helper/inducer and OKT8 suppressor/cytotoxic cells). A similar group of 21 patients undergoing cardiac valvular resection in Albuquerque was studied. The mean age of Indian patients providing valve tissues was 27.7, whereas in those in Albuquerque, it was 52 years. Twenty-five percent of rheumatic heart valves in Indian patients showed significant interstitial lymphoid infiltrates, and one third of the rheumatic valves from patients in Albuquerque showed similar mononuclear cell collections. Lymphoid infiltrates contained a predominance of T cells (70 to 80 percent) and only occasional B cells. Most of the T cells were OKT4-positive, with only a minor representation of suppressor/cytotoxic OKT8-positive T cells. In many instances, OKT4-positive helper T cell collections were closely juxtaposed to fibroblasts and collagen fibrils. These findings suggest that the chronic rheumatic scarring process may involve helper/inducer T cells as an ancillary factor in the indolent contracture and fibrosis of deformed cardiac valvular structures. Attempts to demonstrate residual streptococcal antigens by indirect immunofluorescence using a wide panel of heterologous rabbit F(ab')2 reagents with specificity for group A streptococcal membranes, cell wall mucopeptide, or group A carbohydrate gave negative results.