Protocol for investigating the biogenesis of SARS-CoV-2 S pseudoviruses in HEK293T cells transduced to express the virus-specific intrabodies.

The protocol is designed to investigate the influence of an anti-cleavage site intrabody in modulating the output of LV(CoV-2 S), a lentivirus-based pseudovirus expressing CoV-2 S protein using HEK293T cells. We clone the single-domain antibody sequence into a lentiviral vector (pLenti-GFP) for intracellular expression and assess not only the viral biogenesis but also the fate of the CoV-2 S protein in such cells. For complete details on the use and execution of this protocol, please refer to Singh et al. (2022).1.

The hazards of excessive screen time: Impacts on physical health, mental health, and overall well-being.

Excessive screen time is a growing concern in modern society, with the proliferation of digital devices contributing to increased sedentary behavior and potential hazards to physical health, mental health, and overall well-being. This article explores the potential health and mood deterioration caused by excess screen time. In particular, the article examines how excessive screen time can affect physical health, mental health, and overall well-being. The physical hazards of excess screen time include eye strain, neck and shoulder pain, and back pain. Mental health hazards include increased levels of depression, anxiety, and other mood disorders. Overall well-being can also be impacted by excessive screen time, particularly when it comes to social relationships and cognitive development. The article concludes by recommending the limitation of screen time, particularly for young people, and the incorporation of physical activity and face-to-face interaction into daily routines.

Robust anti-SARS-CoV2 single domain antibodies cross neutralize multiple viruses.

We report robust SARS-CoV2 neutralizing sdAbs targeting the viral peptides encompassing the polybasic cleavage site (CSP) and in the receptor binding domain (RBD) of the spike (S) protein. Both the sdAbs inhibited infectivity of the CoV2 S protein expressing pseudoviruses (LV-CoV2S). Both anti-CSP and RBD intrabodies (IB) inhibited the output of LV(CoV2 S). Anti-CSP IB altered the proteolytic processing and targeted the viral S protein for degradation. Because of cross-reactive CSPs in the entry mediators, the anti-CSP sdAb neutralized in vitro and in vivo the infectivity of SARS-CoV2 unrelated viruses such as herpes simplex virus 1 (HSV1) and pestes des petits ruminants virus (PPRV). Conversely, anti-HSV1 and anti-PPRV sera neutralized LV(CoV2 S) owing to the presence of CSP reactive antibodies indicating that a prior infection with such pathogens could impact on the pattern of COVID-19.

A Mouse Model of PPRV Infection for Elucidating Protective and Pathological Roles of Immune Cells.

The study was aimed at developing an accessible laboratory animal model to elucidate protective and pathological roles of immune mediators during Peste des petits ruminants virus (PPRV) infection. It is because of the critical roles of type I IFNs in anti-viral defense, we assessed the susceptibility of IFN receptor knock out (IFNR KO) mice to PPRV infection. IFNR KO mice were exceedingly susceptible to the infection but WT animals efficiently controlled PPRV. Accordingly, the PPRV infected IFNR KO mice gradually reduced their body weights and succumbed to the infection within 10 days irrespective of the dose and route of infection. The lower infecting doses predominantly induced immunopathological lesions. The viral antigens as well as the replicating PPRV were abundantly present in most of the critical organs such as brain, lungs, heart and kidneys of IFNR KO mice infected with high dose of the virus. Neutrophils and macrophages transported the replicating virus to central nervous system (CNS) and contributed to pathology while the elevated NK and T cell responses directly correlated with the resolution of PPRV infection in WT animals. Using an array of fluorescently labeled H-2Kb tetramers, we discovered four immunogenic epitopes of PPRV. The PPRV-peptides interacted well with H-2Kb in acellular and cellular assay as well as expanded the virus-specific CD8+ T cells in immunized or infected mice. Adoptively transferred CD8+ T cells helped control PPRV in infected mice. Our study therefore established and employed a mouse model for investigating the pathogenesis of PPRV. The model could be useful for elucidating the contribution of immune cells in disease progression as well as to test anti-viral agents.

Transcriptome analysis of buffalo granulosa cells in three dimensional culture systems.

Hanging drop (HD) three-dimensional (3D) culture model for buffalo granulosa cells (GC) was reported to mimic the preovulatory stage of ovarian follicles in our previous study. To further verify its reliability, the present study attempted a comparative transcriptome profile of buffalo GC freshly isolated from ovarian follicles (<8 mm diameter) (FC) and their cultures in normal culture dish (ND or 2D), polyHEMA coated dish (PH) and HD culture systems (3D). Out of 223 significantly (-log2 fold change: >3; p < .0005; false discovery rate [FDR]: <0.1) differentially expressed genes (SDEGs) among different culture systems, 137 were found unannotated, and 94, 29, and 66 were exclusively expressed in FC, PH, and HD, respectively. However, on eliminating the fixed points of p values and FDR from the entire raw data, only 11 genes related to long noncoding RNA, 12 genes related to luteinization, and 3 genes related to follicular maturation were exclusively expressed in FC, PH, and HD culture systems, respectively. The quantitative real time-PCR validation and the next generation sequencing data had more than 90% correlation. Bioinformatics analyses of the exclusively expressed SDEG revealed that the freshly aspirated GCs were a true representative of GCs from small follicles (<8 mm diameter), the GC spheroids under PH maintained mitochondrial function, and those cultured in HD system for 6 days simulated the inflammatory milieu required for ovulation. Therefore, the comparative transcriptome profile also reinforced that HD culture system is better in vitro culture method than the other methods analyzed in this study for buffalo GC.

A Combinatorial in-silico, in-vitro and in-vivo Approach to Quantitatively Study Peptide Induced MHC Stability.

Here, we describe a combinatorial approach in reverse vaccinology to identify immunogenic class I major histocompatibility complex (MHC) displayed epitopes derived from a morbillivirus named pestes des petits ruminants (PPRV). The protocol describes an in silico prediction of immunogenic epitopes using an IEDB tool. The predicted peptides were further analysed by molecular docking with mouse class I MHC (H-2Kb), to assess their binding affinity, and their immunogenicity was validated, using acellular and cellular assays. Finally, an enumeration of the expanded PPRV-specific CD8+ T cells in infected or immunized mice against the immunogenic peptides was performed ex vivo. Synthetic peptide derivatives from different structural and non-structural proteins of PPRV were used to measure the extent of stabilized H2-Kb, using an ELISA based acellular assay and TAP deficient RMA/s cells. Fluorescently labelled H2-Kb-tetramers were generated by displacing a UV photocleavable conditional ligand with the PPRV-peptides. The resulting reagents were used to identify and enumerate virus-specific CD8+ T cells in immunized or PPRV-infected mice. The combinatorial approach described here could be used to identify immunogenic epitopes of any pathogen, autoantigens, as well as cancer antigens. Graphic abstract: Figure 1.General schematic to identify immunogenic peptides and their stabilization on MHC I molecule.

Buffalo liver transcriptome analysis suggests immune tolerance as its key adaptive mechanism during early postpartum negative energy balance.

Females undergo negative energy balance (NEB) during the early postpartum period to meet the lactation demands. The liver, being the key metabolic organ, plays a major role in handling NEB. Dairy animals handling high lactation demands are better models to understand the liver adaptive mechanisms during this phase. Therefore, we analyzed the liver transcriptome of dairy buffaloes during early postpartum. Liver biopsies were performed on three lactating buffaloes on the 15th and the 30th days of early postpartum and three heifers (controls) at the diestrous stage. Paired-end Next Generation Sequencing (NGS) identified 509 significantly differentially expressed genes (SDE) in the liver among the three groups. The SDE with log2 fold change > 3 and the unique SDE revealed the promotion of immune suppression (e.g., TCR), apoptosis (e.g., CCDC103), PGF2α synthesis, fat accumulation (e.g., BGLAP) and liver regeneration (e.g., FGF10) pathways, and the downregulation of antigen presentation (e.g., BOLA-DQA) on the 15th day of lactation. Consistently upregulated genes on the 15th and 30th days of early postpartum indicated the promotion of immune tolerance (e.g., IFITM3), medium and long-chain fatty acids' oxidation (e.g., ACSM3), and lipid accumulation (e.g., INSIG1). However, consistently downregulated genes during early postpartum showed immunosuppression, the downregulation of gluconeogenesis from amino acids (e.g., DDO), and the biosynthesis of taurine (e.g., CSAD) and unsaturated fatty acids (e.g., SCD). Functional annotation and network analyses also indicated the promotion of immune tolerance, fat accumulation and decreased gluconeogenesis from amino acids, and estrogen metabolism on the 15th day of lactation. Overall, the liver showed immune tolerance as an adaptive mechanism during early postpartum of buffaloes.

An efficient method for extracting next-generation sequencing quality RNA from liver tissue of recalcitrant animal species.

The next-generation RNA sequencing technologies expedite the discovery of a large number of novel transcripts and genes associated with various pathophysiological conditions. These technologies involve poly(A) enrichment, which in turn requires micrograms of high-quality total RNA. Unfortunately, the available RNA isolation approaches produce poor quality total RNA from difficult-to-isolate animal tissues, such as the liver with high glycogen content. Moreover, the extraction efficiencies of these approaches vary significantly depending on the animal species. To address this challenge, we optimized a three-step protocol for the extraction of high-yield and high-quality total RNA from the liver tissue (LT). The procedure effectively resolved the problem of glycogen coprecipitation by its stepwise removal. No signs of RNA degradation on gel electrophoresis analysis and RNA integrity number values ≥8.5 indicated that the extracted RNA is suitable for downstream processing, such as poly(A) enrichment and transcriptome profiling. To demonstrate the robustness of the novel protocol, a comparison was made with other currently available RNA extraction approaches from diverse resources. Whereas other protocols yielded partially degraded bands with either decreased or reversed ribosomal RNA (rRNA) ratio, our protocol yielded intact rRNA with a ratio of 2.0 ± 0.1. This optimized protocol was also successfully followed for other animal tissues, such as the bone and muscles. In conclusion, the study has described a highly efficient method for the next-generation sequencing quality RNA isolation from LT across a broad range of animal species, with extended applicability to other difficult-to-isolate tissues.

Galectin-3 Regulates γ-Herpesvirus Specific CD8 T Cell Immunity.

To gain insights into the molecular mechanisms and pathways involved in the activation of γ-herpesvirus (MHV68)-specific T cell receptor transnuclear (TN) CD8+ T cells, we performed a comprehensive transcriptomic analysis. Upon viral infection, we observed differential expression of several thousand transcripts encompassing various networks and pathways in activated TN cells compared with their naive counterparts. Activated cells highly upregulated galectin-3. We therefore explored the role of galectin-3 in influencing anti-MHV68 immunity. Galectin-3 was recruited at the immunological synapse during activation of CD8+ T cells and helped constrain their activation. The localization of galectin-3 to immune synapse was evident during the activation of both naive and memory CD8+ T cells. Galectin-3 knockout mice mounted a stronger MHV68-specific CD8+ T cell response to the majority of viral epitopes and led to better viral control. Targeting intracellular galectin-3 in CD8+ T cells may therefore serve to enhance response to efficiently control infections.

Congenital Pulmonary Vein Stenosis and Pulmonary Artery Branch Stenosis: A Rare Combination.

Congenital pulmonary vein stenosis is a rare entity caused due to failed incorporation of common right and/or left pulmonary vein into the left atrium. Below is a case report of a combination of predominantly left-sided pulmonary vein stenosis with right pulmonary artery branch stenosis. The patient was an adolescent boy with mild symptoms. Clinical examination revealed features of pulmonary artery hypertension. Echocardiography and computed tomography scan were done to confirm the disease.

Late onset hypogonadism in type 2 diabetic and nondiabetic male: a comparative study.

Sexual dysfunction in diabetic men can result from a variety of causes of which late onset hypogonadism is now a recognised entity. The study described here was conducted to determine this entity by comparing diabetic men with age matched controls. A significant number of diabetic men had low levels of testosterone and free testosterone. Among the patients with low testosterone a large percentage had low pituitary gonadotrophic hormones signifying that the disorder was actually hypogonadotrophic hypogonadism. This finding was then correlated with various risk factors present in the diabetic patients and positive correlations were found for many parameters.

Comparison of left ventricular mass in normotensive type 2 diabetes mellitus patients with that in the nondiabetic population.

Cardiovascular disease is increased in individuals with type 1 or type 2 diabetes mellitus (DM). Left ventricular hypertrophy (LVH), which is an ominous prognostic sign and an independent risk factor for cardiac events, is often present in type 2 DM patients. The aim of our cross-sectional study was to evaluate the prevalence of LVH, and risk factors for its development, in normotensive type 2 diabetic patients without antihypertensive medication. The objectives of the study were to find out the prevalence of high left ventricular mass (LVM) in normotensive type 2 diabetic patients and compare it with nondiabetics and to uncover the risk factors for the development of high LVM in normotensive type 2 diabetic patients. A total of 130 age- and sex-matched subjects were selected (65 cases, diabetic normotensive, and 65 controls, nondiabetic normotensive) and baseline data were collected. LVM and left ventricular mass index (LVMI) were calculated using echocardigraphic parameters and body surface area. LVMI was significantly higher in patients with type 2 DM compared with age-, sex-matched healthy population (104.9 ± 21 vs. 78.5 ± 22.7 g/m(2), respectively; P < 0.05). BMI, HbA1c, and duration of diabetes were significantly associated with LVH whereas sexes, age, PPBS, were not.

Randomized, controlled trial of oral ribavirin for Japanese encephalitis in children in Uttar Pradesh, India.

BACKGROUND: Japanese encephalitis is associated with high rates of mortality and disabling sequelae. To date, no specific antiviral has proven to be of benefit for this condition. We attempted to determine the efficacy of oral ribavirin treatment for reducing early mortality among children with Japanese encephalitis in Uttar Pradesh, India. METHODS: Children (age, 6 months to 15 years) who had been hospitalized with acute febrile encephalopathy (a < or =2-week history of fever plus altered sensorium) were tested for the presence of immunoglobulin M antibodies to Japanese encephalitis virus with commercial immunoglobulin M capture enzyme-linked immunosorbent assay. Children with positive results were randomized to receive either ribavirin (10 mg/kg per day in 4 divided doses for 7 days) or placebo syrup through nasogastric tube or by mouth. The primary outcome was early mortality; secondary outcome measures were early (at hospital discharge; normal or nearly normal, independent functioning, dependent, vegetative state, or death) outcome, time to resolution of fever, time to resumption of oral feeding, duration of hospitalization, and late outcome (> or =3 months after hospital discharge). The study was double-blind, and analysis was by intention to treat. RESULTS: A total of 153 patients were enrolled during a 3-year period; 70 patients received ribavirin, and 83 received placebo. There was no statistically significant difference between the 2 groups in the early mortality rate: 19 (27.1%) of 70 ribavirin recipients and 21 (25.3%) of 83 placebo recipients died (odds ratio, 1.10; 95% confidence interval, 0.5-2.4). No statistically significant differences in secondary outcome measures were found. CONCLUSIONS: For the dosage schedule used in our study, oral ribavirin has no effect in reducing early mortality associated with Japanese encephalitis. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00216268 .

Clinical features in children hospitalized during the 2005 epidemic of Japanese encephalitis in Uttar Pradesh, India.

BACKGROUND: Japanese encephalitis is a disease that affects the rural poor in Asia. In August-September 2005, a severe epidemic of Japanese encephalitis occurred in Uttar Pradesh, one of India's poorest states. METHODS: Children admitted to the King George Medical University hospital (Lucknow, Uttar Pradesh, India) with acute febrile encephalopathy (defined as fever plus encephalopathy of