A Framework for Classification of Heterogeneous Calcium Dynamics

G-protein coupled receptors are the target receptors for designing almost 45% of the drug in current drug market. For drug screening using cell-based assays, fluorescent imaging of GPCR mediated calcium dynamics in single cells can be used to obtain the dose-response profile. However, construction of a dose-response function based on single cell responses is rather challenging as the cells in a population respond heterogeneously to the drug. Here we developed a live cell imaging-based approach to quantify the heterogeneity of the HeLa cell population in response to GPCR mediated drugs. First, we found that activation of CXCR4 by SDF-1α induces calcium responses in Hela cells. We measured the temporal dynamics of cytosolic calcium through time-lapse imaging using confocal microscopy for various drug doses

Establishment of Standardization Parameters for Herbal Formulation, Swarasa Bhvit Amalaka Churnam

Herbal formulations are very useful for human health because of their natural and simple processing. A quality herbal formulation must pass the test for their characteristic properties. Inspite of the large number of Ayurvedic formulations are available in the market, standards of their quality are yet to be laid. Amlaka churnam or amalaki rasayanam is a swarasa bhavita, important herbal formulation used for treatment related to immunity systems of human body. In addition to this it is also used for nurturing of skin, heart, eye and stomach. The main ingredient of amalaka churnam is amla (Embalica officinalis) family Euphorbiaceae. Though amalaka churnam is described in many classical Ayurvedic granthas, but there are minor differences in their formulations. Also marketed formulations of amalaki rasayanam by manufacturers do not have description about its standardization.  Therefore, it is necessary to develop standard parameters for establishment of quality of the formulation. In this work, a standardization of herbal formulations of amalaka churnam is reported in terms of organoleptic evaluation of ingredients with rasayana formulation, loss of drying analysis, foreign matter, phytochemical screening, pH estimation, evaluation of ash value, extractive value and rheological evaluation. Keywords: Bhvit, amalaka Churnam, Organoleptic, rheological etc

Standardization Parameters for Herbal Formulation, Avipattikar Churnam

Herbal formulations are being used by human society from a very long period of time. To ensure quality of the herbal formulation, a number of standardization parameters are set. Although there are large number of Ayurvedic formulations, standards of quality are yet to be laid for many of them. Avipattikar Churna is one of the important herbal formulation used for stomach diseases like dyspepsia, gastritis, hyper acidity, loss of appetite and stomach burning sensation. In this work, a standardization of herbal formulations of avipattikar churna is reported in terms of organoleptic evaluation of ingredients with rasayana formulation, loss of drying analysis, foreign matter, phytochemical screening, evaluation of ash value, extractive value and rheological evaluation

Molecular Assessment of Epiretinal Membrane: Activated Microglia, Oxidative Stress and Inflammation

Fibrocellular membrane or epiretinal membrane (ERM) forms on the surface of the inner limiting membrane (ILM) in the inner retina and alters the structure and function of the retina. ERM formation is frequently observed in ocular inflammatory conditions, such as proliferative diabetic retinopathy (PDR) and retinal detachment (RD). Although peeling of the ERM is used as a surgical intervention, it can inadvertently distort the retina. Our goal is to design alternative strategies to tackle ERMs. As a first step, we sought to determine the composition of the ERMs by identifying the constituent cell-types and gene expression signature in patient samples. Using ultrastructural microscopy and immunofluorescence analyses, we found activated microglia, astrocytes, and Muller glia in the ERMs from PDR and RD patients. Moreover, oxidative stress and inflammation associated gene expression was significantly higher in the RD and PDR membranes as compared to the macular hole samples, which are not associated with inflammation. We specifically detected differential expression of hypoxia inducible factor 1-alpha (HIF1-alpha), proinflammatory cytokines, and Notch, Wnt, and ERK signaling pathway-associated genes in the RD and PDR samples. Taken together, our results provide new information to potentially develop methods to tackle ERM formation

Biological and Medical Importance of Cellular Heterogeneity Deciphered by Single-Cell RNA Sequencing

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

Formulation and evaluation of novel stomach specific floating microspheres bearing famotidine for treatment of gastric ulcer and their radiographic study

Objective: To develop and characterize multiple-unit-type oral floating microsphere of famotidine to prolong gastric residence time and to target stomach ulcer. Methods: The floating microspheres were prepared by modified solvent evaporation method. Eudragit S-100 was used as polymer. Microspheres were characterized for the micromeritic properties, floating behavior, entrapment efficiency and scanning electron microscopy. The in-vitro release studies and floating behavior were studied in simulated gastric fluid at pH 1.2. Different drug release kinetics models were also applied for all the batches. Selected formulations were also subjected for X-ray radiographic study. Results: Floating microspheres were successfully prepared by modified solvent evaporation technique. Microspheres showed passable flow properties. The maximum yield of microspheres was up to (95.11±0.35)%. On the basis of optical microscopy particle size range was found to be ranging from (52.18±182.00) to (91.64±5.16) μm. Scanning electron microscopy showed their spherical size, perforated smooth surface and a cavity inside microspheres. Microspheres were capable to float up to 20 h in simulated gastric fluid. X-ray radiographic studies also proved its better retention in the stomach. Conclusions: On the basis of the results, such dosage forms may be a good candidate for stomach targeting and may be dispensed in hard gelatin capsules

<span style="font-size:11.0pt;mso-bidi-font-size: 12.0pt;font-family:"Times New Roman","serif";mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US; mso-bidi-language:HI" lang="EN-GB">Production of α-galactosidase from <i style="mso-bidi-font-style: normal">Aspergillus foetidus</i> MTCC 6322 by solid state fermentation and its application in soymilk hydrolysis</span>

72-75<span style="font-size:11.0pt;mso-bidi-font-size: 12.0pt;font-family:" times="" new="" roman","serif";mso-fareast-font-family:"times="" roman";="" mso-bidi-font-family:mangal;mso-ansi-language:en-gb;mso-fareast-language:en-us;="" mso-bidi-language:hi"="" lang="EN-GB">The production of α-galactosidase from the wild fungal strain Aspergillus foetidus MTCC 6322 using solid state fermentation (SSF), its characterization, and its efficacy in the hydrolysis of soymilk using response surface methodology were studied. The optimum conditions for production of α-galactosidase by SSF were: wheat bran (10 g), moisture content (64%), inoculum volume (1.0 mL; 6 × 107 spores/mL) with a yield of 4.1 × 103 units per gram dry substrate (U/gds) at 96 h. The enzyme showed optimum activity at pH 6.0, temperature 40°C, pH stability between 5.0-8.0, and temperature stability between 30-40°C. The enzyme was stable in the presence of trypsin, lipase, and collagenase and it showed susceptibility of the substrates such as raffinose, melibiose, guar gum and soymilk to hydrolysis in varying degrees. The optimized conditions for soymilk hydrolysis were: soymilk (10 mL) from defatted soybean meal (1.5%), α-galactosidase (0.15 UmL-1) at 30°C, pH 6.0 and duration of 1 h.</span

Identification of Zebrafish Calcium Toolkit Genes and their Expression in the Brain

Zebrafish are well-suited for in vivo calcium imaging because of the transparency of their larvae and the ability to express calcium probes in various cell subtypes. This model organism has been used extensively to study brain development, neuronal function, and network activity. However, only a few studies have investigated calcium homeostasis and signaling in zebrafish neurons, and little is known about the proteins that are involved in these processes. Using bioinformatics analysis and available databases, the present study identified 491 genes of the zebrafish Calcium Toolkit (CaTK). Using RNA-sequencing, we then evaluated the expression of these genes in the adult zebrafish brain and found 380 hits that belonged to the CaTK. Based on quantitative real-time polymerase chain reaction arrays, we estimated the relative mRNA levels in the brain of CaTK genes at two developmental stages. In both 5 dpf larvae and adult zebrafish, the highest relative expression was observed for tmbim4, which encodes a Golgi membrane protein. The present data on CaTK genes will contribute to future applications of zebrafish as a model for in vivo and in vitro studies of Ca2+ signaling

stim2b Knockout Induces Hyperactivity and Susceptibility to Seizures in Zebrafish Larvae

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&minus;/&minus; 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&minus;/&minus; 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

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