The present review discusses recent progress in single-cell RNA sequencing (scRNA-seq), which can describe cellular heterogeneity in various organs, bodily fluids, and pathologies (e.g., cancer and Alzheimer’s disease). We outline scRNA-seq techniques that are suitable for investigating cellular heterogeneity that is present in cell populations with very high resolution of the transcriptomic landscape. We summarize scRNA-seq findings and applications of this technology to identify cell types, activity, and other features that are important for the function of different bodily organs. We discuss future directions for scRNA-seq techniques that can link gene expression, protein expression, cellular function, and their roles in pathology. We speculate on how the field could develop beyond its present limitations (e.g., performing scRNA-seq in situ and in vivo). Finally, we discuss the integration of machine learning and artificial intelligence with cutting-edge scRNA-seq technology, which could provide a strong basis for designing precision medicine and targeted therapy in the future.

G-protein coupled receptor (GPCR) mediated calcium (Ca 2+)-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 Ca 2+-signaling such as the role of IP 3 receptors and Ca 2+-induced-Ca 2+-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 [Ca c 2+ ]oscillations. The central theme of this paper is to present the biochemical pathways, as well as molecular mechanisms underlying the GPCR-mediated Ca 2+-dynamics in order to facilitate a better understanding of how agonist concentration is encoded in Ca 2+-signals for G αq , G αs , and G αi/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 Ca 2+-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 [Ca c 2+ ]-oscillations. Furthermore, we review several examples of dose-response curves obtained from [Ca c 2+ ]-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 Ca 2+dynamics.

In neurons, stromal interaction molecule (STIM) proteins regulate store-operated Ca2+ entry (SOCE) and are involved in calcium signaling pathways. However, STIM activity in neurological diseases is unclear and should be clarified by studies that are performed in vivo rather than in cultured cells in vitro. The present study investigated the role of neuronal Stim2b protein in zebrafish. We generated stim2b knockout zebrafish, which were fertile and had a regular lifespan. Using various behavioral tests, we found that stim2b−/− zebrafish larvae were hyperactive compared with wild-type fish. The mutants exhibited increases in mobility and thigmotaxis and disruptions of phototaxis. They were also more sensitive to pentylenetetrazol and glutamate treatments. Using lightsheet microscopy, a higher average oscillation frequency and higher average amplitude of neuronal Ca2+ oscillations were observed in stim2b−/− larvae. RNA sequencing detected upregulation of the annexin 3a and gpr39 genes and downregulation of the rrm2, neuroguidin, and homer2 genes. The latter gene encodes a protein that is involved in several processes that are involved in Ca2+ homeostasis in neurons, including metabotropic glutamate receptors. We propose that Stim2b deficiency in neurons dysregulates SOCE and triggers changes in gene expression, thereby causing abnormal behavior, such as hyperactivity and susceptibility to seizures.

Imaging cytosolic calcium in neurons is emerging as a new tool in neurological disease diagnosis, drug screening, and toxicity testing. Ca oscillation signatures show a significant variation depending on GPCR targeting agonists. Quantification of Ca spike trains in ligand induced Ca oscillations remains challenging due to their inherent heterogeneity in primary culture. Moreover, there is no framework available for identification of optimal number of clusters and distance metric to cluster Ca spike trains. Using quantitative confocal imaging and clustering analysis, we show the characterization of Ca spiking in GPCR targeting drug-treated primary culture of hippocampal neurons. A systematic framework for selection of the clustering method instead of an intuition-based method was used to optimize the cluster number and distance metric. The results discern neurons with diverse Ca response patterns, including higher amplitude fast spiking and lower spiking responses, and their relative percentage in a neuron population in absence and presence of GPCR-targeted drugs. The proposed framework was employed to show that the  clustering pattern of Ca spiking can be controlled using GABA and mGluR targeting drugs. This approach can be used for unbiased measurement of neural activity and identification of spiking population with varying amplitude and frequencies, providing a platform for high-content drug screening.