Reversible, tunable epigenetic silencing of TCF1 generates flexibility in the T cell memory decision.

The immune system encodes information about the severity of a pathogenic threat in the quantity and type of memory cells it forms. This encoding emerges from lymphocyte decisions to maintain or lose self-renewal and memory potential during a challenge. By tracking CD8+ T cells at the single-cell and clonal lineage level using time-resolved transcriptomics, quantitative live imaging, and an acute infection model, we find that T cells will maintain or lose memory potential early after antigen recognition. However, following pathogen clearance, T cells may regain memory potential if initially lost. Mechanistically, this flexibility is implemented by a stochastic cis-epigenetic switch that tunably and reversibly silences the memory regulator, TCF1, in response to stimulation. Mathematical modeling shows how this flexibility allows memory T cell numbers to scale robustly with pathogen virulence and immune response magnitudes. We propose that flexibility and stochasticity in cellular decisions ensure optimal immune responses against diverse threats.

CD8+ T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor.

Improvements in tumor immunotherapies depend on better understanding of the anti-tumor T cell response. By studying human tumor-draining lymph nodes (TDLNs), we found that activated CD8+ T cells in TDLNs shared functional, transcriptional, and epigenetic traits with TCF1+ stem-like cells in the tumor. The phenotype and TCR overlap suggested that these TDLN cells were precursors to tumor-resident stem-like CD8+ T cells. Murine tumor models revealed that tumor-specific CD8+ T cells were activated in TDLNs but lacked an effector phenotype. These stem-like cells migrated into the tumor, where additional co-stimulation from antigen-presenting cells drove effector differentiation. This model of CD8+ T cell activation in response to cancer is different from that of canonical CD8+ T cell activation to acute viruses, and it proposes two stages of tumor-specific CD8+ T cell activation: initial activation in TDLNs and subsequent effector program acquisition within the tumor after additional co-stimulation.

Proliferating Transitory T Cells with an Effector-like Transcriptional Signature Emerge from PD-1+ Stem-like CD8+ T Cells during Chronic Infection.

T cell dysfunction is a characteristic feature of chronic viral infection and cancer. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection have defined a PD-1+ Tcf-1+ CD8+ T cell subset capable of self-renewal and differentiation into more terminally differentiated cells that downregulate Tcf-1 and express additional inhibitory molecules such as Tim3. Here, we demonstrated that expression of the glycoprotein CD101 divides this terminally differentiated population into two subsets. Stem-like Tcf-1+ CD8+ T cells initially differentiated into a transitory population of CD101-Tim3+ cells that later converted into CD101+ Tim3+ cells. Recently generated CD101-Tim3+ cells proliferated in vivo, contributed to viral control, and were marked by an effector-like transcriptional signature including expression of the chemokine receptor CX3CR1, pro-inflammatory cytokines, and granzyme B. PD-1 pathway blockade increased the numbers of CD101-Tim3+ CD8+ T cells, suggesting that these newly generated transitional cells play a critical role in PD-1-based immunotherapy.

An intra-tumoral niche maintains and differentiates stem-like CD8 T cells.

Tumour-infiltrating lymphocytes are associated with a survival benefit in several tumour types and with the response to immunotherapy1-8. However, the reason some tumours have high CD8 T cell infiltration while others do not remains unclear. Here we investigate the requirements for maintaining a CD8 T cell response against human cancer. We find that CD8 T cells within tumours consist of distinct populations of terminally differentiated and stem-like cells. On proliferation, stem-like CD8 T cells give rise to more terminally differentiated, effector-molecule-expressing daughter cells. For many T cells to infiltrate the tumour, it is critical that this effector differentiation process occur. In addition, we show that these stem-like T cells reside in dense antigen-presenting-cell niches within the tumour, and that tumours that fail to form these structures are not extensively infiltrated by T cells. Patients with progressive disease lack these immune niches, suggesting that niche breakdown may be a key mechanism of immune escape.

2-Deoxy-glucose ameliorates the peritoneal mesothelial and endothelial barrier function perturbation occurring due to Peritoneal Dialysis fluids exposure.

Peritoneal dialysis (PD) and prolonged exposure to PD fluids (PDF) induce peritoneal membrane (PM) fibrosis and hypervascularity, leading to functional PM degeneration. 2-deoxy-glucose (2-DG) has shown potential as PM antifibrotic by inhibiting hyper-glycolysis induced mesothelial-to-mesenchymal transition (MMT). We investigated whether administration of 2-DG with several PDF affects the permeability of mesothelial and endothelial barrier of the PM. The antifibrotic effect of 2-DG was confirmed by the gel contraction assay with embedded mesothelial (MeT-5A) or endothelial (EA.hy926) cells cultured in Dianeal® 2.5 % (CPDF), BicaVera® 2.3 % (BPDF), Balance® 2.3 % (LPDF) with/without 2-DG addition (0.2 mM), and qPCR for αSMA, CDH2 genes. Moreover, 2-DG effect was tested on the permeability of monolayers of mesothelial and endothelial cells by monitoring the transmembrane resistance (RTM), FITC-dextran (10, 70 kDa) diffusion and mRNA expression levels of CLDN-1 to -5, ZO1, SGLT1, and SGLT2 genes. Contractility of MeT-5A cells in CPDF/2-DG was decreased, accompanied by αSMA (0.17 ± 0.03) and CDH2 (2.92 ± 0.29) gene expression fold changes. Changes in αSMA, CDH2 were found in EA.hy926 cells, though αSMA also decreased under LPDF/2-DG incubation (0.42 ± 0.02). Overall, 2-DG mitigated the PDF-induced alterations in mesothelial and endothelial barrier function as shown by RTM, dextran transport and expression levels of the CLDN-1 to -5, ZO1, and SGLT2. Thus, supplementation of PDF with 2-DG not only reduces MMT but also improves functional permeability characteristics of the PM mesothelial and endothelial barrier.

Longitudinal Analysis of the Phenotype, Transcriptional Profile, and Anatomic Location of Memory CD8 T Cell Subsets after Acute Viral Infection.

Increased demand for novel, highly effective vaccination strategies necessitates a better understanding of long-lived memory CD8 T cell differentiation. To achieve this understanding, we used the mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. We reexamined classical memory CD8 T cell subsets and performed in-depth, longitudinal analysis of their phenotype, transcriptional programming, and anatomic location within the spleen. All analyses were performed at multiple time points from 8 days to 1 year postinfection. Memory subsets are conventionally defined by their expression of KLRG1 and IL-7Rα, as follows: KLRG1+IL-7Rα- terminal effectors (TEs) and KLRG1-IL-7Rα+ memory precursors (MPs). But we also characterized a third KLRG1+IL-7Rα+ subset which we refer to as KLRG1+ MPs. In these analyses, we defined a comprehensive memory phenotype that is associated with higher levels of CD28 expression. We also demonstrated that MPs, KLRG1+ MPs, and TEs have distinct localization programs within the spleen. We found that MPs became preferentially enriched in the white pulp as early as 1 to 2 weeks postinfection, and their predominance in the white pulp was maintained throughout the course of a year. On the other hand, KLRG1+ MPs and TEs localized to the red pulp just as early, and they consistently localized to the red pulp thereafter. These findings indicate that location may be crucial for memory formation and that white pulp-derived signals may contribute to long-term memory survival. Achieving robust memory responses following vaccination may require more deliberate consideration of which memory phenotypes are induced, as well as where they traffic, as these factors could impact their longevity. IMPORTANCE CD8 T cells play a critical role in viral immunity and it is important to understand how memory cells are formed and what processes lead to their long-term maintenance. Here, we use a mouse model of acute infection to perform an in-depth, longitudinal analysis of memory CD8 T cell differentiation, examining the phenotype and location of memory cells out to 1 year postinfection.

Does epigenetic markers of HLA gene show association with coronary artery disease in Indian subjects?

BACKGROUND: DNA methylation, one of the most stable forms of epigenetic modification is associated with the development and progression of coronary artery disease (CAD). Our previously reported study on epigenome-wide microarray analysis showed significantly methylated CpG sites. Top 5 significant CpGs from HLA gene were selected and analysed by Pyrosequencing (PSQ) to determine their association with severity of CAD. METHODS: Blood samples of 50-age matched angiographically CAD positive male cases with 50 angiographically CAD negative male controls were subjected to lipid profile estimation and PSQ for methylation level analysis. Findings and subgroup analysis were evaluated by Mann-Whitney U; Kruskal-Wallis' rank test and two-way ANOVA by MedCalc (v19.6). RESULTS: Methylation levels in HLA-DQA1 for cg10217052 was 78.5 (37-85) and 76.5 (24-84); cg09411910 was 81 (72.0 to 93.0) and 81.5 (50.0 to 89.0) in cases and controls respectively. Levels in HLA-DQB1-cg03344051, were 28.88 + 9.41 for cases and 30.36 + 9.37 in controls. For HLA-DRB1-cg07889003, levels in cases and controls were 15.5 (5.00-39.00) and 10.5 (5.00-29.0); while in cg08269402 were 52 (16-65) and 42.5 (17-61) respectively. No association was observed between methylation levels and lipid profile. cg03344051, cg07889003 and cg08269402 were significantly differentiated in double or triple vessel disease (DVD or TVD) as compared to single vessel disease (SVD) suggesting an increase in the extent of methylation with the increase in CAD severity. CONCLUSION: The present study shows significant increase in the extent of methylation in 3 CpG sites in DVD/TVD cases as compared to SVD cases. Additionally, a novel site, cg07889003 identified in our discovery phase has shown association with the severity of CAD.

Differential effects of biocompatible peritoneal dialysis fluids on human mesothelial and endothelial cells in 2D and 3D phenotypes.

INTRODUCTION: Peritoneal dialysis (PD) is a life maintaining treatment in patients with end-stage renal disease. Its chronic application leads to peritoneal mesothelial layer denudation and fibrotic transformation along with vascular activation of inflammatory pathways. The impact of different PD fluids (PDF) on mesothelial and endothelial cell function and repair mechanisms are not comprehensively described. MATERIALS AND METHODS: Mesothelial (MeT-5A) and endothelial cells (EA.hy926) were cultured in 1:1 ratio with cell medium and different PDF (icodextrin-based, amino acid-based, and glucose-based). Cell adhesion, cell migration, and cell proliferation in 2D and spheroid formation and collagen gel contraction assays in 3D cell cultures were performed. RESULTS: Cell proliferation and cell-mediated gel contraction were both significantly decreased in all conditions. 3D spheroid formation was significantly reduced with icodextrin and amino acid PDF, but unchanged with glucose PDF. Adhesion was significantly increased by amino acid PDF in mesothelial cells and decreased by icodextrin and amino acid PDF in endothelial cells. Migration capacity was significantly decreased in mesothelial cells by all three PDF, while endothelial cells remained unaffected. CONCLUSIONS: In 3D phenotypes the effects of PDF are more uniform in both mesothelial and endothelial cells, mitigating spheroid formation and gel contraction. On the contrary, effects on 2D phenotypes are more uniform in the icodextrin and amino acid PDF as opposed to glucose ones and affect mesothelial cells more variably. 2D and 3D comparative assessments of PDF effects on the main peritoneal membrane cell barriers, the mesothelial and endothelial, could provide useful translational information for PD studies.

Post-ICU Care: Why, What, When and How? ISCCM Position Statement.

How to cite this article: Ramakrishnan N, Abraham BK, Barokar R, Chanchalani G, Jagathkar G, Shetty RM, et al. Post-ICU Care: Why, What, When and How? ISCCM Position Statement. Indian J Crit Care Med 2024;28(S2):S279-S287.

Fatal Human Rabies Infection With Suspected Host-Mediated Failure of Post-Exposure Prophylaxis Following a Recognized Zoonotic Exposure-Minnesota, 2021.

BACKGROUND: No human rabies post-exposure prophylaxis (PEP) failure has been documented in the United States using modern cell culture-based vaccines. In January 2021, an 84-year-old male died from rabies 6 months after being bitten by a rabid bat despite receiving timely rabies PEP. We investigated the cause of breakthrough infection. METHODS: We reviewed medical records, laboratory results, and autopsy findings and performed whole-genome sequencing (WGS) to compare patient and bat virus sequences. Storage, administration, and integrity of PEP biologics administered to the patient were assessed; samples from leftover rabies immunoglobulin were evaluated for potency. We conducted risk assessments for persons potentially exposed to the bat and for close patient contacts. RESULTS: Rabies virus antibodies present in serum and cerebrospinal fluid were nonneutralizing. Antemortem blood testing revealed that the patient had unrecognized monoclonal gammopathy of unknown significance. Autopsy findings showed rabies meningoencephalitis and metastatic prostatic adenocarcinoma. Rabies virus sequences from the patient and the offending bat were identical by WGS. No deviations were identified in potency, quality control, administration, or storage of administered PEP. Of 332 persons assessed for potential rabies exposure to the case patient, 3 (0.9%) warranted PEP. CONCLUSIONS: This is the first reported failure of rabies PEP in the Western Hemisphere using a cell culture-based vaccine. Host-mediated primary vaccine failure attributed to previously unrecognized impaired immunity is the most likely explanation for this breakthrough infection. Clinicians should consider measuring rabies neutralizing antibody titers after completion of PEP if there is any suspicion for immunocompromise.

Effects of pharmacological primary cilium disturbance in the context of in vitro 2D and 3D malignant pleura mesothelioma.

INTRODUCTION: Primary cilium (PC) is a single non-motile antenna-like organelle composed of a microtubule core axon originating from the mother centriole of the centrosome. The PC is universal in all mammalian cells and protrudes to the extracellular environment receiving mechanochemical cues that it transmits in the cell. AIM: To investigate the role of PC in mesothelial malignancy in the context of two-dimensional (2D) and three-dimensional (3D) phenotypes. MATERIALS AND METHODS: The effect of pharmacological deciliation [using ammonium sulphate (AS) or chloral hydrate (CH)] and PC elongation [using lithium chloride (LC)] on cell viability, adhesion, and migration (2D cultures) as well as in mesothelial sphere formation, spheroid invasion and collagen gel contraction (3D cultures) was investigated in benign mesothelial MeT-5A cells and in malignant pleural mesothelioma (MPM) cell lines, M14K (epithelioid) and MSTO (biphasic), and primary malignant pleural mesothelioma cells (pMPM). RESULTS: Pharmacological deciliation or elongation of the PC significantly affected cell viability, adhesion, migration, spheroid formation, spheroid invasion and collagen gel contraction in MeT-5A, M14K, MSTO cell lines and in pMPM cells compared to controls (no drug treatment). CONCLUSIONS: Our findings indicate a pivotal role of the PC in functional phenotypes of benign mesothelial cells and MPM cells.

Development and characterization of non-coding RNA-derived simple sequence repeat markers in coconut (Cocos nucifera L.).

Non-coding RNA (ncRNA)-based SSR markers are highly useful in molecular breeding as ncRNAs play a significant role in gene regulation. In the present study, for the first time in coconut, we have identified 597 ncRNA-derived SSR markers, including 509 long non-coding RNASSRs (lncRNASSRs) and 88 micro RNASSRs (miRNASSRs). Of these, 20 primers (10 each from lncRNA-SSR and miRNA-SSR) were selected, screened on 6 coconut accessions, and 50% produced polymorphic fragments. These 10 polymorphic primers were used for genotyping 96 palms of 16 coconut accessions, comprising eight tall and dwarf accessions each. The number of alleles ranged from 2 to 9 per SSR marker, with an average of 4.6 alleles per locus. The average heterozygosity and Shannon index were 0.5 and 1.1, respectively, suggesting that ncRNA-SSRs show high polymorphism level. Distance-based cluster analyses revealed that all the tall and dwarf accessions were differentiated and grouped in different clusters. The study demonstrates the usefulness of ncRNA-based SSR markers for assessing genetic diversity and genetic improvement in coconut.

Integrating bio medical sensors in detecting hidden signatures of COVID-19 with Artificial intelligence.

Today COVID-19 pandemic articulates high stress on clinical resources around the world. At present, physical and viral tests are slowly emerging, and there is a need for robust pandemic detection that biomedical sensors can aid. The utility of biomedical sensors is correlated with the medical instruments with physiological metrics. These Biomedical sensors are integrated with the systematic device to track the target analytes with a biomedical component. The COVID-19 patients' samples are collected, and biomarkers are detected using four sensors: blood pressure sensor, G-FET based biosensor, electrochemical sensor, and potentiometric sensor with different quantifiable measures. The imputed data is then profiled with chest X-ray images from the Covid-19 patients.Multi-Layer Perceptron (MLP), an AI model, is deployed to identify the hidden signatures with biomarkers. The performance of the biosensor is measured with three parameters such as sensitivity, specificity and detection limit by generating the calibration plots that accurately fits the model.

Screening of PCSK9 and LDLR genetic variants in Familial Hypercholesterolemia (FH) patients in India.

Familial Hypercholesterolemia (FH) is an autosomal, dominant, inherited disorder characterized by severely elevated LDL-cholesterol (LDL-C) levels with high risk for Coronary Artery Disease (CAD). There are limited genetic studies especially on genes other than Low Density Lipoprotein receptor (LDLR) conducted in Indian population. Thus, our aim was to screen the entire Proprotein Convertase Subtilisin/Kexin type 9 gene (PCSK9) gene & hotspot exons 3, 4 and 9 of LDLR gene in FH cases and controls. 50 FH cases were categorized into definite, probable and possible cases according to Dutch Lipid Network Criteria (DLNC) who were gender matched with 50 healthy controls. All 12 exons of PCSK9, and hotspot exons 3, 4 & 9 of LDLR gene were screened through High Resolution Melt (HRM) curve analysis. Enzyme linked immunosorbent assay was performed to measure circulating PCSK9 levels. Total cholesterol and LDL-C were significantly high in all three groups of cases. Total 8 nonpathogenic variants in exon 1, 5, 7 and 9 of the PCSK9 gene were detected. In LDLR gene, 3 known pathogenic and 1 benign variant were found in exon 3 & 4. In FH cases, PCSK9 levels were significantly high compared to controls (P = 0.0001), and were directly correlated to LDL-C (P = 0.0001) and Total Cholesterol (P = 0.0001). Our study is first to screen the entire PCSK9 gene in western part of India. Since no pathogenic variants were identified, it is possible that PCSK9 variants are clinically less relevant. However, 3 known pathogenic variants were found in the LDLR gene. These findings support our understanding of the genetic spectrum of FH in India.

HΜGB1/sRAGE levels differ significantly between transudates and exudates.

High mobility group box 1(HMGB1) protein operates as an alarmin with multiple roles in immunity and cell homeostasis. It is highly expressed in epithelial barrier sites and acts via the binding to the receptor for advanced glycation end products (RAGE). Production of HMGB1 and soluble RAGE (sRAGE), a decoy receptor for HMGB1, has been implicated in several pulmonary diseases, but both have been scarcely investigated in pleural diseases. The aim of this study was to determine the levels of HMGB1 and sRAGE in transudative, malignant and parapneumonic pleural effusions (PEs) and to investigate the effect of low and high HMGB1 pleural fluid levels on MeT-5A cell adhesion, migration and spheroid formation, in each group. HMGB1 and sRAGE levels were significantly lower and higher in transudative PEs compared to malignant and parapneumonic PEs, respectively. Patients above 65 years of age had significantly lower HMGB1 and higher sRAGE levels compared to patients below 65 years old. Furthermore, incubation of MeT-5A cells with malignant or parapneumonic PEs bearing low or high levels of HMGB1 yielded significant differential effects on MeT-5A cell adhesion, migration and spheroid formation. In all types of effusions, high HMGB1 levels correlated with more adherence compared to low HMGB1 levels. In transudative and malignant PEs high HMGB1 levels correlated with decreased migration of MeT-5A cells while in parapneumonic ones the effect was the opposite. Only samples from parapneumonic PEs high in HMGB1 achieved uniform spheroid formation. These results reveal a clinical context-dependent effect of the HMGB1/sRAGE axis in PEs.

Cell and extracellular matrix interaction models in benign mesothelial and malignant pleural mesothelioma cells in 2D and 3D in-vitro.

Malignant pleural mesothelioma (MPM) is an aggressive tumour that grows in the pleural cavity. MPM spheroids released in the pleural fluid can form new tumour foci. Cell-cell, cell-extracellular matrix (ECM) interactions in 2D and 3D impact malignant cell behaviour during cell adhesion, migration, proliferation and epithelial-mesenchymal transition (EMT). In this study, epithelioid, biphasic and sarcomatoid MPM cell types as well as benign mesothelial cells were tested with regards to the above phenotypes. Fibronectin (FN) and homologous cell-derived extracellular matrix (hcd-ECM) treated substratum differentially affected the above phenotypes. 3D MPM spheroid invasion was higher in FN-collagen matrices in the epithelioid and biphasic cells, while 3D cell cultures of epithelioid and sarcomatoid MPM cells in FN-collagen showed a higher contractility compared to hcd-ECM-collagen. Cell aggregates demonstrated invasive behaviour in hcd-ECM matrices alone. Our results suggest that ECM and the dimensionality affect malignant cell behaviour during cell culture studies.

Insulin-like growth factor signalling and its significance as a biomarker in fish and shellfish research.

The insulin-like growth factor signalling system comprises insulin-like growth factors, insulin-like growth factor receptors and insulin-like growth factor-binding proteins. Along with the growth hormones, insulin-like growth factor signalling is very pivotal in the growth and development of all vertebrates. In fishes, insulin-like growth factors play an important role in osmoregulation, besides the neuroendocrine regulation of growth. Insulin-like growth factor concentration in plasma can assess the growth in fishes and shellfishes and therefore widely applied in nutritional research as an indicator to evaluate the performance of selected nutrients. The present review summarizes the role of insulin-like growth factor signalling in fishes and shellfishes, its significance in aquaculture and in evaluating growth, reproduction and development, and discusses the utility of this system as biomarkers for early indication of growth in aquaculture.

Evidence for Chloroquine/Hydroxychloroquine in the Treatment of COVID-19.

INTRODUCTION: Given the current lack of an approved and effective treatment or vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), repositioning old drugs for use as an antiviral treatment is an interesting strategy because knowledge about these drugs' safety profile, posology, and drug interactions is already known. Chloroquine and hydroxychloroquine, widely used as antimalarial and autoimmune disease drugs, have recently been reported as a potential broad-spectrum antiviral drug. BACKGROUND: The in vitro antiviral activity of chloroquine has been identified since the late 1960s. However, antiviral mechanisms of chloroquine remain speculative. Several clinical trials have been conducted to test the efficacy and safety of chloroquine or hydroxychloroquine in the treatment of COVID-19-associated pneumonia. The quality of the studies and the outcomes are evaluated in this systematic review and meta-analysis. REVIEW RESULTS: Literature review revealed 23 clinical studies. Only 9 of 23 studies were randomized controlled trials. Of nine randomized controlled trials, only study by Skipper et al. was deemed to be at low risk of bias. All studies evaluated variedwith different outcomes. Mechanical ventilation and virological clearance were the only common outcomes evaluated in more than two studies. Virological clearance odds ratio (OR) was 1.25 (95% confidence interval [CI] of 0.57-2.73; Chi2 = 0.83; I2 = 0%). GRADE quality of evidence was downgraded by three levels to very low due to concerns about the risk of bias, inconsistency, and imprecision. For mechanical ventilation, OR was 1.09 (95% CI 0.80-1.50; Chi2 = 0; I2 = 0). GRADE quality of evidence was downgraded by two levels to low due to concerns about the risk of bias and imprecision. There was no statistically significant difference between the groups for these two outcomes. CONCLUSION: As per the available evidence, based on our review, we conclude that hydroxychloroquine/chloroquine has not shown to be beneficial when used for the treatment of patients with COVID-19 pneumonia. HOW TO CITE THIS ARTICLE: Shetty RM, Namachivayam A. Evidence for Chloroquine/Hydroxychloroquine in the Treatment of COVID-19. Indian J Crit Care Med 2021;25(4):441-452.

Expert Consensus Statements on the Use of Corticosteroids in Non-severe COVID-19.

INTRODUCTION: There is strong evidence for the use of corticosteroid in the management of severe coronavirus disease-2019 (COVID-19). However, there is still uncertainty about the timing of corticosteroids. We undertook a modified Delphi study to develop expert consensus statements on the early identification of a subset of patients from non-severe COVID-19 who may benefit from using corticosteroids. METHODS: A modified Delphi was conducted with two anonymous surveys between April 30, 2021, and May 3, 2021. An expert panel of 35 experts was selected and invited to participate through e-mail. The consensus was defined as >70% votes in multiple-choice questions (MCQ) on Likert-scale type statements, while strong consensus as >90% votes in MCQ or >50% votes for "very important" on Likert-scale questions in the final round. RESULTS: Twenty experts completed two rounds of the survey. There was strong consensus for the increased work of breathing (95%), a positive six-minute walk test (90%), thorax computed tomography severity score of >14/25 (85%), new-onset organ dysfunction (using clinical or biochemical criteria) (80%), and C-reactive protein >5 times the upper limit of normal (70%) as the criteria for patients' selection. The experts recommended using oral or intravenous (IV) low-dose corticosteroids (the equivalent of 6 mg/day dexamethasone) for 5-10 days and monitoring of oxygen saturation, body temperature, clinical scoring system, blood sugar, and inflammatory markers for any "red-flag" signs. CONCLUSION: The experts recommended against indiscriminate use of corticosteroids in mild to moderate COVID-19 without the signs of clinical worsening. Oral or IV low-dose corticosteroids (the equivalent of 6 mg/day dexamethasone) for 5-10 days are recommended for patients with features of disease progression based on clinical, biochemical, or radiological criteria after 5 days from symptom onset under close monitoring. HOW TO CITE THIS ARTICLE: How to cite this article: Nasa P, Chaudhry D, Govil D, Daga MK, Jain R, Chhallani AA, et al. Expert Consensus Statements on the Use of Corticosteroids in Non-severe COVID-19. Indian J Crit Care Med 2021;25(11):1280-1285.

Development of EST-SSR markers for genetic diversity analysis in coconut (Cocos nucifera L.).

Genetic improvement in coconut relies on exploiting the vast existing diversity among coconut accessions. Robust molecular markers are a pre-requisite for efficient characterization of genetic diversity. Microsatellites or simple sequence repeats (SSRs), mined from expressed sequence tags (ESTs), constitute an important resource for analysis of genetic diversity as they are abundant, polymorphic and represent function regions of the genome. We have identified a total of 318,528 putative EST-SSRs from 130,942 unigenes utilizing a leaf transcriptome dataset of coconut. Among the EST-SSRs, dinucleotide repeats were abundant (219,912; 69.04%) followed by trinucleotide (70,722; 22.2%) and tetra-nucleotide repeats (6281; 1.9%). Among the dinucleotide repeat motifs, the dominant repeat was AG/CT (35.87%), followed by AT/AT (18.59%), while the dominant trinucleotide repeat was AAG/CTT (4.59%). One hundred and twenty EST-SSR primer pairs were designed and utilized to amplify six DNA samples of coconut accessions. Fifty primers (41.7%) produced reproducible polymorphic fragments of expected sizes, from which a total of 10 primers were selected for the diversity assessment in 186 palms of 50 coconut accessions, comprising of 25 each of tall and dwarf accessions. A total of 137 alleles were detected with an average of 13.7 alleles per SSR locus. The number of alleles observed at each locus in the data set ranged from 7 to 22. All the loci showed 100% polymorphism with respect to the samples screened. The average observed heterozygosity was 0.46. The PIC values ranged from 0.79 (CnKGDEST129 and CnKGDEST100) to 0.91 (CnKGDEST117 and CnKGDEST122) with a mean value of 0.85, indicating the capacity of the EST-SSR markers to detect high levels of polymorphism. The cluster analysis revealed that accessions were generally clustered based on their relative similarity and irrespective of their geographic origins. The present study demonstrates the usefulness of transcriptome sequencing as a rapid and cost-effective methodology for the development of molecular markers. The EST-SSR markers generated through this study constitute useful and reliable tools for assessment of genetic diversity and marker-assisted selection in coconut.