A computational model to uncover the biophysical underpinnings of neural firing heterogeneity in dissociated hippocampal cultures.

Calcium (Ca2+ ) imaging reveals a variety of correlated firing in cultures of dissociated hippocampal neurons, pinpointing the non-synaptic paracrine release of glutamate as a possible mediator for such firing patterns, although the biophysical underpinnings remain unknown. An intriguing possibility is that extracellular glutamate could bind metabotropic receptors linked with inositol trisphosphate (IP3 ) mediated release of Ca2+ from the endoplasmic reticulum of individual neurons, thereby modulating neural activity in combination with sarco/endoplasmic reticulum Ca2+ transport ATPase (SERCA) and voltage-gated Ca2+ channels (VGCC). However, the possibility that such release may occur in different neuronal compartments and can be inherently stochastic poses challenges in the characterization of such interplay between various Ca2+ channels. Here we deploy biophysical modeling in association with Monte Carlo parameter sampling to characterize such interplay and successfully predict experimentally observed Ca2+ patterns. The results show that the neurotransmitter level at the plasma membrane is the extrinsic source of heterogeneity in somatic Ca2+ transients. Our analysis, in particular, identifies the origin of such heterogeneity to an intrinsic differentiation of hippocampal neurons in terms of multiple cellular properties pertaining to intracellular Ca2+ signaling, such as VGCC, IP3 receptor, and SERCA expression. In the future, the biophysical model and parameter estimation approach used in this study can be upgraded to predict the response of a system of interconnected neurons.

Analytics Pipeline for Visualization of Single Cell RNA Sequencing Data from Brochoaveolar Fluid in COVID-19 Patients: Assessment of Neuro Fuzzy-C-Means and HDBSCAN.

Since the mutation in SARS-COV2 poses new challenges in designing vaccines, it is imperative to develop advanced tools for visualizing the genetic information. Specially, it remains challenging to address the patient-to-patient variability and identify the signature for severe/critical conditions. In this endeavor we analyze the large-scale RNA-sequencing data collected from broncho-alveolar fluid. In this work, we have used PCA and tSNE for the dimension-reduction. The novelty of the current work is to depict a detailed comparison of k-means, HDBSAN and neuro-fuzzy method in visualization of high-dimension data on gene expression. Clinical Relevance- The subpopulation profiling can be used to study the patient-to patient variability when infected by SARS-COV-2 and its variants. The distribution of cell types can be relevant in designing new drugs that are targeted to control the distribution of epithelial cells T cells and macrophages.

Mapping of structural arrangement of cells and collective calcium transients: an integrated framework combining live cell imaging using confocal microscopy and UMAP-assisted HDBSCAN-based approach.

Live cell calcium (Ca2+) imaging is one of the important tools to record cellular activity during in vitro and in vivo preclinical studies. Specially, high-resolution microscopy can provide valuable dynamic information at the single cell level. One of the major challenges in the implementation of such imaging schemes is to extract quantitative information in the presence of significant heterogeneity in Ca2+ responses attained due to variation in structural arrangement and drug distribution. To fill this gap, we propose time-lapse imaging using spinning disk confocal microscopy and machine learning-enabled framework for automated grouping of Ca2+ spiking patterns. Time series analysis is performed to correlate the drug induced cellular responses to self-assembly pattern present in multicellular systems. The framework is designed to reduce the large-scale dynamic responses using uniform manifold approximation and projection (UMAP). In particular, we propose the suitability of hierarchical DBSCAN (HDBSCAN) in view of reduced number of hyperparameters. We find UMAP-assisted HDBSCAN outperforms existing approaches in terms of clustering accuracy in segregation of Ca2+ spiking patterns. One of the novelties includes the application of non-linear dimension reduction in segregation of the Ca2+ transients with statistical similarity. The proposed pipeline for automation was also proved to be a reproducible and fast method with minimal user input. The algorithm was used to quantify the effect of cellular arrangement and stimulus level on collective Ca2+ responses induced by GPCR targeting drug. The analysis revealed a significant increase in subpopulation containing sustained oscillation corresponding to higher packing density. In contrast to traditional measurement of rise time and decay ratio from Ca2+ transients, the proposed pipeline was used to classify the complex patterns with longer duration and cluster-wise model fitting. The two-step process has a potential implication in deciphering biophysical mechanisms underlying the Ca2+ oscillations in context of structural arrangement between cells.

Quantitative Confocal Microscopy for Grouping of Dose-Response Data: Deciphering Calcium Sequestration and Subsequent Cell Death in the Presence of Excess Norepinephrine.

Fluorescent calcium (Ca2+) imaging is one of the preferred methods to record cellular activity during in vitro preclinical studies, high-content drug screening, and toxicity analysis. Visualization and analysis for dose-response data obtained using high-resolution imaging remain challenging, due to the inherent heterogeneity present in the Ca2+ spiking. To address this challenge, we propose measurement of cytosolic Ca2+ ions using spinning-disk confocal microscopy and machine learning-based analytics that is scalable. First, we implemented uniform manifold approximation and projection (UMAP) for visualizing the multivariate time-series dataset in the two-dimensional (2D) plane using Python. The dataset was obtained through live imaging experiments with norepinephrine-induced Ca2+ oscillation in HeLa cells for a large range of doses. Second, we demonstrate that the proposed framework can be used to depict the grouping of the spiking pattern for lower and higher drug doses. To the best of our knowledge, this is the first attempt at UMAP visualization of the time-series dose response and identification of the Ca2+ signature during lytic death. Such quantitative microscopy can be used as a component of a high-throughput data analysis workflow for toxicity analysis.

FDA approved L-type channel blocker Nifedipine reduces cell death in hypoxic A549 cells through modulation of mitochondrial calcium and superoxide generation.

As hypoxia is a major driver for the pathophysiology of COVID-19, it is crucial to characterize the hypoxic response at the cellular and molecular levels. In order to augment drug repurposing with the identification of appropriate molecular targets, investigations on therapeutics preventing hypoxic cell damage is required. In this work, we propose a hypoxia model based on alveolar lung epithelial cells line using chemical inducer, CoCl2 that can be used for testing calcium channel blockers (CCBs). Since recent studies suggested that CCBs may reduce the infectivity of SARS-Cov-2, we specifically select FDA approved calcium channel blocker, nifedipine for the study. First, we examined hypoxia-induced cell morphology and found a significant increase in cytosolic calcium levels, mitochondrial calcium overload as well as ROS production in hypoxic A549 cells. Secondly, we demonstrate the protective behaviour of nifedipine for cells that are already subjected to hypoxia through measurement of cell viability as well as 4D imaging of cellular morphology and nuclear condensation. Thirdly, we show that the protective effect of nifedipine is achieved through the reduction of cytosolic calcium, mitochondrial calcium, and ROS generation. Overall, we outline a framework for quantitative analysis of mitochondrial calcium and ROS using 3D imaging in laser scanning confocal microscopy and the open-source image analysis platform ImageJ. The proposed pipeline was used to visualize mitochondrial calcium and ROS level in individual cells that provide an understanding of molecular targets. Our findings suggest that the therapeutic value of nifedipine may potentially be evaluated in the context of COVID-19 therapeutic trials.

Detection of Specific Templates in Calcium Spiking in HeLa Cells Using Hierarchical DBSCAN: Clustering and Visualization of CellDrug Interaction at Multiple Doses.

One of the major challenges in analyzing large scale intracellular calcium spiking data obtained through fluorescent imaging is to identify various patterns present in time series data. Such an analysis identifying the distinct frequency and amplitude encoding during cell-drug interaction study is expected to provide new insights into the drug action patterns over a time course. Here, we present the HDBSCAN clustering algorithm to find a clustering pattern present in calcium spiking obtained by confocal imaging of single cells. Our methodology uncovers the specific templates present in dynamic responses obtained through treatment with multiple doses of the drug. First, we attempt to visualize the clustering pattern present in time-series data corresponding to various doses of the drug. Secondly, we show that the HDBSCAN can be used for the detection of specific signatures corresponding to low and high cell density regions selected from in vitro experiments. To the best of our knowledge, this is the first attempt to optimize the clustering of calcium dynamics using HDBSCAN. Finally, we emphasize that HDBSCAN offers a high-level grasp on systems biology data, including complex spiking pattern and can be used as a visual analytic tool for drug dose selection.

GPCR mediated control of calcium dynamics: A systems perspective.

G-protein coupled receptor (GPCR) mediated calcium (Ca2+)-signaling transduction remains crucial in designing drugs for various complex diseases including neurodegeneration, chronic heart failure as well as respiratory diseases. Although there are several reviews detailing various aspects of Ca2+-signaling such as the role of IP3 receptors and Ca2+-induced-Ca2+-release, none of them provide an integrated view of the mathematical descriptions of GPCR signal transduction and investigations on dose-response curves. This article is the first study in reviewing the network structures underlying GPCR signal transduction that control downstream [Cac2+]-oscillations. The central theme of this paper is to present the biochemical pathways, as well as molecular mechanisms underlying the GPCR-mediated Ca2+-dynamics in order to facilitate a better understanding of how agonist concentration is encoded in Ca2+-signals for Gαq, Gαs, and Gαi/o signaling pathways. Moreover, we present the GPCR targeting drugs that are relevant for treating cardiac, respiratory, and neuro-diseases. The current paper presents the ODE formulation for various models along with the detailed schematics of signaling networks. To provide a systems perspective, we present the network motifs that can provide readers an insight into the complex and intriguing science of agonist-mediated Ca2+-dynamics. One of the features of this review is to pinpoint the interplay between positive and negative feedback loops that are involved in controlling intracellular [Cac2+]-oscillations. Furthermore, we review several examples of dose-response curves obtained from [Cac2+]-spiking for various GPCR pathways. This paper is expected to be useful for pharmacologists and computational biologists for designing clinical applications of GPCR targeting drugs through modulation of Ca2+-dynamics.