Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories

Occupational risk assessment and selected morbidities among cement brick unit workers in a rural area of Bangalore District, India

Introduction: The labor-intensive nature of cement brick manufacturing, its unorganized nature and internal migration, expose the employees to several occupational health hazards. The objective of the study was to assess the occupational risks in cement brick unit settings and to estimate the prevalence of respiratory and musculoskeletal morbidities among the cement brick unit workers in a rural area of Bangalore urban district. Methods: A cross-sectional study was conducted among cement brick unit workers over two months. A semi-structured questionnaire was used to capture sociodemographic details. Multiple observations on the field and the World Health Organization semi-quantitative risk assessment matrix were used to obtain risk scores of the occupational hazards. A structured questionnaire on respiratory symptoms and Minispir Portable Spirometer were used to assess the respiratory morbidities and lung functions. Musculoskeletal morbidities were assessed using the Modified Nordic questionnaire. Proportions were used to describe respiratory and musculoskeletal morbidities. Chi-square test, Fisher’s exact test and multivariate logistic regressions were done to identify significant variables. Results: Among 120 subjects, 110 (91.6%) were men and 85.8% were migrants. Injury due to falls of heavy objects, back injury, respiratory complaints and slips/falls were found to be high-risk health hazards. The prevalence of respiratory morbidity was 21.7% and that of musculoskeletal morbidity was 51.7%. Workers receiving a higher salary (≥ 1500 Indian rupees) had higher odds of having respiratory morbidity. Conclusion: The prevalence of respiratory and musculoskeletal morbidities was high. Introduction of mechanical equipment, decreasing work hours, periodic medical examinations and appropriate use of personal protective equipment will help in risk reduction as per this study

Feasibility of store-and-forward teledermatology in out-patient care: A prospective study from rural India utilising specialist referral services through an instant messaging platform - "WhatsApp"

Objectives: The COVID-19 pandemic has placed unprecedented demands on the delivery of health care in rural areas of India. We examined the feasibility of store-and-forward mobile teledermatology for outpatient access to specialist dermatologic care in underserved areas in India. Methods: We conducted a prospective study using smartphone-based teledermatology, connecting six underserved clinics manned by primary care physicians (PCP) to three dermatologists, using the instant messaging platform WhatsApp. We assessed the concordance between PCPs and dermatologists (using Cohen’s kappa coefficient), consultation time, the spectrum of conditions, and the outcome. Results: Of the 730 dermatology patients screened in the clinics, (13%) (36 males and 59 females) required teleconsultation, among which 61.1% were non-infective, 34.7% were infective, and the diagnosis could not be ascertained in 4.2 %. The mean time takenwas 13.5 (± 18.4) minutes. Twenty per cent (n=19) required referral, and 80% (n=76) of consultations could be resolved at the clinic, of whom 36.8 % were cured, 38.2% had moderate, 4% had minimal improvement, 13% were lost to follow-up, and 8% refused treatment. Cure was observed in viral infections and eczema. The diagnostic concordance ranged from low values [0.38 (95% CI: 0-0.68)] in infective to moderate [0.66 (95% CI: 0.42-0.83), p=0.033] in non-infective disorders. Conclusion: Asynchronous mobile teledermatology, using specialist referral via instant messaging platforms, is a powerful modality for providing real-time dermatologic care, while offering a very promising alternative for decreasing healthcare disparities and continuity of services even in adverse situations like the Covid-19 pandemic

Feasibility of store-and-forward teledermatology in out-patient care: A prospective study from rural India utilising specialist referral services through an instant messaging platform - "WhatsApp"

Objectives: The COVID-19 pandemic has placed unprecedented demands on the delivery of health care in rural areas of India. We examined the feasibility of store-and-forward mobile teledermatology for outpatient access to specialist dermatologic care in underserved areas in India. Methods: We conducted a prospective study using smartphone-based teledermatology, connecting six underserved clinics manned by primary care physicians (PCP) to three dermatologists, using the instant messaging platform WhatsApp. We assessed the concordance between PCPs and dermatologists (using prevalence adjusted bias adjusted kappa), consultation time, the spectrum of conditions, and the outcome. Results: Of the 730 dermatology patients screened in the clinics, 95 (13%) (36 males and 59 females) required teleconsultation, among which 61.1% were non-infective, 34.7% were infective, and the diagnosis could not be ascertained in 4.2 %. The mean time taken for the first response was 13.5\ub118.4 minutes. Twenty per cent (n=19) required referral, and 80% (n=76) of consultations could be resolved at the clinic, of whom 36.8 % were cured, 38.2% had moderate, 4% had minimal improvement, 13% were lost to follow-up, and 8% refused treatment. Cure was observed in viral infections and eczema. The diagnostic concordance ranged from low values [0.38 (95% CI: 0-0.68)] in infective to moderate [0.66 (95% CI: 0.42 -0.83), p=0.033] in non-infective disorders. Conclusion: Asynchronous mobile teledermatology, using specialist referral via instant messaging platforms, is a powerful modality for providing real-time dermatologic care, while offering a very promising alternative for decreasing healthcare disparities and continuity of services even in adverse situations like the Covid-19 pandemic

TRIM5alpha Restricts Flavivirus Replication by Targeting the Viral Protease for Proteasomal Degradation

Tripartite motif-containing protein 5alpha (TRIM5alpha) is a cellular antiviral restriction factor that prevents early events in retrovirus replication. The activity of TRIM5alpha is thought to be limited to retroviruses as a result of highly specific interactions with capsid lattices. In contrast to this current understanding, we show that both human and rhesus macaque TRIM5alpha suppress replication of specific flaviviruses. Multiple viruses in the tick-borne encephalitis complex are sensitive to TRIM5alpha-dependent restriction, but mosquito-borne flaviviruses, including yellow fever, dengue, and Zika viruses, are resistant. TRIM5alpha suppresses replication by binding to the viral protease NS2B/3 to promote its K48-linked ubiquitination and proteasomal degradation. Importantly, TRIM5alpha contributes to the antiviral function of IFN-I against sensitive flaviviruses in human cells. Thus, TRIM5alpha possesses remarkable plasticity in the recognition of diverse virus families, with the potential to influence human susceptibility to emerging flaviviruses of global concern

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field