Retinopathy of prematurity (ROP) is a neurovascular complication in preterm babies, leading to severe visual impairment, but the underlying mechanisms are yet unclear. The present study aimed at unraveling the molecular mechanisms underlying the pathogenesis of ROP. A comprehensive screening of candidate genes in preterms with ROP (n = 189) and no-ROP (n = 167) was undertaken to identify variants conferring disease susceptibility. Allele and genotype frequencies, linkage disequilibrium and haplotypes were analyzed to identify the ROP-associated variants. Variants in CFH (p = 2.94 × 10−7), CFB (p = 1.71 × 10−5), FBLN5 (p = 9.2 × 10−4), CETP (p = 2.99 × 10−5), and CXCR4 (p = 1.32 × 10−8) genes exhibited significant associations with ROP. Further, a quantitative assessment of 27 candidate proteins and cytokines in the vitreous and tear samples of babies with severe ROP (n = 30) and congenital cataract (n = 30) was undertaken by multiplex bead arrays and further validated by western blotting and zymography. Significant elevation and activation of MMP9 (p = 0.038), CFH (p = 2.24 × 10−5), C3 (p = 0.05), C4 (p = 0.001), IL-1ra (p = 0.0019), vascular endothelial growth factor (VEGF) (p = 0.0027), and G-CSF (p = 0.0099) proteins were observed in the vitreous of ROP babies suggesting an increased inflammation under hypoxic condition. Along with inflammatory markers, activated macrophage/microglia were also detected in the vitreous of ROP babies that secreted complement component C3, VEGF, IL-1ra, and MMP-9 under hypoxic stress in a cell culture model. Increased expression of the inflammatory markers like the IL-1ra (p = 0.014), MMP2 (p = 0.0085), and MMP-9 (p = 0.03) in the tears of babies at different stages of ROP further demonstrated their potential role in disease progression. Based on these findings, we conclude that increased complement activation in the retina/vitreous in turn activated microglia leading to increased inflammation. A quantitative assessment of inflammatory markers in tears could help in early prediction of ROP progression and facilitate effective management of the disease, thereby preventing visual impairment.
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.
MgO nanoparticles are synthesized using water, ethanol and aqueous CTAB solution. The nanoparticles synthesized in ethanol exhibited smallest size, maximum reactive oxygen species generation and maximum antibacterial ability, and low haemolysis.
The development of a minimally invasive, robust, and inexpensive technique that permits real‐time monitoring of cell responses on biomaterial scaffolds can improve the eventual outcomes of scaffold‐based tissue engineering strategies. Towards establishing correlations between in situ biological activity and cell fate, we have developed a comprehensive workflow for real‐time volumetric imaging of spatiotemporally varying cytosolic calcium oscillations in pure microglial cells cultured on electrospun meshes. Live HMC3 cells on randomly oriented electrospun fibers were stained with a fluorescent dye and imaged using a laser scanning confocal microscope. Resonance scanning provided high‐resolution in obtaining the time‐course of intracellular calcium levels without compromising spatial and temporal resolution. Three‐dimensional reconstruction and depth‐coding enabled the visualization of cell location and intracellular calcium levels as a function of sample thickness. Importantly, changes in cell morphology and in situ calcium spiking were quantified in response to a soluble biochemical cue and varying matrix architectures (i.e., randomly oriented and aligned fibers). Importantly, raster plots generated from spiking data revealed calcium signatures specific to culture conditions. In the future, our approach can be used to elucidate correlations between calcium signatures and cell phenotype/activation, and facilitate the rational design of scaffolds for biomedical applications.
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.
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