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.

LY294002 Inhibits Intermediate Conductance Calcium-Activated Potassium (KCa3.1) Current in Human Glioblastoma Cells

Glioblastomas (GBs) are among the most common tumors with high malignancy and invasiveness of the central nervous system. Several alterations in protein kinase and ion channel activity are involved to maintain the malignancy. Among them, phosphatidylinositol 3-kinase (PI3K) activity and intermediate conductance calcium-activated potassium (KCa3.1) current are involved in several aspects of GB biology. By using the electrophysiological approach and noise analysis, we observed that KCa3.1 channel activity is LY294002-sensitive and Wortmannin-resistant in accordance with the involvement of PI3K class IIβ (PI3KC2β). This modulation was observed also during the endogenous activation of KCa3.1 current with histamine. The principal action of PI3KC2β regulation was the reduction of open probability in intracellular free calcium saturating concentration. An explanation based on the “three-gate” model of the KCa3.1 channel by PI3KC2β was proposed. Based on the roles of KCa3.1 and PI3KC2β in GB biology, a therapeutic implication was suggested to prevent chemo- and radioresistance mechanisms.

Genome-Wide Identification and Expression Profile of OSCA Gene Family Members in Triticum aestivum L.

Hyperosmolality and various other stimuli can trigger an increase in cytoplasmic-free calcium concentration ([Ca2+]cyt). Members of the Arabidopsis thaliana (L.) reduced hyperosmolality-gated calcium-permeable channels (OSCA) gene family are reported to be involved in sensing extracellular changes to trigger hyperosmolality-induced [Ca2+]cyt increases and controlling stomatal closure during immune signaling. Wheat (Triticum aestivum L.) is a very important food crop, but there are few studies of its OSCA gene family members. In this study, 42 OSCA members were identified in the wheat genome, and phylogenetic analysis can divide them into four clades. The members of each clade have similar gene structures, conserved motifs, and domains. TaOSCA genes were predicted to be regulated by cis-acting elements such as STRE, MBS, DRE1, ABRE, etc. Quantitative PCR results showed that they have different expression patterns in different tissues. The expression profiles of 15 selected TaOSCAs were examined after PEG (polyethylene glycol), NaCl, and ABA (abscisic acid) treatment. All 15 TaOSCA members responded to PEG treatment, while TaOSCA12/-39 responded simultaneously to PEG and ABA. This study informs research into the biological function and evolution of TaOSCA and lays the foundation for the breeding and genetic improvement of wheat.

Evaluation of the Volume Stability and Resource Benefit of Basic Oxygen Furnace Slag and Its Asphalt Mixture Based on Field Application

Applying basic oxygen furnace (BOF) slag as aggregate in asphalt mixture is continuously investigated due to the increasing shortage of natural aggregate in recent years. However, the negative effect of BOF’s expansion in water greatly limits its further application in pavement construction. To address this problem, this paper studied the volume stability of BOF, and its asphalt mixture relied on actual engineering. The asphalt mixtures contained BOF aggregate was designed by the Marshall method with three different gradation types (AC-16, AC-20, and ATB-25). Besides, both laboratory samples and the core samples from field drilling were investigated in volume expansion rate after curing in a water bath. The economic and resource benefits of BOF replacement of natural aggregates were also analyzed. The results showed that the free calcium oxide content of BOF slag is positively related to the particle sizes. Nevertheless, the expansion rates of both the BOF aggregate and its asphalt mixture were less than 1%, which meant the BOF aggregate applied to the asphalt mixture meets the practical engineering requirements. The maximum allowable free calcium oxide content for large-grain size of steel slag is the smallest; it is also recommended that the expansibility of large-grain steel slag should be the first concern in the application. The resource assessment indicated that the use of steel slag for the construction of a trial section of one kilometer of single lane can save 967 tons of natural aggregates. The economic evaluation showed that the use of steel slag instead of natural aggregates for surface course construction could reduce the investment by 16.87%. The experimental methods and conclusions mentioned in this article provide stable references to enhance the development of sustainable pavement by recycling metallurgical slag in highway construction.

Damage Signaling by Extracellular Nucleotides: A Role for Cyclic Nucleotides in Elevating Cytosolic Free Calcium?

Extracellular ATP (eATP) is now held to be a constitutive damage-associated molecular pattern (DAMP) that is released by wounding, herbivory or pathogen attack. The concentration of eATP must be tightly regulated as either depletion or overload leads to cell death. In Arabidopsis thaliana, sensing of eATP is by two plasma membrane legume-like lectin serine–threonine receptor kinases (P2K1 and P2K2), although other receptors are postulated. The transcriptional response to eATP is dominated by wound- and defense-response genes. Wounding and pathogen attack can involve the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) which, in common with eATP, can increase cytosolic-free Ca2+ as a second messenger. This perspective on DAMP signaling by eATP considers the possibility that the eATP pathway involves production of cyclic nucleotides to promote opening of cyclic nucleotide-gated channels and so elevates cytosolic-free Ca2+. In silico analysis of P2K1 and P2K2 reveals putative adenylyl and guanylyl kinase sequences that are the hallmarks of “moonlighting” receptors capable of cAMP and cGMP production. Further, an Arabidopsis loss of function cngc mutant was found to have an impaired increase in cytosolic-free Ca2+ in response to eATP. A link between eATP, cyclic nucleotides, and Ca2+ signaling therefore appears credible.

Glycine Ameliorates Endoplasmic Reticulum Stress Induced by Thapsigargin in Porcine Oocytes

The endoplasmic reticulum (ER) is a multifunctional organelle in the cytoplasm that plays important roles in female mammalian reproduction. The endoplasmic reticulum and mitochondria interact to maintain the normal function of cells by maintaining intracellular calcium homeostasis. As proven by previous research, glycine (Gly) can regulate the intracellular free calcium concentration ([Ca2+]i) and enhance mitochondrial function to improve oocyte maturation in vitro. The effect of Gly on ER function during oocyte in vitro maturation (IVM) is not clear. In this study, we induced an ER stress model with thapsigargin (TG) to explore whether Gly can reverse the ER stress induced by TG treatment and whether it is associated with calcium regulation. The results showed that the addition of Gly could improve the decrease in the average cumulus diameter, the first polar body excretion rate caused by TG-induced ER stress, the cleavage rate and the blastocyst rate. Gly supplementation could reduce the ER stress induced by TG by significantly improving the ER levels and significantly downregulating the expression of genes related to ER stress (Xbp1, ATF4, and ATF6). Moreover, Gly also significantly alleviated the increase in reactive oxygen species (ROS) levels and the decrease in mitochondrial membrane potential (ΔΨ m) to improve mitochondrial function in porcine oocytes exposed to TG. Furthermore, Gly reduced the [Ca2+]i and mitochondrial Ca2+ ([Ca2+]m) levels and restored the ER Ca2+ ([Ca2+]ER) levels in TG-exposed porcine oocytes. Moreover, we found that the increase in [Ca2+]i may be caused by changes in the distribution and expression of inositol 1,4,5-triphosphate receptor (IP3R1) and voltage-dependent anion channel 1 (VDAC1), while Gly can restore the distribution and expression of IP3R1 and VDAC1 to normal levels. Apoptosis-related indexes (Caspase 3 activity and Annexin-V) and gene expression Bax, Cyto C, and Caspase 3) were significantly increased in the TG group, but they could be restored by adding Gly. Our results suggest that Gly can ameliorate ER stress and apoptosis in TG-exposed porcine oocytes and can further enhance the developmental potential of porcine oocytes in vitro.

Therapeutic Deep Brain Stimulation Disrupts Subthalamic Nucleus Activity Dynamics in Parkinsonian Mice

Subthalamic nucleus deep brain stimulation (STN DBS) relieves many motor symptoms of Parkinson Disease (PD), but its underlying therapeutic mechanisms remain unclear. Since its advent, three major theories have been proposed: (1) DBS inhibits the STN and basal ganglia output; (2) DBS antidromically activates motor cortex; and (3) DBS disrupts firing dynamics within the STN. Previously, stimulation-related electrical artifacts limited mechanistic investigations using electrophysiology. We used electrical artifact-free calcium imaging to investigate activity in basal ganglia nuclei during STN DBS in parkinsonian mice. To test whether the observed changes in activity were sufficient to relieve motor symptoms, we then combined electrophysiological recording with targeted optical DBS protocols. Our findings suggest that STN DBS exerts its therapeutic effect through the disruption of STN dynamics, rather than inhibition or antidromic activation. These results provide insight into optimizing PD treatments and establish an approach for investigating DBS in other neuropsychiatric conditions.

Construction of a bioluminescence-based assay for bitter taste receptors (TAS2Rs)

In humans, a family of 25 bitter taste receptors (TAS2Rs) mediates bitter taste perception. A common approach to characterize bitter causative agents involves expressing TAS2Rs and the appropriate signal transducers in heterologous cell systems, and monitoring changes in the intracellular free calcium levels upon ligand exposure using a fluorescence-based modality. However, a fluorescence-based assay typically suffers from a low signal window, and is susceptible to interference by autofluorescence, therefore limiting its application to screening of plant or food extracts, which are likely to contain autofluorescent compounds. Here, we report the development and validation of a bioluminescence-based intracellular calcium release assay for TAS2Rs that has a better assay performance than a fluorescence-based assay. Furthermore, the bioluminescence-based assay enables the evaluation of TAS2R agonists within an autofluorescent matrix, highlighting its potential utility in the assessment of the bitterness-inducing properties of plant or food fractions by the food industry. Additionally, further improvement to the bioluminescence-based assay for some TAS2Rs was achieved by altering their N-terminal signal sequences, leading to signal window enhancement. Altogether, the bioluminescence-based TAS2R assay can be used to perform functional studies of TAS2Rs, evaluate TAS2R-modulating properties of autofluorescent samples, and facilitate the discovery of compounds that can function as promising bitter taste modulators.

Revisiting the Large-Conductance Calcium-Activated Potassium (BKCa) Channels in the Pulmonary Circulation

Potassium ion concentrations, controlled by ion pumps and potassium channels, predominantly govern a cell′s membrane potential and the tone in the vessels. Calcium-activated potassium channels respond to two different stimuli-changes in voltage and/or changes in intracellular free calcium. Large conductance calcium-activated potassium (BKCa) channels assemble from pore forming and various modulatory and auxiliary subunits. They are of vital significance due to their very high unitary conductance and hence their ability to rapidly cause extreme changes in the membrane potential. The pathophysiology of lung diseases in general and pulmonary hypertension, in particular, show the implication of either decreased expression and partial inactivation of BKCa channel and its subunits or mutations in the genes encoding different subunits of the channel. Signaling molecules, circulating humoral molecules, vasorelaxant agents, etc., have an influence on the open probability of the channel in pulmonary arterial vascular cells. BKCa channel is a possible therapeutic target, aimed to cause vasodilation in constricted or chronically stiffened vessels, as shown in various animal models. This review is a comprehensive collation of studies on BKCa channels in the pulmonary circulation under hypoxia (hypoxic pulmonary vasoconstriction; HPV), lung pathology, and fetal to neonatal transition, emphasising pharmacological interventions as viable therapeutic options.