Transcriptional profiling of single fiber cells in a transgenic paradigm of an inherited childhood cataract reveals absence of molecular heterogeneity.

Our recent single-cell transcriptomic analysis has demonstrated that heterogeneous transcriptional activity attends molecular transition from the nascent to terminally differentiated fiber cells in the developing mouse lens. To understand the role of transcriptional heterogeneity in terminal differentiation and the functional phenotype (transparency) of this tissue, here we present a single-cell analysis of the developing lens, in a transgenic paradigm of an inherited pathology, known as the lamellar cataract. Cataracts hinder transmission of light into the eye. Lamellar cataract is the most prevalent bilateral childhood cataract. In this disease of early infancy, initially, the opacities remain confined to a few fiber cells, thus presenting an opportunity to investigate early molecular events that lead to cataractogenesis. We used a previously established paradigm that faithfully recapitulates this disease in transgenic mice. About 500 single fiber cells, manually isolated from a 2-day-old transgenic lens were interrogated individually for the expression of all known 17 crystallins and 78 other relevant genes using a Biomark HD (Fluidigm). We find that fiber cells from spatially and developmentally discrete regions of the transgenic (cataract) lens show remarkable absence of the heterogeneity of gene expression. Importantly, the molecular variability of cortical fiber cells, the hallmark of the WT lens, is absent in the transgenic cataract, suggesting absence of specific cell-type(s). Interestingly, we find a repetitive pattern of gene activity in progressive states of differentiation in the transgenic lens. This molecular dysfunction portends pathology much before the physical manifestations of the disease

The Anti-Inflammatory and Antibacterial Basis of Human Omental Defense: Selective Expression of Cytokines and Antimicrobial Peptides

BACKGROUND: The wound healing properties of the human omentum are well known and have extensively been exploited clinically. However, the underlying mechanisms of these effects are not well understood. We hypothesize that the omentum tissue promotes wound healing via modulation of anti-inflammatory pathways, and because the omentum is rich in adipocytes, the adipocytes may modulate the anti-inflammatory response. Factors released by human omentum may affect healing, inflammation and immune defense. METHODOLOGY: Six human omentum tissues (non obese, free from malignancy, and any other systemic disorder) were obtained during diagnostic laparoscopies having a negative outcome. Healthy oral mucosa (obtained from routine oral biopsies) was used as control. Cultured adipocytes derived from human omentum were exposed to lipopolysaccharide (LPS) (1-50 ng/mL) for 12-72 hours to identify the non-cytotoxic doses. Levels of expression (mRNA and protein) were carried out for genes associated with pro- and anti-inflammatory cytokine responses and antibacterial/antimicrobial activity using qRT-PCR, western blotting, and cell-based ELISA assays. RESULTS: The study shows significant higher levels of expression (mRNA and protein) of several specific cytokines, and antibacterial peptides in the omentum tissues when compared to oral sub-mucosal tissues. In the validation studies, primary cultures of adipocytes, derived from human omentum were exposed to LPS (5 and 10 ng/mL) for 24 and 48 h. The altered expressions were more pronounced in cultured adipocytes cells when exposed to LPS as compared to the omentum tissue. CONCLUSIONS/SIGNIFICANCE: Perhaps, this is the first report that provides evidence of expressional changes in pro- and anti-inflammatory cytokines and antibacterial peptides in the normal human omentum tissue as well as adipocytes cultured from this tissue. The study provides new insights on the molecular and cellular mechanisms of healing and defense by the omentum, and suggests the potential applicability of cultured adipocytes derived from the omentum for future therapeutic applications

Physics Potential of the ICAL detector at the India-based Neutrino Observatory (INO)

The upcoming 50 kt magnetized iron calorimeter (ICAL) detector at the India-based Neutrino Observatory (INO) is designed to study the atmospheric neutrinos and antineutrinos separately over a wide range of energies and path lengths. The primary focus of this experiment is to explore the Earth matter effects by observing the energy and zenith angle dependence of the atmospheric neutrinos in the multi-GeV range. This study will be crucial to address some of the outstanding issues in neutrino oscillation physics, including the fundamental issue of neutrino mass hierarchy. In this document, we present the physics potential of the detector as obtained from realistic detector simulations. We describe the simulation framework, the neutrino interactions in the detector, and the expected response of the detector to particles traversing it. The ICAL detector can determine the energy and direction of the muons to a high precision, and in addition, its sensitivity to multi-GeV hadrons increases its physics reach substantially. Its charge identification capability, and hence its ability to distinguish neutrinos from antineutrinos, makes it an efficient detector for determining the neutrino mass hierarchy. In this report, we outline the analyses carried out for the determination of neutrino mass hierarchy and precision measurements of atmospheric neutrino mixing parameters at ICAL, and give the expected physics reach of the detector with 10 years of runtime. We also explore the potential of ICAL for probing new physics scenarios like CPT violation and the presence of magnetic monopoles.Comment: 139 pages, Physics White Paper of the ICAL (INO) Collaboration, Contents identical with the version published in Pramana - J. Physic

Fluid challenges in intensive care: the FENICE study A global inception cohort study

Fluid challenges (FCs) are one of the most commonly used therapies in critically ill patients and represent the cornerstone of hemodynamic management in intensive care units. There are clear benefits and harms from fluid therapy. Limited data on the indication, type, amount and rate of an FC in critically ill patients exist in the literature. The primary aim was to evaluate how physicians conduct FCs in terms of type, volume, and rate of given fluid; the secondary aim was to evaluate variables used to trigger an FC and to compare the proportion of patients receiving further fluid administration based on the response to the FC.This was an observational study conducted in ICUs around the world. Each participating unit entered a maximum of 20 patients with one FC.2213 patients were enrolled and analyzed in the study. The median [interquartile range] amount of fluid given during an FC was 500 ml (500-1000). The median time was 24 min (40-60 min), and the median rate of FC was 1000 [500-1333] ml/h. The main indication for FC was hypotension in 1211 (59 %, CI 57-61 %). In 43 % (CI 41-45 %) of the cases no hemodynamic variable was used. Static markers of preload were used in 785 of 2213 cases (36 %, CI 34-37 %). Dynamic indices of preload responsiveness were used in 483 of 2213 cases (22 %, CI 20-24 %). No safety variable for the FC was used in 72 % (CI 70-74 %) of the cases. There was no statistically significant difference in the proportion of patients who received further fluids after the FC between those with a positive, with an uncertain or with a negatively judged response.The current practice and evaluation of FC in critically ill patients are highly variable. Prediction of fluid responsiveness is not used routinely, safety limits are rarely used, and information from previous failed FCs is not always taken into account

Safety, immunogenicity, and reactogenicity of BNT162b2 and mRNA-1273 COVID-19 vaccines given as fourth-dose boosters following two doses of ChAdOx1 nCoV-19 or BNT162b2 and a third dose of BNT162b2 (COV-BOOST): a multicentre, blinded, phase 2, randomised trial

Background Some high-income countries have deployed fourth doses of COVID-19 vaccines, but the clinical need, effectiveness, timing, and dose of a fourth dose remain uncertain. We aimed to investigate the safety, reactogenicity, and immunogenicity of fourth-dose boosters against COVID-19.Methods The COV-BOOST trial is a multicentre, blinded, phase 2, randomised controlled trial of seven COVID-19 vaccines given as third-dose boosters at 18 sites in the UK. This sub-study enrolled participants who had received BNT162b2 (Pfizer-BioNTech) as their third dose in COV-BOOST and randomly assigned them (1:1) to receive a fourth dose of either BNT162b2 (30 µg in 0·30 mL; full dose) or mRNA-1273 (Moderna; 50 µg in 0·25 mL; half dose) via intramuscular injection into the upper arm. The computer-generated randomisation list was created by the study statisticians with random block sizes of two or four. Participants and all study staff not delivering the vaccines were masked to treatment allocation. The coprimary outcomes were safety and reactogenicity, and immunogenicity (antispike protein IgG titres by ELISA and cellular immune response by ELISpot). We compared immunogenicity at 28 days after the third dose versus 14 days after the fourth dose and at day 0 versus day 14 relative to the fourth dose. Safety and reactogenicity were assessed in the per-protocol population, which comprised all participants who received a fourth-dose booster regardless of their SARS-CoV-2 serostatus. Immunogenicity was primarily analysed in a modified intention-to-treat population comprising seronegative participants who had received a fourth-dose booster and had available endpoint data. This trial is registered with ISRCTN, 73765130, and is ongoing.Findings Between Jan 11 and Jan 25, 2022, 166 participants were screened, randomly assigned, and received either full-dose BNT162b2 (n=83) or half-dose mRNA-1273 (n=83) as a fourth dose. The median age of these participants was 70·1 years (IQR 51·6–77·5) and 86 (52%) of 166 participants were female and 80 (48%) were male. The median interval between the third and fourth doses was 208·5 days (IQR 203·3–214·8). Pain was the most common local solicited adverse event and fatigue was the most common systemic solicited adverse event after BNT162b2 or mRNA-1273 booster doses. None of three serious adverse events reported after a fourth dose with BNT162b2 were related to the study vaccine. In the BNT162b2 group, geometric mean anti-spike protein IgG concentration at day 28 after the third dose was 23 325 ELISA laboratory units (ELU)/mL (95% CI 20 030–27 162), which increased to 37 460 ELU/mL (31 996–43 857) at day 14 after the fourth dose, representing a significant fold change (geometric mean 1·59, 95% CI 1·41–1·78). There was a significant increase in geometric mean anti-spike protein IgG concentration from 28 days after the third dose (25 317 ELU/mL, 95% CI 20 996–30 528) to 14 days after a fourth dose of mRNA-1273 (54 936 ELU/mL, 46 826–64 452), with a geometric mean fold change of 2·19 (1·90–2·52). The fold changes in anti-spike protein IgG titres from before (day 0) to after (day 14) the fourth dose were 12·19 (95% CI 10·37–14·32) and 15·90 (12·92–19·58) in the BNT162b2 and mRNA-1273 groups, respectively. T-cell responses were also boosted after the fourth dose (eg, the fold changes for the wild-type variant from before to after the fourth dose were 7·32 [95% CI 3·24–16·54] in the BNT162b2 group and 6·22 [3·90–9·92] in the mRNA-1273 group).Interpretation Fourth-dose COVID-19 mRNA booster vaccines are well tolerated and boost cellular and humoral immunity. Peak responses after the fourth dose were similar to, and possibly better than, peak responses after the third dose

Spatial Analysis of Single Fiber Cells of the Developing Ocular Lens Reveals Regulated Heterogeneity of Gene Expression

Summary: The developing eye lens presents an exceptional paradigm for spatial transcriptomics. It is composed of highly organized long, slender transparent fiber cells, which differentiate from the edges of the anterior epithelium of the lens (equator), attended by high expression of crystallins, which generates transparency. Every fiber cell, therefore, is an optical unit whose refractive properties derive from its gene activity. Here, we probe this tangible relationship between the gene activity and the phenotype by studying the expression of all known 17 crystallins and 77 other non-crystallin genes in single fiber cells isolated from three states/regions of differentiation, allowing us to follow molecular progression at the single-cell level. The data demonstrate highly variable gene activity in cortical fibers, interposed between the nascent and the terminally differentiated fiber cell transcription. These data suggest that the so-called stochastic, highly heterogeneous gene activity is a regulated intermediate in the realization of a functional phenotype. : Cell Biology; Developmental Biology; Omics; Biological Sciences Research Methodologies Subject Areas: Cell Biology, Developmental Biology, Omics, Biological Sciences Research Methodologie

Transcriptional profiling of single fiber cells in a transgenic paradigm of an inherited childhood cataract reveals absence of molecular heterogeneity.

Our recent single-cell transcriptomic analysis has demonstrated that heterogeneous transcriptional activity attends molecular transition from the nascent to terminally differentiated fiber cells in the developing mouse lens. To understand the role of transcriptional heterogeneity in terminal differentiation and the functional phenotype (transparency) of this tissue, here we present a single-cell analysis of the developing lens, in a transgenic paradigm of an inherited pathology, known as the lamellar cataract. Cataracts hinder transmission of light into the eye. Lamellar cataract is the most prevalent bilateral childhood cataract. In this disease of early infancy, initially, the opacities remain confined to a few fiber cells, thus presenting an opportunity to investigate early molecular events that lead to cataractogenesis. We used a previously established paradigm that faithfully recapitulates this disease in transgenic mice. About 500 single fiber cells, manually isolated from a 2-day-old transgenic lens were interrogated individually for the expression of all known 17 crystallins and 78 other relevant genes using a Biomark HD (Fluidigm). We find that fiber cells from spatially and developmentally discrete regions of the transgenic (cataract) lens show remarkable absence of the heterogeneity of gene expression. Importantly, the molecular variability of cortical fiber cells, the hallmark of the WT lens, is absent in the transgenic cataract, suggesting absence of specific cell-type(s). Interestingly, we find a repetitive pattern of gene activity in progressive states of differentiation in the transgenic lens. This molecular dysfunction portends pathology much before the physical manifestations of the disease

An open-label pilot study to explore usefulness of Homoeopathic treatment in nonerosive gastroesophageal reflux disease

Background and Aim: Nonerosive gastroesophageal reflux disease or nonerosive reflux disease (NERD) is characterized by troublesome reflux-related symptoms in the absence of esophageal erosions/breaks at conventional endoscopy. There are a number of medicines cited in homoeopathic literature which can be used for treatment of symptoms such as heartburn and regurgitation. A pilot study was undertaken to explore usefulness of homoeopathic medicines in treatment of NERD. Methodology: In this study, 78 patients were screened and 34 were enrolled, having symptoms of heartburn and/or regurgitation at least twice a week, having a gastroesophageal reflux disease (GERD) symptom score of more than 4. Homoeopathic medicine was prescribed on the basis of presenting symptoms. Response to treatment was assessed on GERD symptom score, visual analog scale (VAS) for heartburn, and World Health Organization quality of life-BREF (WHO-QOL) questionnaire evaluated at baseline and at end of 8 weeks of treatment . Results: Significant difference was found in pre- and post-treatment GERD symptom score (8.79 ± 2.7 vs. 0.76 ± 1.8; P = 0.001) and VAS for heartburn (47.47 ± 19.6 vs. 5.06 ± 11.8; P = 0.001). Statistically significant improvement was seen in three domains of WHO-QOL score, i.e. psychological health, social relationship, and environmental domain. Conclusion: The findings are encouraging to open avenues for further studies on reflux disease

Iron Catalyzed Hydroformylation of Alkenes under Mild Conditions: Evidence of an Fe(II) Catalyzed Process

Earth abundant, first row transition metals offer a cheap and sustainable alternative to the rare and precious metals. However, utilization of first row metals in catalysis requires harsh reaction conditions, suffers from limited activity, and fails to tolerate functional groups. Reported here is a highly efficient iron catalyzed hydroformylation of alkenes under mild conditions. This protocol operates at 10–30 bar syngas pressure below 100 °C, utilizes readily available ligands, and applies to an array of olefins. Thus, the iron precursor [HFe­(CO)₄]⁻[Ph₃PNPPh₃]⁺ (<b>1</b>) in the presence of triphenyl phosphine catalyzes the hydroformylation of 1-hexene (S2), 1-octene (S1), 1-decene (S3), 1-dodecene (S4), 1-octadecene (S5), trimethoxy­(vinyl)­silane (S6), trimethyl­(vinyl)­silane (S7), cardanol (S8), 2,3-dihydrofuran (S9), allyl malonic acid (S10), styrene (S11), 4-methylstyrene (S12), 4-<i>i</i>Bu-styrene (S13), 4-<i>t</i>Bu-styrene (S14), 4-methoxy styrene (S15), 4-acetoxy styrene (S16), 4-bromo styrene (S17), 4-chloro styrene (S18), 4-vinylbenzonitrile (S19), 4-vinylbenzoic acid (S20), and allyl benzene (S21) to corresponding aldehydes in good to excellent yields. Both electron donating and electron withdrawing substituents could be tolerated and excellent conversions were obtained for S11–S20. Remarkably, the addition of 1 mol % acetic acid promotes the reaction to completion within 16–24 h. Detailed mechanistic investigations revealed <i>in situ</i> formation of an iron-dihydride complex [H₂Fe­(CO)₂(PPh₃)₂] (<b>A</b>) as an active catalytic species. This finding was further supported by cyclic voltammetry investigations and intermediacy of an Fe(0)–Fe­(II) species was established. Combined experimental and computational investigations support the existence of an iron-dihydride as the catalyst resting state, which then follows a Fe­(II) based catalytic cycle to produce aldehyde