Biology subjects

Malhotra, D.

Publications and source records attributed to Malhotra, D..

11 recordsLinked to original sources

bioRxiv2025

Microcystin-LR aerosol exposure increases inflammatory drivers of asthma, Evidence of an NF-κB amplification mechanism

Microcystin-LR (MC-LR) is one of a large family of cyanotoxins which are naturally produced by cyanobacteria within harmful algal blooms occurring in bodies of water globally. Early findings of the toxicity of such blooms stemmed from fatalities of livestock drinking from affected water. Since then, various toxins have been identified such as the microcystins. Microcystin-LR has been studied as a representative congener due to its abundance and toxicity. While there have been extensive studies of microcystin-LR by oral route exposure, we have turned our attention to inhalation route exposure due to the recent findings of microcystin-containing lake and sea-spray aerosol. We have shown inflammatory outcomes in the airways of mice and in human cell culture models after microcystin aerosol exposure, and have found a consistent molecular patterns similar to those of Type 1/Type 17 driven neutrophilic asthma. Here we address the hypothesis that MC-LR will increase the inflammatory mediators of neutrophilic asthma leading to worsening symptoms. This is tested and characterized in both in vitro and in vivo models. We found that asthma symptoms and molecular signatures of inflammation are both worsened by MC-LR exposure in a mouse model of neutrophilic asthma. We found that 3D human airway cell culture models reconstructed from asthmatic donor cells are similarly affected, however healthy donor cells are nearly unaltered by comparison. Aggregating these findings with RNA sequencing data from all models, we developed a hypothetical molecular mechanism which relies on MC-LR mediated amplification of existing inflammatory signaling. We test this in a human reporter cell line of NF-{kappa}B activity and further demonstrate the mechanism by inhibitor testing. This study sheds light on the risk to asthmatic patients living near or recreating on affected bodies of water. Beyond asthma, we believe this study provides crucial insight into the findings over the last 40 years concerning disparate outcomes of MC-LR exposure as the result of exposure will be dependent on the signaling state of the tissue upon exposure.

molecular biology↗

bioRxiv2024

Single-nucleus and spatial transcriptomic profiling of human temporal cortex and white matter reveals novel associations with AD pathology

Alzheimers disease (AD) is a neurodegenerative disorder with complex pathological manifestations and is the leading cause of cognitive decline and dementia in elderly individuals. A major goal in AD research is to identify new therapeutic pathways by studying the molecular and cellular changes in the disease, either downstream or upstream of the pathological hallmarks. In this study, we present a comprehensive investigation of cellular heterogeneity from the temporal cortex region of 40 individuals, comprising healthy donors and individuals with differing tau and amyloid burden. Using single-nucleus transcriptome analysis of 430,271 nuclei from both gray and white matter of these individuals, we identified cell type-specific subclusters in both neuronal and glial cell types with varying degrees of association with AD pathology. In particular, these associations are present in layer specific glutamatergic (excitatory) neuronal types, along with GABAergic (inhibitory) neurons and glial subtypes. These associations were observed in early as well as late pathological progression. We extended this analysis by performing multiplexed in situ hybridization using the CARTANA platform, capturing 155 genes in 13 individuals with varying levels of tau pathology. By modeling the spatial distribution of these genes and their associations with the pathology, we not only replicated key findings from our snRNA data analysis, but also identified a set of cell type-specific genes that show selective enrichment or depletion near pathological inclusions. Together, our findings allow us to prioritize specific cell types and pathways for targeted interventions at various stages of pathological progression in AD.

neuroscience↗

bioRxiv2022

Single-cell Mendelian randomisation identifies cell-type specific genetic effects on human brain disease and behaviour

Translating genome-wide association loci to therapies requires knowledge of the causal genes, their directionality of effect and the cell-types in which they act. To infer these relationships in the human brain, we implemented Mendelian randomisation using single cell-type expression quantitative trait loci (eQTLs) as genetic anchors. Expression QTLs were mapped across 8 major cell-types in brain tissue exclusively ascertained from donors with no history of brain disease. We report evidence for a causal association between the change in expression of 118 genes and one or more of 16 brain phenotypes, revealing candidate targets for risk mitigation and opportunities for shared preventative therapeutic strategies. We highlight key causal genes for neurodegenerative and neuropsychiatric disease and for each, we report its cellular context and the therapeutic directionality required for risk mitigation. Our use of control samples establishes a new resource for the causal interpretation of GWAS risk alleles for human brain phenotypes.

neuroscience↗

bioRxiv2022

Single nuclei RNAseq stratifies multiple sclerosis patients into three distinct white matter glia responses

The lack of understanding of the cellular and molecular basis of clinical and genetic heterogeneity in progressive multiple sclerosis (MS) has hindered the search for new effective therapies. Here, to address this gap, we analysed 632,000 single nuclei RNAseq profiles of 156 brain tissue samples, comprising white matter (WM) lesions, normal appearing WM, grey matter (GM) lesions and normal appearing GM from 54 MS patients and 26 controls. We observed the expected changes in overall neuronal and glial numbers previously described within the classical lesion subtypes. We found highly cell type-specific gene expression changes in MS tissue, with distinct differences between GM and WM areas, confirming different pathologies. However, surprisingly, we did not observe distinct gene expression signatures for the classical different WM lesion types, rather a continuum of change. This indicates that classical lesion characterization better reflects changes in cell abundance than changes in cell type gene expression, and indicates a global disease effect. Furthermore, the major biological determinants of variability in gene expression in MS WM samples relate to individual patient effects, rather than to lesion types or other metadata. We identify four subgroups of MS patients with distinct WM glial gene expression signatures and patterns of oligodendrocyte stress and/or maturation, suggestive of engagement of different pathological processes, with an additional more variable regenerative astrocyte signature. The discovery of these patterns, which were also found in an independent MS patient cohort, provides a framework to use molecular biomarkers to stratify patients for optimal therapeutic approaches for progressive MS, significantly advances our mechanistic understanding of progressive MS, and highlights the need for precision-medicine approaches to address heterogeneity among MS patients.

neuroscience↗

bioRxiv2021

Defining the architecture of cerebrospinal fluid cellular communities in neuroinflammatory diseases

Cerebrospinal fluid (CSF) biomarkers are important for multiple sclerosis (MS) diagnosis. Moreover, absent of autopsy or biopsy tissue, CSF is the most relevant source for studying the immune cells involved in MS pathophysiology. Single-cell RNA sequencing (scRNA-seq) provides new opportunities to advance our understanding of disease-associated changes in CSF immune cells. Here, using scRNA-seq data generated from 58 CSF and 10 PBMC samples, we provide an updated atlas of the immune cells present in human CSF in MS and other neuroinflammatory conditions, including novel lymphoid and myeloid cell clusters. Our atlas can thus serve as a reference for future studies of immune cells in neuroinflammation. Our further characterization of CSF myeloid cells suggests that most CSF microglia-like cells resemble two of the previously-described brain microglia signatures. Additionally, our data from a sex-mismatched bone marrow transplant recipient suggest that CSF microglia-like cells are of peripheral origin. Our comparisons between MS and other neuroinflammatory disorders show a highly-specific increase in plasma cells, along with reductions in the proportion of microglia-like cells in MS CSF. Furthermore, our analyses on MS patients receiving anti-CD20 therapy ocrelizumab suggest that the treatment effects are not limited to B cell depletion, and ocrelizumab appears to reverse some MS-associated T and myeloid changes in CSF. Finally, we utilized our atlas to prioritize (1) CSF cell types expressing genes associated with MS susceptibility, and (2) ligand-receptor gene pairs that are differentially expressed in MS CSF, providing targets for further mechanistic and causal investigations in pathophysiology and treatment of MS.

immunology↗

bioRxiv2021

A structured evaluation of cryopreservation in generating single cell transcriptomes from cerebrospinal fluid

ImportanceA robust cerebrospinal fluid (CSF) cell cryopreservation protocol using high resolution single-cell (sc) transcriptomic data would enable the deployment of this important modality in multi-center translational research studies and clinical trials in which many sites do not have the expertise or resources to produce data from fresh samples. It would also serve to reduce technical variability in larger projects. ObjectiveTo test a reliable cryopreservation protocol adapted for CSF cells, facilitating the characterization of these rare, fragile cells in moderate to large scale studies. DesignDiagnostic lumbar punctures were performed on twenty-one patients at two independent sites. Excess CSF was collected and cells were isolated. Each cell sample was split into two fractions for single cell analysis using one of two possible chemistries: 3 sc-RNA-Sequencing or 5sc-RNA-Sequencing. One cell fraction was processed fresh while the second sample was cryopreserved and profiled at a later time after thawing. SettingThe research protocol was deployed at two academic medical centers taking care of multiple sclerosis and other neurological conditions. Participants21 subjects (age 24 - 72) were recruited from individuals undergoing a diagnostic lumbar puncture for suspected neuroinflammatory disease or another neurologic illness; they donated excess CSF. FindingsOur comparison of fresh and cryopreserved data from the same individuals demonstrates highly efficient recovery of all known CSF cell types. The proportion of all cell types was similar between the fresh and the cryopreserved cells processed, and RNA expression was not significantly different. Results were comparable at both performance sites, and with different single cell sequencing chemistries. Cryopreservation also did not affect recovery of T and B cell clonotype diversity. Conclusion and relevanceOur cryopreservation protocol for CSF-cells provides an important alternative to fresh processing of fragile CSF cells: cryopreservation enables the involvement of sites with limited capacity for experimental manipulation and reduces technical variation by enabling batch processing and pooling of samples. Key pointsO_ST_ABSQuestionC_ST_ABSHow efficient is CSF cryopreservation for single-cell transcriptome analysis and can it be implemented in large multi-center translational and clinical trial settings? FindingsWe compared single-cell transcriptomes of paired fresh and cryopreserved CSF from 21 patients at two independent sites. We validate the efficacy of a simple and cost effective CSF cryopreservation method that preserves the composition and the transcriptomes of CSF cells stored for weeks-months. The protocol is deployed in a large multicenter Phase 4 MS clinical trial. MeaningA validated CSF cryopreservation method that would significantly advance basic science and biomarker research in neurological disorders by implementing single-cell transcriptome analyses in multi-center research and clinical trials.

genetics↗

bioRxiv2021

Ischemic tolerance and cardiac repair in the African spiny mouse

Ischemic heart disease and by extension myocardial infarction is the primary cause of death worldwide, necessitating regenerative therapies to restore heart function. Current models of heart regeneration are restricted in that they are not of adult mammalian origin, precluding the study of class-specific traits that have emerged throughout evolution, and reducing translatability of research findings to humans. Here, we overcome those restrictions by introducing the African spiny mouse (Acomys spp.), a murid rodent that has recently been found to exhibit bona fide regeneration of the back skin and ear pinna. We show that spiny mice exhibit tolerance to myocardial infarction through superior survivability, improved ventricular conduction, smaller scar size, and near-absence of cardiac remodeling. Critically, spiny mice display increased vascularization and cardiomyocyte expansion, with an associated improvement in heart function. These findings present new avenues for mammalian heart research by leveraging unique tissue properties of the spiny mouse.

developmental biology↗

bioRxiv2020

CellMixS: quantifying and visualizing batch effects in single cell RNA-seq data

A key challenge in single cell RNA-sequencing (scRNA-seq) data analysis are dataset- and batch-specific differences that can obscure the biological signal of interest. While there are various tools and methods to perform data integration and correct for batch effects, their performance can vary between datasets and according to the nature of the bias. Therefore, it is important to understand how batch effects manifest in order to adjust for them in a reliable way. Here, we systematically explore batch effects in a variety of scRNA-seq datasets according to magnitude, cell type specificity and complexity. We developed a cell-specific mixing score (cms) that quantifies how well cells from multiple batches are mixed. By considering distance distributions (in a lower dimensional space), the score is able to detect local batch bias and differentiate between unbalanced batches (i.e., when one cell type is more abundant in a batch) and systematic differences between cells of the same cell type. We implemented cms and related metrics to detect batch effects or measure structure preservation in the CellMixS R/Bioconductor package. We systematically compare different metrics that have been proposed to quantify batch effects or bias in scRNA-seq data using real datasets with known batch effects and synthetic data that mimic various real data scenarios. While these metrics target the same question and are used interchangeably, we find differences in inter- and intra-dataset scalability, sensitivity and in a metrics ability to handle batch effects with differentially abundant cell types. We find that cell-specific metrics outperform cell type-specific and global metrics and recommend them for both method benchmarks and batch exploration.

bioinformatics↗

bioRxiv2020

Impact of Comorbidities on the Expression of SARS-CoV-2 Viral Entry-Related Genes

Viral entry mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are an important aspect of virulence. Proposed mechanisms involve host cell membrane-bound angiotensin-converting enzyme 2 (ACE2) and type II transmembrane serine proteases (TTSPs), such as transmembrane serine protease isoform 2 (TMPRSS2). The distribution of expression of these genes across cell types representing multiple organ systems in healthy individuals has been recently demonstrated. However, comorbidities such as diabetes and cardiovascular disease are highly prevalent in patients with Coronavirus Disease 2019 (COVID-19) and associated with worse outcomes. Whether these conditions contribute directly to SARS-CoV-2 virulence remain unclear. Here we show that the expression levels of ACE2, TMPRSS2 and other viral entry-related genes are modulated in target organs of select disease states. In tissues such as heart, which normally express ACE2 but minimal TMPRSS2, we found that TMPRSS2 as well as other TTSPs are elevated in individuals with comorbidities vs healthy individuals. Additionally, we found increased expression of viral entry-related genes in the settings of hypertension, cancer or smoking across target organ systems. Our results demonstrate that common comorbidities may contribute directly to SARS-CoV-2 virulence and suggest new therapeutic targets to improve outcomes in vulnerable patient populations.

microbiology↗

bioRxiv2019

On the discovery of population-specific state transitions from multi-sample multi-condition single-cell RNA sequencing data

Single-cell RNA sequencing (scRNA-seq) has quickly become an empowering technology to profile the transcriptomes of individual cells on a large scale. Many early analyses of differential expression have aimed at identifying differences between subpopulations, and thus are focused on finding subpopulation markers either in a single sample or across multiple samples. More generally, such methods can compare expression levels in multiple sets of cells, thus leading to cross-condition analyses. However, given the emergence of replicated multi-condition scRNA-seq datasets, an area of increasing focus is making sample-level inferences, termed here as differential state analysis. For example, one could investigate the condition-specific responses of cell subpopulations measured from patients from each condition; however, it is not clear which statistical framework best handles this situation. In this work, we surveyed the methods available to perform cross-condition differential state analyses, including cell-level mixed models and methods based on aggregated "pseudobulk" data. We developed a flexible simulation platform that mimics both single and multi-sample scRNA-seq data and provide robust tools for multi-condition analysis within the muscat R package.

bioinformatics↗

bioRxiv2018

A junction coverage compatibility score to quantify the reliability of transcript abundance estimates and annotation catalogs

Most methods for statistical analysis of RNA-seq data take a matrix of abundance estimates for some type of genomic features as their input, and consequently the quality of any obtained results are directly dependent on the quality of these abundances. Here, we present the junction coverage compatibility (JCC) score, which provides a way to evaluate the reliability of transcript-level abundance estimates as well as the accuracy of transcript annotation catalogs. It works by comparing the observed number of reads spanning each annotated splice junction in a genomic region to the predicted number of junction-spanning reads, inferred from the estimated transcript abundances and the genomic coordinates of the corresponding annotated transcripts. We show that while most genes show good agreement between the observed and predicted junction coverages, there is a small set of genes that do not. Genes with poor agreement are found regardless of the method used to estimate transcript abundances, and the corresponding transcript abundances should be treated with care in any downstream analyses.

bioinformatics↗