Co-Silencing of the Voltage-Gated Calcium Channel β Subunit and High-Voltage Activated α1 Subunit by dsRNA Soaking Resulted in Enhanced Defects in Locomotion, Stylet Thrusting, Chemotaxis, Protein Secretion, and Reproduction in Ditylenchus destructor

The voltage-gated calcium channel (VGCC) β subunit (Cavβ) protein is a kind of cytosolic auxiliary subunit that plays an important role in regulating the surface expression and gating characteristics of high-voltage-activated (HVA) calcium channels. Ditylenchus destructor is an important plant-parasitic nematode. In the present study, the putative Cavβ subunit gene of D. destructor, namely, DdCavβ, was subjected to molecular characterization. In situ hybridization assays showed that DdCavβ was expressed in all nematode tissues. Transcriptional analyses showed that DdCavβ was expressed during each developmental stage of D. destructor, and the highest expression level was recorded in the third-stage juveniles. The crucial role of DdCavβ was verified by dsRNA soaking-mediated RNA interference (RNAi). Silencing of DdCavβ or HVA Cavα1 alone and co-silencing of the DdCavβ and HVA Cavα1 genes resulted in defective locomotion, stylet thrusting, chemotaxis, protein secretion and reproduction in D. destructor. Co-silencing of the HVA Cavα1 and Cavβ subunits showed stronger interference effects than single-gene silencing. This study provides insights for further study of VGCCs in plant-parasitic nematodes.

Modification of mesenchymal stem cells by HMGB1 promotes the activity of Cav3.2 T-type calcium channel via PKA/β-catenin/γ-cystathionase pathway

Background Mesenchymal stem cells (MSC) hold great promise for treating cardiovascular disease. Recently, we genetically modified MSCs with high mobility group box 1 (HMGB1), and these cells demonstrated high mobility by efficient migrating and homing to target neointima. The possible mechanism was investigated in the current study. Methods Rat MSCs were transfected with lentivirus containing HMGB1 cDNA to yield MSC-H cell line stably overexpressing HMGB1. The MSC-C cells which were transfected with empty lentivirus served as negative control, and the differentially expressed genes were analyzed by microarray. The cell mobility was determined by transwell migration assay. Intracellular free calcium and the expression of Cav3.2 T-type calcium channel (CACNA1H) were assayed to analyze activity of CACNA1H-mediated calcium influx. H2S production and γ-cystathionase expression were examined to assess the activity of γ-cystathionase/H2S signaling. The interaction of HMGB1 with γ-cystathionase in MSC-H cells was analyzed by co-immunoprecipitation. Luciferase reporter assay was performed to determine whether the promoter activity of γ-cystathionase was regulated by interaction of β-catenin and TCF/LEF binding site. Intercellular cAMP, PKA activity, phosphorylation of β-catenin, and GSK3β were investigated to reveal cAMP/PKA mediated β-catenin activation. Result Microarray analysis revealed that differentially expressed genes were enriched in cAMP signaling and calcium signaling. CACNA1H was upregulated to increase intracellular free calcium and MSC-H cell migration. Blockage of CACNA1H by ABT-639 significantly reduced intracellular free calcium and cell migration. The γ-cystathionase/H2S signaling was responsible for CACNA1H activation. H2S production was increased with high expression of γ-cystathionase in MSC-H cells, which was blocked by γ-cystathionase inhibitor DL-propargylglycine. Upregulation of γ-cystathionase was not attributed to interaction with HMGB1 overexpressed in MSC-H cells although γ-cystathionase was suggested to co-immunoprecipitate with oxidized HMGB1. Bioinformatics analysis identified a conserved TCF/LEF binding site in the promoter of γ-cystathionase gene. Luciferase reporter assay confirmed that the promoter had positive response to β-catenin which was activated in MSC-H cells. Finally, cAMP/PKA was activated to phosphorylate β-catenin at Ser657 and GSK3β, enabling persisting activation of Wnt/β-catenin signaling in MSC-H cells. Conclusion Our study revealed that modification of MSCs with HMGB1 promoted CACNA1H-mediated calcium influx via PKA/β-catenin/γ-cystathionase pathway. This was a plausible mechanism for high mobility of MSC-H cell line.

Oral Antihypertensives for Nonsevere Pregnancy Hypertension: Systematic Review, Network Meta- and Trial Sequential Analyses

Background: We aimed to address which antihypertensives are superior to placebo/no therapy or another antihypertensive for controlling nonsevere pregnancy hypertension and provide future sample size estimates for definitive evidence. Methods: Randomized trials of antihypertensives for nonsevere pregnancy hypertension were identified from online electronic databases, to February 28, 2021 (registration URL: https://www.crd.york.ac.uk/PROSPERO/ ; unique identifier: CRD42020188725). Our outcomes were severe hypertension, proteinuria/preeclampsia, fetal/newborn death, small-for-gestational age infants, preterm birth, and admission to neonatal care. A Bayesian random-effects model generated estimates of direct and indirect treatment comparisons. Trial sequential analysis informed future trials needed. Results: Of 1246 publications identified, 72 trials were included; 61 (6923 women) were informative. All commonly prescribed antihypertensives (labetalol, other β-blockers, methyldopa, calcium channel blockers, and mixed/multi-drug therapy) versus placebo/no therapy reduced the risk of severe hypertension by 30% to 70%. Labetalol decreased proteinuria/preeclampsia (odds ratio, 0.73 [95% credible interval, 0.54–0.99]) and fetal/newborn death (odds ratio, 0.54 [0.30–0.98]) compared with placebo/no therapy, and proteinuria/preeclampsia compared with methyldopa (odds ratio, 0.66 [0.44–0.99]) and calcium channel blockers (odds ratio, 0.63 [0.41–0.96]). No other differences were identified, but credible intervals were wide. Trial sequential analysis indicated that 2500 to 10 000 women/arm (severe hypertension or safety outcomes) to >15 000/arm (fetal/newborn death) would be required to provide definitive evidence. Conclusions: In summary, all commonly prescribed antihypertensives in pregnancy reduce the risk of severe hypertension, but labetalol may also decrease proteinuria/preeclampsia and fetal/newborn death. Evidence is lacking for many other safety outcomes. Prohibitive sample sizes are required for definitive evidence. Real-world data are needed to individualize care.