Localized Calcium Signals in Early Zebrafish Development

AbstractActivation of the phosphoinositide (PI) pathway has been shown to be involved in the compaction of blastomeres in mouse embryos and in embryonic axis formation in Xenopus and in zebrafish embryos. Here we investigate Ca2+ signals in individual blastomeres of zebrafish embryos with the goal to better understand the role of PI and Ca2+ signaling for early vertebrate embryogenesis. Initial studies showed that the inositol 1,4,5-trisphosphate (IP3) concentration increases after the 32-cell stage of development, suggesting that IP3-mediated Ca2+ signals may be present during the blastula stage. Ca2+ signals were measured by identifying individual cells using confocal imaging of a nuclear localized Ca2+ indicator. Using this in situ indicator, changes in Ca2+ concentration were measured over several hours in each cell of a series of sections through the developing embryo. Transient increases in Ca2+ concentration that lasted 20-50 sec (Ca2+ spikes) were first triggered during the 32- to 128-cell stage in cells of the outer embryonic cell layer. These cells develop epithelial characteristics and specialize into the enveloping layer (EVL). No Ca2+ activity was observed during the earlier cleavage cycles or in deep blastomeres. Ca2+ spikes remained restricted to the EVL until the end of the blastula stage. Ca2+ spikes in neighboring EVL cells often occurred in the same short time interval, indicating that small groups of EVL cells can synchronize their activity. When averaged over several cell cycles, Ca2+ activity showed an even distribution in the EVL and did not indicate future polarities

RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease Modeling and Therapy

Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human-induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogramming may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation, and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA-based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic, and translational research. J. Cell. Physiol. 232: 1262–1269, 2017. © 2016 Wiley Periodicals, Inc

A Comparison of Adverse Effect Profiles of Two Anti-IL-5 Therapies in Adults with Uncontrolled Asthma―A Network Meta-analysis of Phase 3 Trials―

The aim of this study was to compare the adverse effect profiles of mepolizumab（MPZ）and benralizumab（BRZ）in adults with uncontrolled asthma. A network meta-analysis of phase 3 trials was conducted to compare the adverse effects of MPZ and BRZ in patients with uncontrolled asthma. The MEDLINE-PubMed, Scopus and the Cochrane library databases were searched to identify any relevant articles. The outcome measures of fatal adverse events, headache, and injection site reaction are presented as odds ratios（ORs）with 95％ confidence intervals（CIs）. The surface under the cumulative curve（SUCRA）for each outcome was also compared among MPZ, BRZ, and placebo treatments. Four randomized controlled trials of MPZ（100mg s/c every four weeks）（100-MPZ）or BRZ（30mg s/c every eight weeks）（30-BRZ）met the criteria for inclusion in the study. The ORs and 95％ CIs of 100-MPZ compared with BRZ for fatal adverse events, headache, and injection site reaction were 0.26（0.01-4.90）, 0.79（0.40-1.54）, and 2.32（0.79-6.80）, respectively. SUCRAs for 100-MPZ, 30-BRZ, and placebo were 0.8, 0.3, and 0.4 for fatal adverse events, 0.5, 0.1, and 0.8 for headache, and 0.0, 0.6, and 0.8 for injection site reaction, respectively. There were no significant differences in the incidence of fatal adverse events, headache, and injection site reaction between MPZ and BRZ treatment. However, the SUCRA values indicate an association between administration of BRZ and the occurrence of fatal adverse event or headache, or between administration of MPZ and the occurrence of injection site reaction. Moreover, the incidence odds of injection site reaction were significantly higher in the MPZ group than in the placebo group. Further analysis will be needed to clarify the details of safety profiles of these anti-IL-5 therapies

Inhibitory Effects of Caffeic Acid Phenethyl Ester Derivatives on Replication of Hepatitis C Virus

<div><p>Caffeic acid phenethyl ester (CAPE) has been reported as a multifunctional compound. In this report, we tested the effect of CAPE and its derivatives on hepatitis C virus (HCV) replication in order to develop an effective anti-HCV compound. CAPE and CAPE derivatives exhibited anti-HCV activity against an HCV replicon cell line of genotype 1b with EC₅₀ values in a range from 1.0 to 109.6 µM. Analyses of chemical structure and antiviral activity suggested that the length of the n-alkyl side chain and catechol moiety are responsible for the anti-HCV activity of these compounds. Caffeic acid n-octyl ester exhibited the highest anti-HCV activity among the tested derivatives with an EC₅₀ value of 1.0 µM and an SI value of 63.1 by using the replicon cell line derived from genotype 1b strain Con1. Treatment with caffeic acid n-octyl ester inhibited HCV replication of genotype 2a at a similar level to that of genotype 1b irrespectively of interferon signaling. Caffeic acid n-octyl ester could synergistically enhance the anti-HCV activities of interferon-alpha 2b, daclatasvir, and VX-222, but neither telaprevir nor danoprevir. These results suggest that caffeic acid n-octyl ester is a potential candidate for novel anti-HCV chemotherapy drugs.</p></div

Effect of CAPE derivatives on the interferon-signaling pathway.

<p>(A) Plasmids pISRE-TA-Luc and phRG-TK were co-transfect into Huh7 OK1 cells. The transfected cells were cultured with 1, 10, 100, or 1000 U/mL of interferon-alpha 2b, and compounds <b>1</b>, <b>6</b> and <b>10</b>. Treatment with DMSO corresponds to ‘0’. After 48 h of treatment, luciferase activities were measured, and the value were normalized against <i>Renilla</i> luciferase activities. Error bars indicate standard deviation. The data represent three independent experiments. (B) Huh7 replicon cell line of genotype 1b was treated with 1, 10, 100, or 1000 U/mL of interferon-alpha 2b, and compounds <b>1</b>, <b>6</b> and <b>10</b> for 48 h. Treatment with DMSO corresponds to the control. The mRNAs of Mx1, MxA, IFIT4, ISG15, OAS1, OAS2, OAS3, and GAPDH as an internal control were detected by RT-PCR.</p

Effect of caffeic acid esters 7and 8 on HCV replication.

<p>Chemical structures of both compounds are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082299#pone.0082299.s003" target="_blank">Figure S3</a></p><p>a: Fifty percent effective concentration based on the inhibition of HCV replication.</p><p>b: Fifty percent cytotoxicity concentration based on the reduction in cell viability.</p><p>c: Selectivity index (CC₅₀/EC₅₀).</p><p>d: Determined with ChemDraw software (Chem Bio Office Ultra, 2008).</p

Correlation between the inhibitory effect on HCV replication <i>and C</i>log <i>P</i> of CAPE analogues.

<p>Values of <i>x</i>-axis indicate EC₅₀ values of CAPE analogues, while values of <i>y</i>-axis show <i>C</i>log <i>P</i> values. (A) Correlation between the inhibitory effect on HCV replication and <i>C</i>log <i>P</i> of CAPE analogues (Compound 7–11). (B) Correlation between the inhibitory effect on HCV replication and <i>C</i>log <i>P</i> of CAPE analogues (Compound 10 and 13–16).</p

Anti-HCV activity of compound 10 in replicon cell lines of genotypes 1b and 2a.

<p>a: Fifty percent effective concentration based on the inhibition of HCV replication.</p><p>b: Fifty percent cytotoxicity concentration based on the inhibition of HCV replication.</p><p>c: Selectivity Index (CC₅₀/EC₅₀).</p

Effect of octyl esters 10 and 13–16 on HCV replication.

<p>The basic structure and side moieties are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082299#pone.0082299.s004" target="_blank">Figure S4</a>.</p><p>a: Fifty percent effective concentration based on the inhibition of HCV replication.</p><p>b: Fifty percent cytotoxicity concentration based on the reduction in cell viability.</p><p>c: Selectivity index (CC₅₀/EC₅₀).</p><p>d: Determined with ChemDraw software (Chem Bio Office Ultra, 2008).</p