Increase In Free Calcium Research Articles

Microbiome profile and nutritional benefits of traditional overnight soaked cooked rice

This study explores the microbial composition, nutrient content, and health benefits of "Panta Bhat," a traditional dish made from cooked rice soaked overnight, commonly consumed in Southeast Asia, particularly in Bangladesh and India. The research aims to evaluate its potential as an affordable food to enhance nutrition and combat malnutrition in population where rice is a staple. The findings reveal that soaking cooked rice overnight initiates mild fermentation, leading to the consistent growth of beneficial bacteria from the Bacillota phylum. 16S rRNA sequencing identified two Gram-positive bacteria, Leuconostoc lactis and Weissella confusa, known for their probiotic and lactic acid-producing capabilities. Furthermore, mineral analysis via ICP-MS showed significant increases in free iron, zinc, manganese, boron, calcium, magnesium, and potassium in soaked rice compared to unsoaked rice. Notably, there was a significant reduction in glycaemic response after consuming soaked rice (P < 0.01). This study suggests that overnight-soaked cooked rice is a promising source of probiotics and enhanced micronutrients, making it a valuable and cost-effective, low-glycaemic food option.

СОДЕРЖАНИЕ ИОНИЗИРОВАННОГО КАЛЬЦИЯ В РОТОВОЙ ЖИДКОСТИ У ДЕТЕЙ В ГОРОДЕ С ЭКОТОКСИКАНТАМИ

The study of the pathogenesis of enamel demineralization in children living in a city with ecotoxicants is an urgent task of modern dentistry. We examined 183 schoolchildren aged 7 and 12 in Ufa. The children were divided into groups depending on where they live. The first group included 94 children from an area located in an industrial district with an environmentally unfavorable situation. The second group (control) included 89 schoolchildren living in an ecologically good district of the city of Ufa. We studied the content of free calcium in the saliva of schoolchildren in two districts of the city of Ufa. When determining the activity of caries according to Vinogradova T.F. (I, II, III degree) among 7 and 12-year-old children, we did not reveal a high correlation between the district where the children live and the concentration of free calcium in the oral fluid. In the mixed saliva of 7-year-old schoolchildren of the first and second groups, the concentration of free calcium was 0.645 ± 0.018 and 0.638 ± 0.011 mm/l (p &gt; 0.05), in 12-year-olds it was 0.603 ± 0.050 and 0.597 ± 0.040 mm/l (p &gt; 0.05). In the industrial district, we noted the maximum increase in free calcium at the III degree of caries activity. This indicator was 0.851 ± 0.006 mmol/l (p &lt; 0.001) in 7-year-old children, and 0.760 ± 0.005 mmol/l in 12-year-old schoolchildren (p &lt; 0.001). While in the district without ecotoxicants, free calcium in 7-year-old schoolchildren was 0.846 ± 0.026 mmol/l (p &lt; 0.001), and in 12-year-old children it was 0.774 ± 0.010 mmol/l (p &lt; 0.001). Thus, the third degree of caries activity is highly correlated with an increase in the concentration of free calcium in saliva, which is proved by studies of oral fluid in schoolchildren from different districts of Ufa.

Oocyte activation deficiency and assisted oocyte activation: mechanisms, obstacles and prospects for clinical application.

BACKGROUNDOocyte activation deficiency (OAD) is attributed to the majority of cases underlying failure of ICSI cycles, the standard treatment for male factor infertility. Oocyte activation encompasses a series of concerted events, triggered by sperm-specific phospholipase C zeta (PLCζ), which elicits increases in free cytoplasmic calcium (Ca2+) in spatially and temporally specific oscillations. Defects in this specific pattern of Ca2+ release are directly attributable to most cases of OAD. Ca2+ release can be clinically mediated via assisted oocyte activation (AOA), a combination of mechanical, electrical and/or chemical stimuli which artificially promote an increase in the levels of intra-cytoplasmic Ca2+. However, concerns regarding safety and efficacy underlie potential risks that must be addressed before such methods can be safely widely used.OBJECTIVE AND RATIONALERecent advances in current AOA techniques warrant a review of the safety and efficacy of these practices, to determine the extent to which AOA may be implemented in the clinic. Importantly, the primary challenges to obtaining data on the safety and efficacy of AOA must be determined. Such questions require urgent attention before widespread clinical utilization of such protocols can be advocated.SEARCH METHODSA literature review was performed using databases including PubMed, Web of Science, Medline, etc. using AOA, OAD, calcium ionophores, ICSI, PLCζ, oocyte activation, failed fertilization and fertilization failure as keywords. Relevant articles published until June 2019 were analysed and included in the review, with an emphasis on studies assessing large-scale efficacy and safety.OUTCOMESContradictory studies on the safety and efficacy of AOA do not yet allow for the establishment of AOA as standard practice in the clinic. Heterogeneity in study methodology, inconsistent sample inclusion criteria, non-standardized outcome assessments, restricted sample size and animal model limitations render AOA strictly experimental. The main scientific concern impeding AOA utilization in the clinic is the non-physiological method of Ca2+ release mediated by most AOA agents, coupled with a lack of holistic understanding regarding the physiological mechanism(s) underlying Ca2+ release at oocyte activation.LIMITATIONS, REASONS FOR CAUTIONThe number of studies with clinical relevance using AOA remains significantly low. A much wider range of studies examining outcomes using multiple AOA agents are required.WIDER IMPLICATIONSIn addition to addressing the five main challenges of studies assessing AOA safety and efficacy, more standardized, large-scale, multi-centre studies of AOA, as well as long-term follow-up studies of children born from AOA, would provide evidence for establishing AOA as a treatment for infertility. The delivery of an activating agent that can more accurately recapitulate physiological fertilization, such as recombinant PLCζ, is a promising prospect for the future of AOA. Further to PLCζ, many other avenues of physiological oocyte activation also require urgent investigation to assess other potential physiological avenues of AOA.STUDY FUNDING/COMPETING INTERESTSD.G. was supported by Stanford University’s Bing Overseas Study Program. J.K. was supported by a Healthcare Research Fellowship Award (HF-14-16) made by Health and Care Research Wales (HCRW), alongside a National Science, Technology, and Innovation plan (NSTIP) project grant (15-MED4186-20) awarded by the King Abdulaziz City for Science and Technology (KACST). The authors have no competing interests to declare.

Acute Csk inhibition hinders B cell activation by constraining the PI3 kinase pathway

T cell antigen receptor (TCR) and B cell antigen receptor (BCR) signaling are initiated and tightly regulated by Src-family kinases (SFKs). SFKs positively regulate TCR signaling in naïve T cells but have both positive and negative regulatory roles in BCR signaling in naïve B cells. The proper regulation of their activities depends on the opposing actions of receptor tyrosine phosphatases CD45 and CD148 and the cytoplasmic tyrosine kinase C-terminal Src kinase Csk. Csk is a major negative regulator of SFKs. Using a PP1-analog-sensitive Csk (CskAS) system, we have previously shown that inhibition of CskAS increases SFK activity, leading to augmentation of responses to weak TCR stimuli in T cells. However, the effects of Csk inhibition in B cells were not known. In this study, we surprisingly found that inhibition of CskAS led to marked inhibition of BCR-stimulated cytoplasmic free calcium increase and Erk activation despite increased SFK activation in B cells, contrasting the effects observed in T cells. Further investigation revealed that acute CskAS inhibition suppressed BCR-mediated phosphatidylinositol 3,4,5-trisphosphate (PIP3) production in B cells. Restoring PIP3 levels in B cells by CD19 cross-linking or SHIP1 deficiency eliminated the negative regulatory effect of CskAS inhibition. This reveals the critical role of Csk in maintaining an appropriate level of SFK activity and regulating PIP3 amounts as a means of compensating for SFK fluctuations to prevent inappropriate B cell activation. This regulatory mechanism controlling PIP3 amounts may also contribute to B cell anergy and self-tolerance.

CPL207280, a Novel G Protein-Coupled Receptor 40/Free Fatty Acid Receptor 1-Specific Agonist, Shows a Favorable Safety Profile and Exerts Antidiabetic Effects in Type 2 Diabetic Animals.

G protein-coupled receptor (GPR) 40 is a free fatty acid receptor mainly expressed in pancreatic β-cells activated by medium- and long-chain fatty acids and regulating insulin secretion via an increase in cytosolic free calcium ([Ca2+]i). Activation of GPR40 in pancreatic β-cells may improve glycemic control in type 2 diabetes through enhancement of glucose-stimulated insulin secretion. However, the most clinically advanced GPR40 agonist-TAK-875 (fasiglifam)-was withdrawn from phase III because of its hepatotoxicity resulting from the inhibition of pivotal bile acid transporters. Here, we present a new, potent CPL207280 agonist and compare it with fasiglifam in numerous in vitro and in vivo studies. CPL207280 showed greater potency than fasiglifam in a Ca2+ influx assay with a human GPR40 protein (EC50 = 80 vs. 270 nM, respectively). At the 10 µM concentration, it showed 3.9 times greater enhancement of glucose-stimulated insulin secretion in mouse MIN6 pancreatic β-cells. In Wistar Han rats and C57BL6 mice challenged with glucose, CPL207280 stimulated 2.5 times greater insulin secretion without causing hypoglycemia at 10 mg/kg compared with fasiglifam. In three diabetic rat models, CPL207280 improved glucose tolerance and increased insulin area under the curve by 212%, 142%, and 347%, respectively. Evaluation of potential off-target activity (Safety47) and selectivity of CPL207280 (at 10 μM) did not show any significant off-target activity. We conclude that CPL207280 is a potent enhancer of glucose-stimulated insulin secretion in animal disease models with no risk of hypoglycemia at therapeutic doses. Therefore, we propose the CPL207280 compound as a compelling candidate for type 2 diabetes treatment. SIGNIFICANCE STATEMENT: GPR40 is a well-known and promising target for diabetes. This study is the first to show the safety and effects of CPL207280, a novel GPR40/free fatty acid receptor 1 agonist, on glucose homeostasis both in vitro and in vivo in different diabetic animal models. Therefore, we propose the CPL207280 compound as a novel, glucose-lowering agent, overcoming the unmet medical needs of patients with type 2 diabetes.

Live Animal Imaging of Calcium Signaling During Transendothelial Migration Demonstrates Tight Temporospatial Regulation Coordinated by Endothelial Cell TRPC6, IQGAP1, and CaMKIIδ

Proper regulation of inflammatory cell recruitment is critical to fighting infection and maintaining organism homeostasis. Misdirected inflammation can exacerbate pathology and underlies the pathology of virtually all diseases including arthritis, atherosclerosis, and multiple sclerosis. The transendothelial migration (TEM) of leukocytes out of the bloodstream and into tissue is the rate-limiting and putative committed step of the inflammatory process. As such, the regulation of TEM represents a key target for potential therapeutic intervention. Our lab has previously reported that the divalent cation channel TRPC6 was responsible for the increase in cytosolic free calcium (↑[Ca2+]i) in endothelial cells that is required for TEM. TRPC6 is activated downstream of PECAM engagement and is concentrated at the site of leukocyte-endothelial contact during TEM. However, the temporal and spatial regulation of ↑[Ca2+]i during TEM was not known. To visualize calcium signaling during TEM in live animals we developed an intravital microscopy system using the GCaMP3 reporter mouse. In this model, the fluorescence of a modified GFP construct increases with calcium concentration. Indeed, during leukocyte TEM we observed a highly localized increase in endothelial calcium signal surrounding the transmigrating leukocyte that corresponded temporally to the transmigration event. This is the first demonstration of endothelial cell calcium signaling during TEM in vivo. Our lab has also previously reported that the scaffolding protein IQ-motif containing GTPase Activating Protein 1 (IQGAP1) in endothelial cells is required for TEM and functions downstream of PECAM engagement. Here we show that the signals propagated by TRPC6 and IQGAP1 converge into one pathway that results in the activation of calmodulin kinase II δ (CaMKIIδ) that is required for TEM. More specifically, we show that the IQ domain of IQGAP1 binds calmodulin (CaM), a known calcium binding protein. This interaction is required for the downstream activation of CaMKIIδ, which binds to CaM in a calcium-dependent manner. Disruption of either the IQGAP1-CaM or the CaM-CaMKIIδ interaction substantially reduces TEM in vitro. To further validate these findings, we developed two separate genetically-altered mouse lines: one with an inducible endothelial-specific deletion of CaMKIIδ, the other with an inducible endothelial-specific expression an intracellular CaMKIIN inhibitory peptide. Both mice showed significantly reduced TEM in the croton oil dermatitis model of inflammation compared to uninduced controls, confirming that endothelial CaMKIIδ is required for efficient leukocyte TEM in vivo. Overall, these findings uncover a new role for CaMKIIδ and novel connections between it and TRPC6, IQGAP1, and CaM in coordinating the calcium signal that facilitates leukocyte TEM. These results also identify CAMKIIδ as a potential target for anti-inflammatory therapeutics.

Calcium Nanodomains at Spindles

The role of calcium signaling in specific events of animal cell meiosis or mitosis (M-phase) is a subject of enduring controversy. Early efforts suggested that increases in intracellular free calcium ([Ca2+]i) promote spindle disassembly while subsequent work suggested that global [Ca2+]i increases trigger nuclear envelope breakdown, spindle assembly, the metaphase-anaphase transition, and cytokinesis. However, further studies led to the conclusion that elevation of [Ca2+]i either has no role in these events, plays a permissive role in these events, or functions as an auxiliary signaling pathway that supplements other mechanisms. One potential explanation of the controversy is that specific M-phase events might depend on highly localized increases in [Ca2+]i, variously referred to as microdomains or nanodomains, as proposed recently. Such domains are hypothesized to arise from rapid shuttling of calcium between closely positioned sources and sinks, rendering them potentially difficult to detect with traditional dyes and largely insensitive to slow chelators such as EGTA. Here a novel microtubule-binding calcium sensor, TubeCamp, was used to test the hypothesis that spindles are associated with calcium nanodomains. TubeCamp imaging revealed that spindles in Xenopus eggs, Xenopus embryos, and HeLa cells were all associated with calcium nanodomains at the spindle poles. Calcium nanodomains also formed in spindles assembled in cell extracts and at the center of monopolar spindles, suggesting that they are a basic feature of spindle self-assembly. Disruption of calcium nanodomains via perturbation of inositol-1,4,5-trisphosphate signaling or rapid chelation of [Ca2+]i resulted in spindle disassembly in vivo and vitro. The results demonstrate the existence of spindle-associated calcium nanodomains and indicate that such domains are an essential and common feature of spindles in vertebrates.

Alzheimer's Risk Gene TREM2 Determines Functional Properties of New Type of Human iPSC-Derived Microglia.

Microglia are key in the homeostatic well-being of the brain and microglial dysfunction has been implicated in neurodegenerative disorders such as Alzheimer’s disease (AD). Due to the many limitations to study microglia in situ or isolated for large scale drug discovery applications, there is a high need to develop robust and scalable human cellular models of microglia with reliable translatability to the disease. Here, we describe the generation of microglia-like cells from human induced pluripotent stem cells (iPSC) with distinct phenotypes for mechanistic studies in AD. We started out from an established differentiation protocol to generate primitive macrophage precursors mimicking the yolk sac ontogeny of microglia. Subsequently, we tested 36 differentiation conditions for the cells in monoculture where we exposed them to various combinations of media, morphogens, and extracellular matrices. The optimized protocol generated robustly ramified cells expressing key microglial markers. Bulk mRNA sequencing expression profiles revealed that compared to cells obtained in co-culture with neurons, microglia-like cells derived from a monoculture condition upregulate mRNA levels for Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), which is reminiscent to the previously described disease-associated microglia. TREM2 is a risk gene for AD and an important regulator of microglia. The regulatory function of TREM2 in these cells was confirmed by comparing wild type with isogenic TREM2 knock-out iPSC microglia. The TREM2-deficient cells presented with stronger increase in free cytosolic calcium upon stimulation with ATP and ADP, as well as stronger migration towards complement C5a, compared to TREM2 expressing cells. The functional differences were associated with gene expression modulation of key regulators of microglia. In conclusion, we have established and validated a work stream to generate functional human iPSC-derived microglia-like cells by applying a directed and neuronal co-culture independent differentiation towards functional phenotypes in the context of AD. These cells can now be applied to study AD-related disease settings and to perform compound screening and testing for drug discovery.

The common diabetes drug metformin can diminish the action of citral against Rhabdomyosarcoma cells in vitro.

Rhabdomyosarcoma (RMS) is a rare type of soft tissue sarcoma most commonly found in pediatric patients. Despite progress, new and improved drug regimens are needed to increase survival rates. Citral, a natural product plant oil can induce cell death in cancer cells. Another compound, metformin, isolated originally from French lilac and used by diabetics, has been shown to reduce the incidence of cancer in these patients. Application of citral to RMS cells showed increase in cell death, and RD and RH30 cells showed half maximal inhibitory concentration (IC50 ) values as low as 36.28 μM and 62.37 μM, respectively. It was also shown that the citral initiated cell apoptosis through an increase in reactive oxygen species (ROS) and free calcium. In comparison, metformin only showed moderate cell death in RMS cell lines at a very high concentration (1,000 μM). Combinatorial experiments, however, indicated that citral and metformin worked antagonistically when used together. In particular, the ability of metformin to quench the ROS induced by citral could lead to the suppression of activity. These results clearly indicate that while clinical use of citral is a promising anti-tumor therapy, caution should be exercised in patients using metformin for diabetes.

Phosphate Deprivation Can Impair Mechano-Stimulated Cytosolic Free Calcium Elevation in Arabidopsis Roots.

The root tip responds to mechanical stimulation with a transient increase in cytosolic free calcium as a possible second messenger. Although the root tip will grow through a heterogeneous soil nutrient supply, little is known of the consequence of nutrient deprivation for such signalling. Here, the effect of inorganic phosphate deprivation on the root’s mechano-stimulated cytosolic free calcium increase is investigated. Arabidopsis thaliana (cytosolically expressing aequorin as a bioluminescent free calcium reporter) is grown in zero or full phosphate conditions, then roots or root tips are mechanically stimulated. Plants also are grown vertically on a solid medium so their root skewing angle (deviation from vertical) can be determined as an output of mechanical stimulation. Phosphate starvation results in significantly impaired cytosolic free calcium elevation in both root tips and whole excised roots. Phosphate-starved roots sustain a significantly lower root skewing angle than phosphate-replete roots. These results suggest that phosphate starvation causes a dampening of the root mechano-signalling system that could have consequences for growth in hardened, compacted soils.

DORN1/P2K1 and purino-calcium signalling in plants: making waves with extracellular ATP

Background and AimsExtracellular ATP governs a range of plant functions, including cell viability, adaptation and cross-kingdom interactions. Key functions of extracellular ATP in leaves and roots may involve an increase in cytosolic free calcium as a second messenger (‘calcium signature’). The main aim here was to determine to what extent leaf and root calcium responses require the DORN1/P2K1 extracellular ATP receptor in Arabidopsis thaliana. The second aim was to test whether extracellular ATP can generate a calcium wave in the root.MethodsLeaf and root responses to extracellular ATP were reviewed for their possible links to calcium signalling and DORN1/P2K1. Leaves and roots of wild type and dorn1 plants were tested for cytosolic calcium increase in response to ATP, using aequorin. The spatial abundance of DORN1/P2K1 in the root was estimated using green fluorescent protein. Wild type roots expressing GCaMP3 were used to determine the spatial variation of cytosolic calcium increase in response to extracellular ATP.Key ResultsLeaf and root ATP-induced calcium signatures differed markedly. The leaf signature was only partially dependent on DORN1/P2K1, while the root signature was fully dependent. The distribution of DORN1/P2K1 in the root supports a key role in the generation of the apical calcium signature. Root apical and sub-apical calcium signatures may operate independently of each other but an apical calcium increase can drive a sub-apical increase, consistent with a calcium wave.ConclusionDORN1 could underpin several calcium-related responses but it may not be the only receptor for extracellular ATP in Arabidopsis. The root has the capacity for a calcium wave, triggered by extracellular ATP at the apex.

Differential actions of second messenger systems in the corpus luteum

Our current working hypothesis for the intracellular mechanism of action for LH and PGF-2 alpha is shown in Fig. 8. Luteinizing hormone appears to act primarily on the small luteal cell through the cAMP/protein kinase A effector system and thereby stimulates secretion of progesterone. Activation of the protein kinase C effector pathway is inhibitory to progesterone secretion from stimulated small luteal cells but it is not clear which hormones, if any, activate this effector system. Results from studies in whole animals suggest that LH may also stimulate differentiation of small luteal cells into large luteal cells (Donaldson & Hansel, 1965; Farin et al., 1988). Although there are LH receptors on large luteal cells, LH treatment does not stimulate progesterone secretion and does not appear to activate any of the second messenger pathways which we have examined. Prostaglandin F-2 alpha appears to act on the large luteal cell through free intracellular calcium and protein kinase C effector systems. Apparently, PGF-2 alpha-induced activation of protein kinase C results in the acute inhibition of progesterone production seen in the first 8 h after PGF-2 alpha treatment. The cytotoxic effects of PGF-2 alpha on the large luteal cell (Fitz et al., 1984; Braden et al., 1988) may be caused by a sustained elevation in free intracellular calcium concentrations. No direct effects of PGF-2 alpha on small luteal cells have been detected (no inhibition of progesterone production, no activation of protein kinase C, no increase in free intracellular calcium), which is consistent with an absence of high affinity PGF-2 alpha receptors on this cell type. The cytotoxic effects of PGF-2 alpha on small luteal cells and endothelial cells (Braden et al., 1988) may be caused by decreases in luteal blood flow (Niswender et al., 1975; Wiltbank et al., 1990b), actions of cytotoxic agents released by large luteal cells, or increases in cytotoxic white blood cells (Murdoch, 1987; Bagavandoss et al., 1988).

Endothelial Calmodulin and CaMKII Play a Role in Leukocyte Transmigration

Inflammation results in the dilation of blood vessels and recruitment of circulating leukocytes into damaged tissues for repair. A transient increase in endothelial cytosolic free calcium (↑[Ca2+]i) is required for leukocyte transendothelial migration (TEM). We have also found that IQ‐domain GTPase‐activating protein 1 (IQGAP1) is essential for TEM. Specifically, the calmodulin‐binding isoleucine‐glutamine (IQ) motif domain is critical for its function during TEM. Calmodulin (CaM) is an intracellular calcium‐binding protein, however, its role in TEM is undescribed. Therefore, we hypothesized that it is involved in the endothelial calcium signaling cascade essential for supporting leukocyte transmigration.In endothelial cells IQGAP1 knockdown reduced TEM substantially. Re‐expressing IQGAP1 truncation mutants containing both the actin‐binding and IQ‐motif domains rescued TEM but truncation mutants lacking the IQ motif domain but did not support TEM.Human endothelial cells treated with the calmodulin inhibitor trifluoperazine (TFP) demonstrated a marked reduction in leukocyte TEM. Only 13% of leukocytes transmigrated through endothelial monolayers pretreated with 100 mM TFP compared to 84% TEM in control, untreated monolayers (p&lt;0.05). We also looked at the role of calmodulin kinase II (CaMKII). Treating endothelial monolayers with 10 mM KN‐93, a CaMKII inhibitor, substantially reduced leukocyte TEM. Here 22% of leukocytes transmigrated which was significantly reduced compared to control TEM rates (p&lt;0.05). Calmodulin pulldowns also showed that without the IQ‐motif domain IQGAP1 cannot substantially interact with calmodulin further suggesting a link between endothelial calcium, IQGAP1 and CaM.Current experiments focus on inhibiting CaM and CaMKII in endothelial cells using genetically encoded selective inhibitory peptides and inducible endothelial‐specific CaMKII‐deficient mice. Identifying the mechanism by which endothelial Ca2+ influx mediates TEM will provide novel insight into the signaling and also provide potential targets for anti‐inflammatory therapy.Support or Funding InformationR01 HL046849, R01 HL064774, F30 HL134202This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acaricidal Mechanism of Scopoletin Against Tetranychus cinnabarinus.

Scopoletin is a promising acaricidal botanical natural compound against Tetranychus cinnabarinus, and its acaricidal mechanism maybe involve calcium overload according to our previous study. To seek potential candidate target genes of calcium overload induced by scopoletin in T. cinnabarinus, RNA-seq was utilized to detect changes in transcription levels. 24 and 48 h after treatment, 70 and 102 differentially expressed genes were obtained, respectively. Target genes included 3 signal transduction genes, 4 cell apoptosis genes, 4 energy metabolism genes, and 2 transcription factor genes. The role of 3 calcium signaling pathway-related genes, namely, G-protein-coupled neuropeptide receptor, Bcl-2 protein and guanylate kinase (designated TcGPCR, TcBAG, and TcGUK, respectively) in the calcium overload were investigated in this study. RT-qPCR detection showed that scopoletin treatment upregulated the expression level of TcGPCR and downregulated the expression level of TcBAG and TcGUK. The result of RNAi indicated that downregulation of TcGPCR decreased susceptibility to scopoletin, and downregulation of TcBAG and TcGUK enhanced susceptibility to scopoletin. Functional expression in Chinese hamster ovary cells showed that scopoletin induced a significant increase in intracellular free calcium [Ca2+]i levels by activating TcGPCR. These results demonstrated that the acaricidal mechanism of scopoletin was via disrupting intracellular Ca2+ homeostasis and calcium signaling pathway mediated by GPCR, BAG, and GUK.

Aspirin eugenol ester attenuates oxidative injury of vascular endothelial cells by regulating NOS and Nrf2 signalling pathways.

Aspirin eugenol ester (AEE) is a new drug compound synthesized by combining aspirin with eugenol. It was reported to possess anti-thrombotic, anti-atherosclerotic, and anti-oxidative effects. However, its molecular mechanism against oxidative injury is unclear. This study investigated how AEE affected the oxidative injury of vascular endothelial cells in vivo and in vitro. A hamster model of atherosclerosis induced by a high fat diet (HFD) and an in vitro model of oxidative stress, H2 O2 -induced apoptosis of HUVECs, were used to investigate the anti-oxidative effects of AEE. AEE significantly reduced the stimulatory effect of HFD on malondialdehyde, the inhibitory effect of HFD on SOD activity and GSH/GSSG ratio, and the overexpression of inducible NOS (iNOS) in the aorta. In vitro, incubation of HUVECs with H2 O2 led their apoptosis, dysfunctions of the NO systems (including increased iNOS activity, decreased endothelial NOS activity, and increased production of NO), an imbalance in calcium homeostasis and energy metabolism with an increase in intracellular free calcium and decrease in ATP, and a down-regulation of Nrf2. In contrast, in the HUVECs pretreated with 1 μM AEE for 24hr, the above adverse effects induced by H2 O2 were significantly ameliorated. Moreover, the decrease in NO production and activity of iNOS induced by AEE was significantly attenuated in Nrf2-inhibited HUVECs. AEE protects vascular endothelial cells from oxidative injury by regulating NOS and Nrf2 signalling pathways. This suggests that AEE is a novel potential agent for the prevention of atherosclerosis.

Danger signals activate a putative innate immune system during regeneration in a filamentous fungus.

The ability to respond to injury is a biological process shared by organisms of different kingdoms that can even result in complete regeneration of a part or structure that was lost. Due to their immobility, multicellular fungi are prey to various predators and are therefore constantly exposed to mechanical damage. Nevertheless, our current knowledge of how fungi respond to injury is scarce. Here we show that activation of injury responses and hyphal regeneration in the filamentous fungus Trichoderma atroviride relies on the detection of two danger or alarm signals. As an early response to injury, we detected a transient increase in cytosolic free calcium ([Ca2+]c) that was promoted by extracellular ATP, and which is likely regulated by a mechanism of calcium-induced calcium-release. In addition, we demonstrate that the mitogen activated protein kinase Tmk1 plays a key role in hyphal regeneration. Calcium- and Tmk1-mediated signaling cascades activated major transcriptional changes early following injury, including induction of a set of regeneration associated genes related to cell signaling, stress responses, transcription regulation, ribosome biogenesis/translation, replication and DNA repair. Interestingly, we uncovered the activation of a putative fungal innate immune response, including the involvement of HET domain genes, known to participate in programmed cell death. Our work shows that fungi and animals share danger-signals, signaling cascades, and the activation of the expression of genes related to immunity after injury, which are likely the result of convergent evolution.

Both gravistimulation onset and removal trigger an increase of cytoplasmic free calcium in statocytes of roots grown in microgravity

Gravity is a permanent environmental signal guiding plant growth and development. Gravity sensing in plants starts with the displacement of starch-filled plastids called statoliths, ultimately leading to auxin redistribution and organ curvature. While the involvement in gravity sensing of several actors such as calcium is known, the effect of statolith displacement on calcium changes remains enigmatic. Microgravity is a unique environmental condition offering the opportunity to decipher this link. In this study, roots of Brassica napus were grown aboard the International Space Station (ISS) either in microgravity or in a centrifuge simulating Earth gravity. The impact of short simulated gravity onset and removal was measured on statolith positioning and intracellular free calcium was assessed using pyroantimonate precipitates as cytosolic calcium markers. Our findings show that a ten-minute onset or removal of gravity induces very low statolith displacement, but which is, nevertheless, associated with an increase of the number of pyroantimonate precipitates. These results highlight that a change in the cytosolic calcium distribution is triggered in absence of a significant statolith displacement.

The early signaling pathway of live yeast cell derivative in THP-1 monocytes

Live yeast cell derivative (LYCD) is a medicinal yeast extract that has been used in the treatment of burns, wounds and hemorrhoids for over 70 years. It has been shown to enhance the closure of skin wounds in diabetic mice by increasing inflammation, angiogenesis, formation of granulation tissue and epithelial migration. An active fraction of LYCD has been identified as a mixture of peptides ranging in size from 5 kDA to 17 kDA. Despite its widespread use over many years, understanding of the mechanism by which LYCD acts to effect tissue repair responses is very limited. In this study, we have used a human monocyte-derived cell line, THP-1, as a representative of the inflammatory component of the wound healing process. We have identified two of the earliest responses to LYCD as an increase in cytoplasmic free calcium ([Ca2+]i) and the transcripts for c-fos. We have found that the increase in [Ca2+]i is both necessary and sufficient to account for the LYCD-induced elevation of c-fos. Furthermore, we have shown that the signaling pathway by which LYCD elevates [Ca2+]i involves both mobilization of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular medium. Mobilization of store Ca2+ occurs first via activation of phospholipase C and this is followed by influx through activation of store operated calcium channels. These results constitute the first delineation of the early steps of the LYCD signaling pathway.

Exploring the Role of Calmodulin and Calcium Signaling in Leukocyte Transmigration

Inflammation results in the dilation of local blood vessels, the activation of endothelium by cytokines and the recruitment of circulating leukocytes into areas of tissue damage. A transient increase in endothelial cytosolic free calcium (Ca2+) is required for leukocyte transendothelial migration (TEM) and transient receptor potential canonical 6 (TRPC6) has been found to mediate this required Ca2+ influx. Furthermore, we have also found that IQ‐domain GTPase‐activating protein 1 (IQGAP1) is essential for TEM. Specifically, the actin‐binding domain and the calmodulin‐binding isoleucine‐glutamine (IQ) domain of IQGAP1 are critical for its function during TEM. In endothelial cells re‐expression of IQGAP1 truncation mutants lacking the N‐terminal actin‐binding domain did not localize to endothelial borders and did not support TEM. Re‐expression of truncated IQGAP1 constructs containing the actin‐binding domain and the IQ domain localized to cell borders and supported TEM while constructs containing the actin‐binding domain but lacking the IQ domain localized to the cell borders but did not restore TEM.Calmodulin (CaM) is a small, highly conserved intracellular calcium‐binding protein, which has been shown to interact with IQGAP1 through the IQ domain. It has also been previously found to regulate TRPC channel activation; however, not much is known regarding its role in TEM. Therefore, we hypothesized that it may be involved in the calcium signaling cascade within endothelial cells essential for supporting leukocyte transmigration.Human endothelial cells treated with calmodulin inhibitor trifluoperazine (TFP) demonstrated a marked reduction in leukocyte TEM. Endothelial monolayers pretreated with 100 mM TFP for 1 hour drastically reduced leukocyte TEM. Only about 13% of leukocytes were able to transmigrate compared to 84% TEM in control, untreated monolayers (p=0.0025).After identifying the potential role of calmodulin, we generated CaM shRNA to deplete endogenous CaM within endothelial cells, wildtype Flag‐tagged CaM re‐expression constructs and CaM calcium‐binding mutants that we are currently in the process of testing. Identifying the specific mechanism by which endothelial Ca2+ influx through TRPC6 mediates the events of TEM will provide novel insight into the signaling involved in TEM and also provide potential therapeutic targets for anti‐inflammatory therapy.Support or Funding InformationF30HL134202, R01HL046849, R01HL064774This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Non-canonical pathway induced by Wnt3a regulates β-catenin via Pyk2 in differentiating human neural progenitor cells

Wnt/β-catenin and Wnt/Ca2+ pathways are involved in cellular processes during embryonic development and the interaction between them in the same cell decides the outcome of cellular functions. In this study, we showed that Wnt3a triggers the Wnt/Ca2+ signaling pathway, indicated by an increase of cytosolic free calcium ([Ca2+]i) and activation of calmodulin dependent kinase II (CaMKII) during the differentiation of human neuronal progenitor cells (hNPCs). Wnt3a via the increase of [Ca2+]i activates proline-rich tyrosine kinase 2 (Pyk2), which subsequently regulates phosphorylation of glycogen synthase kinase 3β (GSK3β) and β-catenin stabilization. Our findings suggest that Pyk2 plays an important role in the coordination of stabilization of β-catenin in the crosstalk between Wnt/β-catenin and Wnt/Ca2+ signaling pathways upon Wnt3a stimulation in differentiating hNPCs.