Biology subjects

Nayak, K.

Publications and source records attributed to Nayak, K..

12 recordsLinked to original sources

bioRxiv2025

Human Milk Oligosaccharides Modulate Inflammatory Responses and Lipid Metabolism in a Human Intestinal Organoid Model

Human milk oligosaccharides (HMOs) are a major component of human breast milk and have significant protective effects on infant gut health. HMOs are also present in the amniotic fluid surrounding the developing fetus in the womb. Beginning at 10 weeks of gestational age, HMOs enter the fetal gut through the swallowing of amniotic fluid. The effects of this earliest exposure to HMOs on the gut epithelium, which pre-term infants partially miss, remains unknown. In this study, we investigate the effect of a blend of HMOs, including 2-fucosyllactose (2FL), 3-sialyllactose (3SL), and difucosyllactose (DFL), on intestinal epithelial cells, examining their role in steady-state conditions and during inflammation. By utilizing advanced intestinal epithelial organoid models derived from fetal and pediatric donors, we reveal developmental-stage-specific responses to this HMO blend, as well as its potential to mitigate inflammatory damage. These findings offer important insights into the role of HMOs in early-life nutrition and gut health, lending compelling evidence to support their inclusion in infant nutrition strategies.

cell biology↗

bioRxiv2024

Reconstructing bat antiviral immunity using epithelial organoids

Bats are remarkably resilient to viruses with pandemic potential. To resolve largely unknown molecular mechanisms governing their exceptional antiviral immunity, we established an organoid platform to model the entire respiratory airway and intestinal epithelium of the important viral reservoir species Rousettus aegyptiacus (Egyptian fruit bat). These bat organoids exhibit an unexpected diversity of cell types and support replication of highly pathogenic zoonotic viruses including Marburg virus (MARV) and MERS-Coronavirus. Following virus infection, bat organoids unleash a strong interferon response, uniquely regulated through virus-dependent and virus-independent mechanisms. By contrast, MARV infected human organoids fail to induce an antiviral gene response and express pro-inflammatory cytokines after interferon stimulation, revealing important molecular differences between bats and humans with implications for lethal Marburg virus infections in primates. These data provide the most comprehensive organoid platform in bats to decode species-specific differences and uncover fundamental principles of bat disease resilience to emerging viruses with pandemic potential.

immunology↗

bioRxiv2023

Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR

Organoids are currently one of the most widely used ex vivo models in epithelial biology. Combined with genetic editing strategies, organoids offer a promise of rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has been only up to 30%, with implications for the subsequent need for selection including including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method of intestinal organoid editing using a Ribonucleoprotein CRISPR-based approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different anatomical gut locations and developmental timepoints from multiple patient samples with no off-target editing. The method allowed us to study the effect of the loss of the tumour suppressor gene, PTEN, in normal human intestinal cells. Analysis of PTEN deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss including mTORC1 activation.

cell biology↗

bioRxiv2022

Transcriptional profiles of functionally distinct HLADR+CD38+ CD8 T cells subsets from acute febrile dengue patients

Previous studies showed that a discrete population of the CD8 T cells with HLADR+CD38+ phenotype expand massively during the acute febrile phase of dengue natural infection. Although about a third of these massively expanding HLADR+CD38+ CD8 T cells were of CD69high phenotype, only a small fraction of them produced IFN{gamma} upon in vitro peptide stimulation. What other cytokines/ chemokines do these peptides stimulated HLADR+CD38+ CD8 T cells express, what transcriptional profiles distinguish the CD69+IFN{gamma}+, CD69+IFN{gamma}-, and CD69-IFN{gamma}- subsets, and whether the expansion of the total HLADR+CD38+ CD8 T cells or the IFN{gamma} producing CD8 T cells differ depending on disease severity remained unclear. This study addresses these knowledge gaps. We find that the CD69+IFN{gamma}+ subset uniquely expressed key genes involved in protein translation, cellular metabolism, proliferation and dendritic cell cross talk. Both the CD69+IFN{gamma}+ and CD69+IFN{gamma}- subsets had an antigen responsive gene signature with genes involved in cytotoxic effector functions, regulation of T cell receptor signaling, signaling by MAPK, chemotaxis and T cell trafficking to inflamed tissues with the expression being more robust in the IFN{gamma}+ CD69+ subset. On the other hand, the CD69- IFN{gamma}- subset was biased towards expression of genes that both augment and dampen T cell responses. Lastly, the expansion of total HLADR+ CD38+ CD8 T cells and also the IFN{gamma} producing HLADR+ CD38+ CD8 T cells was similar in patients with different grades of disease. Taken together, this study provides valuable insights into the inherent diversity of the effector CD8 T cell response during dengue.

immunology↗

bioRxiv2022

Structural insights for neutralization of BA.1 and BA.2 Omicron variants by a broadly neutralizing SARS-CoV-2 antibody

The SARS-CoV-2 BA.1 and BA.2 (Omicron) variants contain more than 30 mutations within the spike protein and evade therapeutic monoclonal antibodies (mAbs). Here, we report a receptor-binding domain (RBD) targeting human antibody (002-S21F2) that effectively neutralizes live viral isolates of SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Gamma, Delta, and Omicron (BA.1 and BA.2) with IC50 ranging from 0.02 - 0.05 g/ml. This near germline antibody 002-S21F2 has unique genetic features that are distinct from any reported SARS-CoV-2 mAbs. Structural studies of the full-length IgG in complex with spike trimers (Omicron and WA.1) reveal that 002-S21F2 recognizes an epitope on the outer face of RBD (class-3 surface), outside the ACE2 binding motif and its unique molecular features enable it to overcome mutations found in the Omicron variants. The discovery and comprehensive structural analysis of 002-S21F2 provide valuable insight for broad and potent neutralization of SARS-CoV-2 Omicron variants BA.1 and BA.2.

immunology↗

bioRxiv2021

Immunophenotyping and transcriptional profiling of human plasmablasts in dengue.

Previous studies have shown that plasmablasts expand massively in dengue patients as compared to many other situations such as influenza infection or vaccination. However, a detailed understanding of the phenotypes and transcriptional features of these cells is lacking. Moreover, despite India having nearly a third of global dengue disease burden, there is virtually no information on plasmablasts responses in dengue patients from India. Here, we provide a detailed characterization of plasmablast responses from dengue confirmed febrile children in India. Immunophenotyping and RNA seq analysis showed that in addition to secreting dengue specific antibodies, these massively expanding cells expressed several adhesion molecules, chemokines and chemokine receptors that are involved in endothelial interactions, homing to skin or mucosal tissues including intestine. Surprisingly, we found that these cells also upregulated expression of several cytokine genes that are involved in angiogenesis, leukocyte extravasation and vascular permeability. These transcriptional features were qualitatively similar to plasmablasts from influenza vaccinees. Interestingly, the expansion of the plasmablasts in dengue patients was significantly lower in patients with primary dengue infection compared to those with secondary dengue. Moreover, within the primary dengue patients, their expansion was significantly lower in patients with mild dengue infection (DI) compared to patients with dengue with warning signs (DW) or severe dengue (SD). These results significantly improve our understanding of human plasmablast responses in dengue. Importance Dengue is a globally spreading with over 100 million clinical cases annually with symptoms ranging from mild self-limiting febrile illness to more severe and sometimes life-threatening dengue hemorrhagic fever or shock, especially among children. India contributes nearly a third of global dengue disease burden. The pathophysiology of dengue is complex and remains poorly understood despite many advances indicating a key role for antibody dependent enhancement of infection. While serum antibodies have been extensively studied, the characteristics of the cellular factories responsible for antibody production, i.e., plasmablasts, are only beginning to emerge. This study provides a comprehensive understanding of the magnitude, phenotype, functional and transcriptional profiles of human plasmablasts from dengue patients in India.

immunology↗

bioRxiv2021

Cells of the human intestinal tract mapped across space and time

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. To comprehensively map cell lineages in the healthy developing, pediatric and adult human gut from ten distinct anatomical regions, as well as draining lymph nodes, we used singlecell RNA-seq and VDJ analysis of roughly one third of a million cells. This reveals the presence of BEST4+ absorptive cells throughout the human intestinal tract, demonstrating the existence of this cell type beyond the colon for the first time. Furthermore, we implicate IgG sensing as a novel function of intestinal tuft cells, and link these cells to the pathogenesis of inflammatory bowel disease. We define novel glial and neuronal cell populations in the developing enteric nervous system, and predict cell-type specific expression of Hirschsprungs disease-associated genes. Finally, using a systems approach, we identify key cell players across multiple cell lineages driving secondary lymphoid tissue formation in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. These data provide an unprecedented catalogue of intestinal cells, and new insights into cellular programs in development, homeostasis and disease.

cell biology↗

bioRxiv2020

Characterization of neutralizing versus binding antibodies and memory B cells in COVID-19 recovered individuals from India

India is one of the countries most affected by the recent COVID-19 pandemic. Characterization of humoral responses to SARS-CoV-2 infection, including immunoglobulin isotype usage, neutralizing activity and memory B cell generation, is necessary to provide critical insights on the formation of immune memory in Indian subjects. In this study, we evaluated SARS-CoV-2 receptor-binding domain (RBD)-specific IgG, IgM, and IgA antibody responses, neutralization of live virus, and RBD-specific memory B cell responses in pre-pandemic healthy versus convalescent COVID-19 individuals from India. We observed substantial heterogeneity in the formation of humoral and B cell memory post COVID-19 recovery. While a vast majority (38/42, 90.47%) of COVID-19 recovered individuals developed SARS-CoV-2 RBD-specific IgG responses, only half of them had appreciable neutralizing antibody titers. RBD-specific IgG titers correlated with these neutralizing antibody titers as well as with RBD-specific memory B cell frequencies. In contrast, IgG titers measured against SARS-CoV-2 whole virus preparation, which includes responses to additional viral proteins besides RBD, did not show robust correlation. Our results suggest that assessing RBD-specific IgG titers can serve as a surrogate assay to determine the neutralizing antibody response. These observations have timely implications for identifying potential plasma therapy donors based on RBD-specific IgG in resource-limited settings where routine performance of neutralization assays remains a challenge. ImportanceOur study provides an understanding of SARS-CoV-2-specific neutralizing antibodies, binding antibodies and memory B cells in COVID-19 convalescent subjects from India. Our study highlights that PCR-confirmed convalescent COVID-19 individuals develop SARS-CoV-2 RBD-specific IgG antibodies, which correlate strongly with their neutralizing antibody titers. RBD-specific IgG titers, thus, can serve as a valuable surrogate measurement for neutralizing antibody responses. These finding have timely significance for selection of appropriate individuals as donors for plasma intervention strategies, as well as determining vaccine efficacy.

immunology↗

bioRxiv2020

CD8+ T-cell transcription and DNA methylation show age specific differences and lack correlation with clinical outcome in pediatric Inflammatory Bowel Disease

Background & AimsCD8+ T-cell gene expression has been implicated in the pathogenesis of Inflammatory Bowel Diseases (IBD) and has been shown to correlate with disease outcome in adult patients. Moreover, CD8+ T-cell exhaustion was identified as a contributing mechanism that impacts on disease behaviour. We aimed to explore CD8+ T-cell gene expression and DNA methylation in children newly diagnosed with IBD and test their correlation with disease outcome. MethodsWe prospectively recruited 112 children with IBD at the point of diagnosis and 19 non-IBD controls. Follow-up samples were obtained from a subset of patients at 3-month intervals (n=62). CD8+ T-cells were purified from peripheral blood samples using magnetic bead sorting and genome-wide transcriptional (n=192) and DNA methylation (n=66) profiles were generated using Affymetrix and Illumina arrays respectively. Publicly available adult CD8+ T-cell transcriptomes and DNA methylomes were included in data analyses to investigate age dependant differences. ResultsVariation amongst CD8+ T-cell transcriptomes obtained from children showed association with disease, systemic inflammation, age and gender but lacked correlation with disease outcome in pediatric IBD. In contrast to CD8+ T-cell transcriptomes in adult Crohns Disease (CD), samples from pediatric patients did not show variation within genes forming part of the previously reported prognostic expression or T-cell exhaustion signatures. Pediatric CD patient derived DNA methylation profiles also lacked correlation with disease outcome but in comparison to adult CD8+ methylomes showed a higher predicted proportion of CD8+ naive T-cells. ConclusionsOur findings indicate age-related differences in IBD pathogenesis and highlight the importance of validating adult clinical biomarkers in pediatric cohorts.

immunology↗

bioRxiv2020

Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohns disease

Human gut development requires the orchestrated interaction of various differentiating cell types. Here we generate an in-depth single-cell map of the developing human intestine at 6-10 weeks post-conception, a period marked by crypt-villus formation. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells, which are distinct from LGR5-expressing cells. We use computational analyses to show that these cells contribute to differentiated cell subsets directly and indirectly via the generation of LGR5-expressing stem cells and receive signals from the surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNAseq profiles from paediatric Crohns disease epithelium alongside matched healthy controls to reveal disease associated changes in epithelial composition. Contrasting these with the fetal profiles reveals re-activation of fetal transcription factors in Crohns disease epithelium. Our study provides a unique resource, available at www.gutcellatlas.org, and underscores the importance of unravelling fetal development in understanding disease.

cell biology↗

bioRxiv2019

Norovirus replication in human intestinal epithelial cells is restricted by the interferon-induced JAK/STAT signalling pathway and RNA Polymerase II mediated transcriptional responses.

Human noroviruses (HuNoV) are a leading cause of viral gastroenteritis worldwide and a significant cause of morbidity and mortality in all age groups. The recent finding that HuNoV can be propagated in B cells and mucosa derived intestinal epithelial organoids (IEOs), has transformed our capability to dissect the life cycle of noroviruses. Using RNA-Seq of HuNoV infected intestinal epithelial cells (IECs), we have found that replication of HuNoV in IECs results in interferon-induced transcriptional responses and that HuNoV replication in IECs is sensitive to IFN. This contrasts with previous studies that suggest that the innate immune response may play no role in the restriction of HuNoV replication in immortalised cells. We demonstrate that the inhibition of JAK1/JAK2 enhances HuNoV replication in IECs. Surprisingly, targeted inhibition of cellular RNA polymerase II-mediated transcription was not detrimental to HuNoV replication, but enhanced replication to a greater degree compared to blocking of JAK signalling directly. Furthermore, we demonstrate for the first time that IECs generated from genetically modified intestinal organoids, engineered to be deficient in the interferon response, are more permissive to HuNoV infection. Together our work identifies the IFN-induced transcriptional responses restrict HuNoV replication in IECs and demonstrates that the inhibition of these responses by modifications to the culture conditions can greatly enhance the robustness of the norovirus culture system.\n\nImportanceNoroviruses are a major cause of gastroenteritis worldwide yet the challenges associated with their growth culture has greatly hampered the development of therapeutic approaches and has limited our understanding of cellular pathways that control infection. Here we show that human intestinal epithelial cells, the first point of entry of human noroviruses into the host, limit virus replication by the induction of the innate responses. Furthermore we show that modulating the ability of intestinal epithelial cells to induce transcriptional responses to HuNoV infection can significantly enhance human norovirus replication in culture. Collectively our findings provide new insights into the biological pathways that control norovirus infection but also identify mechanisms to enhance the robustness of norovirus culture.

microbiology↗

bioRxiv2019

Analysis of physiological noise in quantitative cardiac magnetic resonance

PurposeTo determine the impact of imaging parameters on the temporal signal-to-noise ratio (TSNR) of quantitative cardiac magnetic resonance (MR) in humans, and to determine applicability of the physiological noise covariance (PNC) model for physiological noise (PN).\n\nMethodsWe conducted MRI experiments in four healthy volunteers, and obtained series of short-axis cardiac images acquired with snapshot balanced steady-state free precession (bSSFP) and snapshot gradient echo (GRE) using a broad range of spatial resolutions and parallel imaging acceleration factors commonly used in quantitative cardiac MR. We measured regional SNR and TSNR in these datasets and fit the measurements to the PNC model for PN, which assumes that PN scales with signal strength.\n\nResultsThe relationship between SNR and TSNR in human cardiac MR without contrast preparation was well modeled by the PNC model. SNR consistently decreased as the spatial resolution (matrix size) and acceleration factor (R) increased for both GRE and bSSFP imaging. TSNR varied linearly with SNR using GRE imaging, when SNR was low (SNR < 20), and approached an asymptotic limit using bSSFP imaging, when SNR was high (SNR > 40).\n\nConclusionsThe PNC model can be used to guide the choice of matrix size and acceleration factor to optimize TSNR in stable contrast cardiac MR, such as T2-prepared Blood-Oxygen-Level-Dependent (BOLD) and several variants of Arterial Spin Labeled (ASL) cardiac MR.

bioengineering↗