Initial Ca2 Research Articles

Protease-activated receptor-1 mediated calcium oscillations in renal mesangial cells

INTRODUCTION. Mesangial cells provide structural support to the glomerular tuft and modulate the glomerular capillary flow via their contractile properties. Mesangial cells undergo phenotypic changes into myofibroblast-like cells, such as proliferation, mesangial expansion, abnormal glomerular tuft formation, and reduced numbers of capillary loops, are present in several glomerular diseases, including diabetic nephropathy and glomerulonephritis. In addition, thrombin-induced mesangial remodeling was found in diabetic patients, and expression of the corresponding protease-activated receptors (PARs) in the renal mesangium was reported. However, the functional PAR-mediated signaling and mechanisms in mesangial cells were not examined. This study aims to investigate protease-activated mechanisms regulating mesangial cell contraction and glomerular capillary flow. METHODS. We used primary human renal mesangial cells (HRMC) to determine the signaling mechanisms mediated by PAR1 thrombin-activated receptors. Confocal fluorescent microscopy was utilized to detect changes in intracellular Ca 2+ response to specific PAR1 modulators. Pharmacology and patch clamp electrophysiology was further applied to reveal downstream signaling mechanisms responsible for intracellular Ca 2+ oscillations. RESULTS. PAR1-mediated Ca 2+ response displayed high sensitivity to specific agonist (TFLLR-NH 2 ) with EC 50 values of 3 and 6.3 nM for male and female-derived cultured cells, respectively (the competition of a ligand for receptor binding fit converged; adj. R 2 =0.98). The response to PAR1 activation promoted initial cytosolic Ca 2+ increase followed by synchronized, damped Ca 2+ oscillations with a lag of 6.74±0.84 min between peaks. The pre-application of a specific inhibitor (RWJ56110) eliminated PAR1-mediated response, and oscillations were blocked by the changes of an extracellular solution to zero Ca 2+ . The specific inhibitors for store-operated calcium (SOCs) (Pyr6) and TRPC3 (GSK 2833503A) channels strongly attenuated oscillation behavior (up to 40% when added separately and up to 65% when both were applied; two-way ANOVA, * p<0.0001). In addition, the effect of a specific inhibitor of TRPC6 channels (BI-749327) had a minimal impact on Ca 2+ flux. Further single-channel electrophysiology experiments in HRMC cells confirmed the involvement of SOC and TRPC3 channels in PAR1-mediated GPCR activation. CONCLUSION. Our results indicate that coagulation proteases like thrombin may strongly regulate mesangial cell contraction and corresponding glomerular capillary flow by PAR1 GPCRs-related activation. The contraction mechanism is mediated presumably through SOCs entry and TRPC3 channels. Since high thrombin levels are linked to poor diabetic control, the described signaling may play a crucial role in the development of diabetic glomerular disease. DK126720 (to OP), DK129227 (to AS and OP), Veterans Affairs Support Veterans Affairs (Merit Award I01 BX000820 to JL), research grant from Dialysis Clinic, Inc (to JL). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhalable Biomineralized Liposomes for Cyclic Ca2+-Burst-Centered Endoplasmic Reticulum Stress Enhanced Lung Cancer Ferroptosis Therapy

Lung cancer with the highest mortality poses a great threat to human health. Ferroptosis therapy has recently been raised as a promising strategy for lung cancer treatment by boosting the reactive species (ROS) production and lipid peroxidation (LPO) accumulation intracellularly. However, the insufficient intracellular ROS level and the unsatisfactory drug accumulation in lung cancer lesions hamper the efficacy of ferroptosis therapy. Here, an inhalable biomineralized liposome LDM co-loaded with dihydroartemisinin (DHA) and pH-responsive calcium phosphate (CaP) was constructed as a ferroptosis nanoinducer for achieving Ca2+-burst-centered endoplasmic reticulum (ER) stress enhanced lung cancer ferroptosis therapy. Equipped with excellent nebulization properties, about 6.80-fold higher lung lesions drug accumulation than intravenous injection made the proposed inhalable LDM an ideal nanoplatform for lung cancer treatment. The Fenton-like reaction mediated by DHA with peroxide bridge structure could contribute to intracellular ROS production and induce ferroptosis. Assisted by DHA-mediated sarco-/endoplasmic reticulum calcium ATPase (SERCA) inhibition, the initial Ca2+ burst caused by CaP shell degradation triggered the Ca2+-mediated intense ER stress and subsequently induced mitochondria dysfunction to further boost ROS accumulation, which strengthens ferroptosis. The second Ca2+ burst occurred as a result of Ca2+ influx through ferroptotic pores on cell membranes, thus sequentially constructing the lethal "Ca2+ burst-ER stress-ferroptosis" cycle. Consequently, the Ca2+-burst-centered ER stress enhanced ferroptosis process was confirmed as a cell swelling and cell membrane disruption process driven by notable intracellular ROS and LPO accumulation. The proposed LDM showed an encouraging lung retention property and extraordinary antitumor ability in an orthotropic lung tumor murine model. In conclusion, the constructed ferroptosis nanoinducer could be a potential tailored nanoplatform for nebulization-based pulmonary delivery and underscore the application of Ca2+-burst-centered ER stress enhanced lung cancer ferroptosis therapy.

Constraints of vigilance-dependent noradrenergic signaling to mouse cerebellar Bergmann glia.

Behavioral state plays an important role in determining astroglia Ca2+ signaling. In particular, locomotion-mediated elevated vigilance has been found to trigger norepinephrine-dependent whole cell Ca2+ elevations in astroglia throughout the brain. For cerebellar Bergmann glia it has recently been found that locomotion-induced transient Ca2+ elevations depend on their α1A -adrenergic receptors. With increasing availability and implementation of locomotion as behavioral parameter it becomes important to understand the constraints of noradrenergic signaling to astroglia. Here we evaluated the effect of speed, duration and interval of locomotion on Ca2+ signals in Bergmann glia as well as cerebellar noradrenergic axon terminals. We found almost no dependence on locomotion speed, but following the initial Ca2+ transient prolonged locomotion events revealed a steady-state Ca2+ elevation. Comparison of time course and recovery of transient Bergmann glia and noradrenergic terminal Ca2+ dynamics suggested that noradrenergic terminal Ca2+ activity determines Bergmann glia Ca2+ activation and does not require noradrenergic receptor desensitization to account for attenuation during prolonged locomotion. Further, analyzing the correlation among Ca2+ dynamics within regions within the field of observation we found that coordinated activity among noradrenergic terminals accounts for fluctuations of steady-state Bergmann glia Ca2+ activity. Together, our findings will help to better understand astroglia Ca2+ dynamics during less controlled awake behavior and may guide the identification of behavioral contexts preferably dependent on astroglia Ca2+ signaling.

Dynamic communication between TRPC3 and IP3R shape Ca2+ signaling signatures.

Physical and functional coupling between TRPC channels and IP3 receptors (IP3R) has been proposed as a pivotal mechanism in cellular Ca2+ signaling. Here we set out to explore the impact of TRPC3-IP3R communication at the level of spatiotemporal organisation of Ca2+ signaling. The low affinity Ca2+ sensor R-GECO1.2 was fused as a reporter to the C-terminus of TRPC3 and the channel function of this construct was modified specifically by point mutations. Cytoplasmic Ca2+ changes at the TRPC3 channels expressed in HEK293 cells originated from both the entry of Ca2+ through the TRPC channel and Ca2+ mobilization via IP3R. Local Ca2+ changes within the TRPC3 signaling domain were predominantly biphasic with IP3R-dependent initial Ca2+ transients, while exclusively monophasic signals were recorded when all three IP3R isoforms were lacking. Abrogation of Ca2+ entry through TRPC3 by mutational impairment of Ca2+ permeability (E630Q), cation permeation (E630K), or DAG sensitivity (G652A), promoted local Ca2+ oscillations. Ca2+ signals at E630Q, E630K, and G652A channels featured initial Ca2+ transients along with oscillatory activity. By contrast, oscillations and initial Ca2+ transients, were virtually lacking, when the TRPC3 channels were sensitized by subthreshold stimulation of phospholipase C. TIRF imaging provided evidence for dynamic colocalization of TRPC3 and IP3R. Our results suggest that TRPC3-mediated Ca2+ entry controls IP3R activity at ER-PM junctions to shape Ca2+ signaling signatures and enable specificity of downstream signaling.

The Spatial Distribution of Microbially Induced Carbonate Precipitation in Sand Column with Different Grouting Strategies

For ground improvement via microbially induced carbonate precipitation (MICP), the spatial distribution of biocementation considerably affects the mechanical performance of soil. This study systematically assessed the CaCO3 spatial distribution in sand columns with various grouting strategies. CaCO3 dispersion along the radial and vertical directions was quantitatively evaluated. Furthermore, a series of biochemical monitoring experiments was conducted to investigate the MICP process in sand. The results indicated that the reversed and downward chemical solution grouting and high rates of microbial grouting all promoted CaCO3 vertical uniformity. Moreover, the grouting strategy slightly affected the dispersion degree of radially distributed CaCO3. The variation coefficient basically ranged from 0.1 to 0.3. However, the variation coefficient for radially distributed CaCO3 along the vertical direction was significantly affected by the chemical grouting direction. The biochemical monitoring results indicated that 38% Ca2+ was even consumed near the chemical solution outlet in the chemical solution grouting process. The initial Ca2+ concentration during the intermittent period gradually decreased along the chemical solution grouting direction. The CaCO3 vertical distribution was controlled by a combination of the total MICP reaction time, biomass, and Ca2+ availability in the intermittent phase. Different CaCO3 distributions resulted in each grouting strategy exhibiting specific applicable engineering requirements. Furthermore, the carbon footprint was preliminarily evaluated. The results in this study provide references for the selection of grouting strategies for the MICP technique in engineering applications.

TRPC3 governs the spatiotemporal organization of cellular Ca2+ signatures by functional coupling to IP3 receptors

Communication between TRPC channels and IP3 receptors (IP3R) is considered pivotal in the generation of spatiotemporal Ca2+signaling patterns. Here we revisited the role of TRPC3-IP3R coupling for local Ca2+ signaling within TRPC3-harbouring micro/nanodomains using R-GECO as a reporter, fused to the channel´s C-terminus. Cytoplasmic Ca2+ changes at TRPC3 originated from both the entry of Ca2+ through the TRPC channel and Ca2+ mobilization via IP3R. Local Ca2+ changes at TRPC3 channels expressed in HEK293 cells were predominantly biphasic with IP3R-dependent initial Ca2+ transients, while exclusively monophasic signals were recorded when all three IP3R isoforms were lacking. Abrogation of Ca2+ entry through TRPC3 by point mutations, which impair Ca2+ permeability (E630Q), cation permeation (E630K), or DAG sensitivity (G652A), promoted microdomain Ca2+ oscillations. Ca2+ signals at E630Q, E630K, and G652A channels featured initial Ca2+ transients along with oscillatory activity. Similarly, when extracellular Ca2+ was omitted, IP3R-mediated Ca2+ transients and Ca2+ oscillations were promoted at the cytoplasmic face of wild-type TRPC3 channels. By contrast, oscillations, as well as initial Ca2+ transients, were virtually lacking, when the TRPC3 channels were sensitized by preexposure to low-level PLC activity. TIRF imaging provided evidence for dynamic colocalization of TRPC3 and IP3R. We suggest that TRPC3-mediated Ca2+ entry controls IP3R activity at ER-PM junctions to determine Ca2+ signaling signatures and enable specificity of downstream signaling.

Litter Decomposition of Qinghai Spruce (Picea crassifolia) Is Dependent on Mn Concentration in the Qilian Mountains, Northwest China

The factors determining litter decomposition incorporated into C and nutrient cycles were examined as part of a broader study investigating the biogeochemical cycle in forest ecosystems. Litter was collected from five altitudes of Qinghai spruce (Picea crassifolia) woodland stands in the Qilian Mountains and placed in litterbags. These litterbags were installed at the crown center (CC) and crown edge (CE) at different altitudes in Qinghai spruce forests during the growing season to study the effect of litter substrate quality on litter decomposition. Results indicate that at varying altitudes in the growing season, the initial mass loss rate and initial decomposition rate of Qinghai spruce litter showed a nonlinear relationship with altitude. The Olson exponential regression equation showed that the decomposition coefficient (k) was the largest at 3050 m (k = 0.709), and the decomposition coefficient (k) was the smallest at 3250 m (k = 0.476). Meanwhile, the initial decomposition rate was highly correlated with initial litter Ca and Mn concentrations. At the CC and CE at different altitudes in the growing season, the initial mass loss rate of CE was significantly higher than that of CC (p < 0.01), and the initial decomposition rate of CE was markedly faster than that of CC (p < 0.01). The Olson exponential regression equation showed that CE’s decomposition coefficients (k) were larger than those of CC. The initial decomposition rate of CE was highly correlated with initial litter C and Mn concentrations. However, the initial decomposition rate at CC was independent of the litter substrate quality. Finally, we realize that litter decomposition in the early stages is not ultimately determined by a single common factor, but rather the result of multiple factors working together in different orders and strengths. The results lay a foundation for understanding the process and mechanism of litter decomposition in the alpine mountain forest ecosystem and further understanding the structure and function of the ecosystem.

Misfolded protein oligomers induce an increase of intracellular Ca2+ causing an escalation of reactive oxidative species

Alzheimer's disease is characterized by the accumulation in the brain of the amyloid β (Aβ) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aβ also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aβ oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca2+ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca2+ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aβ42-derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca2+ influx in human neuroblastoma SH-SY5Y cells and rat primary cortical neurons in a variety of conditions. In all cases we found a faster increase of intracellular Ca2+ than ROS levels, and a lag phase in the latter process. A Ca2+-deprived cell medium prevented the increase of intracellular Ca2+ ions and abolished ROS production. By contrast, treatment with antioxidant agents prevented ROS formation, did not prevent the initial Ca2+ flux, but allowed the cells to react to the initial calcium dyshomeostasis, restoring later the normal levels of the ions. These results reveal a mechanism in which the entry of Ca2+ causes the production of ROS in cells challenged by aberrant protein oligomers.

Integrating divided electrolysis-microfiltration process for energy-efficient phosphorus recovery in the form of calcium phosphate

To make up the gap of phosphorus emissions being too much as a contaminant and phosphorus being too little as a fertilizer resource, an energy-efficient electrochemically mediated precipitation-microfiltration (EMP-MF) system was rationally developed for P removal and recovery as calcium phosphate. In this system, the electrolysis cell was divided by a polytetrafluoroethylene (PTFE) membrane, allowing for the productions of acidic and alkaline effluents. The alkaline effluent was fed directly into the crystallizer for the precipitation of calcium phosphate crystals, which was well intercepted by the following Ti tubular microfilter. In this system, phosphorus recovery efficiency increased from 54 % to 90 % by elevating current density from 1 to 6 mA cm−2, accompanying the specific energy consumption increasing from 3.92 to 22.44 kWh (kgP)-1 or from 0.039 to 0.38 kWh m−3 water treated. The phosphorus recovery efficiency by ECP-MF system was promoted at high initial solution pH. The phosphorus recovery efficiency was 67 %, 85 %, and 91 %, when initial solution pH was 4.0, 8.0 and 10.0. High initial Ca2+ concentration favored the phosphorus recovery, whereas it was negatively influenced with the presence of HCO3–. In addition, the coexistence of magnesium had little effect on the recovery of phosphorus but inhibited the Ca2+ removal. Natural organic matter could enhance the phosphorus recovery, but affected the purity of the recovered product. Generally, the established ECP-MF system exhibited significant prospect in promoting the widespread applications for phosphorus recovery from the wastewater.

Nutrient recovery from digested sludge centrate using alkali metals from steel-making slag

Results in this study highlighted the potential of nutrient recovery from anaerobically digested sludge centrate using calcium and other alkali metals from steel-making slag. Up to 96% phosphate and 71% ammonia could be recovered from sludge centrate at the optimal conditions. Mass balance calculation confirmed precipitation and volatilisation as the main mechanisms for phosphorus and ammonia recovery, respectively. Morphology and elemental analysis of obtained precipitates confirmed that phosphorus was recovered in the form of dicalcium phosphate dihydrate (CaHPO4·2H2O). The results also showed that sludge centrate pre-treatment by sand filtration and forward osmosis (FO) enrichment was essential to achieve high nutrient recovery. Sand filtration pre-treatment decreased the total suspended solid of sludge centrate by eightfold, leading to mitigated membrane fouling and reduced nutrient loss during FO pre-concentration. The production of slag liquor with high calcium and alkaline content from steel-making slag for nutrient recovery was demonstrated. Slag liquor with high pH increased ammonia recovery significantly, but only enhanced phosphate recovery slightly. Phosphate recovery was more dependent on the initial Ca:PO4 molar ratio than the final pH. The process demonstrated in this study has potential and significant practical implications to nutrient recovery from wastewater and beneficial use of steel-making slag.

A Novel Role of PP2A Methylation in the Regulation of Tight Junction Assembly and Integrity.

Tight junctions (TJs) are multiprotein complexes essential for cell polarity and the barrier function of epithelia. The major signaling molecule, protein serine/threonine phosphatase 2A (PP2A), interacts with the TJ and modulates the phosphorylation state of TJ proteins. An important PP2A regulatory mechanism involves leucine carboxyl methyltransferase-1 (LCMT1)-dependent methylation and protein phosphatase methylesterase-1 (PME1)-mediated demethylation of its catalytic subunit on Leu309. Here, using MDCK cells, we show that overexpression of LCMT1, which enhances cellular PP2A methylation, inhibits TJ formation, induces TJ ruffling, and decreases TJ barrier function. Conversely, overexpression of PME1 accelerates TJ assembly and enhances TJ barrier function. PME1-dependent PP2A demethylation increases during early Ca2+-dependent junctional assembly. Inhibition of endogenous PME1 delays the initial Ca2+-mediated redistribution of TJ proteins to cell-cell contacts and affects TJ morphology and barrier function. Manipulating one-carbon metabolism modulates TJ assembly, at least in part by affecting PP2A methylation state. The integrity of PP2A methylation is critical for proper targeting of PP2A to the TJ. It is necessary for PP2A complex formation with the TJ proteins, occludin and ZO-1, and proteins of the PAR complex, Par3 and atypical protein kinase C ζ (aPKCζ), which play a key role in development of cell polarity. Expression of a methylation incompetent PP2A mutant induces defects in TJ assembly and barrier function. aPKCζ-mediated Par3 phosphorylation is also required for targeting of the PP2A ABαC holoenzyme to the TJ. Our findings provide the first evidence for a role of LCMT1, PME1 and PP2A methylation/demethylation processes in modulating TJ assembly and functional integrity. They also position PP2A at the interface of one-carbon metabolism and the regulation of key TJ and polarity proteins that become deregulated in many human diseases.

Binary droplet interactions in shear water-in-oil emulsion: A molecular dynamics study

The shear field and binary droplet interaction regimes of water-in-oil emulsions between parallel gold plates were investigated by molecular dynamics (MD) methods. The developed MD model was subjected to dimensional analysis and the dimensionless numbers that guarantee the similarity between the model and the actual system were identified as the capillary number (Ca), Reynolds number (Re), Weber number (We), and dimensionless time (τ). The effects of different pressures were compared by applying different forces perpendicular to the shear direction to the parallel plates. The heptane formed adsorbed layers on the gold plates, exhibited a shear thinning characteristic, and formed a no-slip uniform shear field driven by the parallel plates. The interactions between gold atoms and a heptane molecule have been investigated in depth by quantum chemical methods and it has been found that the nature of the adsorption is a van der Waals interaction. Three main binary droplet interaction regimes are produced in the presence of shear fields: coalescence, a temporary bridge formation, and sliding. The effects of capillary forces and shear effects on liquid bridges were discussed. Droplet trajectories and deformations under different pressure were analyzed. Finally, the effect of initial offset (ΔYin/2R) and Ca on the droplet interaction regime was investigated through regime maps. It was found that the coalescence regime has upper critical points for ΔYin/2R and Ca; droplet slipping occurs mainly at higher ΔYin/2R; the temporary bridge formation regime appears mainly at higher Ca. This study provides a new idea for understanding binary droplet interactions in shear emulsions.

Trials of Commercial- and Wild-Type Saccharomyces cerevisiae Strains under Aerobic and Microaerophilic/Anaerobic Conditions: Ethanol Production and Must Fermentation from Grapes of Santorini (Greece) Native Varieties

In modern wine-making technology, there is an increasing concern in relation to the preservation of the biodiversity, and the employment of “new”, “novel” and wild-type Saccharomyces cerevisiae strains as cell factories amenable for the production of wines that are not “homogenous”, expressing their terroir and presenting interesting and “local” sensory characteristics. Under this approach, in the current study, several wild-type Saccharomyces cerevisiae yeast strains (LMBF Y-10, Y-25, Y-35 and Y-54), priorly isolated from wine and grape origin, selected from the private culture collection of the Agricultural University of Athens, were tested regarding their biochemical behavior on glucose-based (initial concentrations ca 100 and 200 g/L) shake-flask experiments. The wild yeast strains were compared with commercial yeast strains (viz. Symphony, Cross X and Passion Fruit) in the same conditions. All selected strains rapidly assimilated glucose from the medium converting it into ethanol in good rates, despite the imposed aerobic conditions. Concerning the wild strains, the best results were achieved for the strain LMBF Y-54 in which maximum ethanol production (EtOHmax) up to 68 g/L, with simultaneous ethanol yield on sugar consumed = 0.38 g/g were recorded. Other wild strains tested (LMBF Y-10, Y-25 and Y-35) achieved lower ethanol production (up to ≈47 g/L). Regarding the commercial strains, the highest ethanol concentration was achieved by S. cerevisiae Passion Fruit (EtOHmax = 91.1 g/L, yield = 0.45 g/g). Subsequently, the “novel” strain that presented the best technological characteristics regards its sugar consumption and alcohol production properties (viz. LMBF Y-54) and the commercial strain that equally presented the best previously mentioned technological characteristics (viz. Passion Fruit) were further selected for the wine-making process. The selected must originated from red and white grapes (Assyrtiko and Mavrotragano, Santorini Island; Greece) and fermentation was performed under wine-making conditions showing high yields for both strains (EtOHmax = 98–106 g/L, ethanol yield = 0.47–0.50 g/g), demonstrating the production efficiency under microaerophilic/anaerobic conditions. Molecular identification by rep-PCR carried out throughout fermentations verified that each inoculated yeast was the one that dominated during the whole bioprocess. The aromatic compounds of the produced wines were qualitatively analyzed at the end of the processes. The results highlight the optimum technological characteristics of the selected “new” wild strain (S. cerevisiae LMBF Y-54), verifying its suitability for wine production while posing great potential for future industrial applications.

Fulvic acid-like substance-Ca(II) complexes improved the utilization of calcium in rice: Chelating and absorption mechanism

Water-soluble chelated calcium has been widely used in agriculture as a fertilizer to improve the absorption and utilization of calcium by plants. This paper prepared a new organic mineral fertilizer, based on fulvic acid-like substance chelated calcium (PFA-Ca2+ complex), using optimal parameters (i.e., pH, time, temperature, and Ca2+ concentration) to achieve a high chelation efficiency. The absorption, utilization, and distribution of the PFA-Ca2+ complex in rice roots were analyzed using laser scanning confocal microscopy (LSCM). Our results demonstrated that the optimal PFA-Ca2+ complex chelating efficiency (87%) was achieved at an initial Ca2+ concentration of 0.1 mol L−1, an equilibration time of 120 min, a pH of 5.0, and a temperature of 40 °C. The chelating reaction of a fulvic acid-like substance with Ca2+ primarily occurred on phenol hydroxyl, alcohol hydroxyl, and carboxyl groups. The PFA-Ca2+ complex was primarily enriched in the roots' pericycle, cortical, and epidermis cells, in both chelating and non-chelating forms. To our knowledge, this is the first report investigating how the PFA-Ca2+complex affects transformation in plants, which has significant implications for research on plant nutrition and nutrient distribution.

The allosteric gating mechanism of the MthK channel.

Allostery is a fundamental element during channel gating in response to an appropriate stimulus by which events occurring at one site are transmitted to distal sites to regulate activity. To address how binding of the first Ca2+ ion at one of the eight chemically identical subunits facilitates the other Ca2+-binding events in MthK, a Ca2+-gated K+ channel containing a conserved ligand-binding RCK domain, we analysed a large collection of MthK structures and performed the corresponding thermodynamic and electrophysiological measurements. These structural and functional studies led us to conclude that the conformations of the Ca2+-binding sites alternate between two quaternary states and exhibit significant differences in Ca2+ affinity. We further propose an allosteric model of the MthK-gating mechanism by which a cascade of structural events connect the initial Ca2+-binding to the final changes of the ring structure that open the ion-conduction pore. This mechanical model reveals the exquisite design that achieves the allosteric gating and could be of general relevance for the action of other ligand-gated ion channels containing the RCK domain.

Crosstalk between ERO1α and ryanodine receptor in arsenite-dependent mitochondrial ROS formation

Arsenite, a well-established human carcinogen and toxic compound, promotes the formation of mitochondrial superoxide (mitoO2−) via a Ca2+-dependent mechanism, in which an initial stimulation of the inositol 1, 4, 5-trisphosphate receptor (IP3R) is followed by the activation of the ryanodine receptor (RyR), critical for providing Ca2+ to the mitochondria. We now report that, under the same conditions, arsenite triggers endoplasmic reticulum (ER) stress and a threefold increase in ER oxidoreductin 1α (ERO1 α) levels in proliferating U937 cells. EN460, an inhibitor of ERO1 α, recapitulated all the effects associated with RyR inhibition or downregulation, including prevention of RyR-induced Ca2+ accumulation in mitochondria and the resulting O2–. formation. Quantitatively similar results were obtained in inhibitor studies performed in terminally differentiated wild type C2C12 cells. Moreover, ERO1 α knockout C2C12 myotubes responded to arsenite as their wild type counterpart supplemented with EN460. As a final note, arsenite enhanced the expression of ERO1 α via a mechanism mediated by Ca2+ release from both the IP3R and RyR.We therefore conclude that arsenite activates a positive feedback amplification cycle between Ca2+ levels and ERO1 α in the ER, by which IP3R-dependent Ca2+ induces ERO1 α and ERO1 α promotes Ca2+ release via RyR, thereby amplifying the initial Ca2+ load and causing the mitochondrial accumulation of the cation, critical for mitoO2− formation.

Microbial induced calcium precipitation based anaerobic immobilized biofilm reactor for fluoride, calcium, and nitrate removal from groundwater

In this study, the anaerobic quartz sand fixed biofilm reactor containing Cupriavidus sp. W12 was established to simultaneously remove calcium (Ca2+), fluoride (F−) and nitrate (NO3–N) from groundwater. After 84 days of continuous operation, the optimum operating parameters and defluoridation mechanism were explored, and the microbial community structure under different pH environments were compared and analyzed. Under the optimal operation conditions (HRT of 6 h, initial Ca2+ concentration of 180 mg L−1, and pH of 7.0), the removal efficiencies of Ca2+, F−, and NO3–N were 58.97%, 91.93%, and 100%, respectively. Gas chromatography (GC) results indicate that N2 is the main gas produced by the bioreactor. Three−dimension excitation emission matrix fluorescence spectroscopy (3D−EEM) showed that extracellular polymers (EPS) are produced during bacterial growth and metabolism. The results of Scanning electron microscopy−energy dispersive spectrometer (SEM−EDS), X−ray diffraction (XRD), and Fourier transform infrared spectrometer (FTIR) demonstrated that the defluoridation mechanism is attributed to the synergetic effects of ion exchange, co−precipitation, and chemisorption. The comparative analysis of the microbial community structure under different pH conditions show that Cupriavidus is the dominant bacteria in the bioreactor throughout the experiment, and it shows a prominent advantage at pH of 7.0. This research provides an application foundation for anaerobic microbial induced calcium precipitation (MICP) bioremediation of Ca2+, F−, and NO3–N from groundwater.

Oxidative Refining of Metallurgical Grade Silicon: Lab-Scale Measurements on the Overarching Refining Behavior of Ca and Al

Oxidative ladle refining (OLR) is the most widely used refining process for removal of dissolved Ca and Al in the industrial production of metallurgical grade silicon (MG-Si). Refining occurs by purging the silicon alloy with an oxygen–air mixture at temperatures preferably between 1823 K and 1873 K, forming a SiO_{2}–CaO–Al_{2}O_{3} slag, which is then separated from the liquid alloy before casting. To meet higher quality demands and customer specifications, it is paramount to achieve greater process control of the industrial system, which requires a deeper understanding of the Ca and Al mass transfer kinetics, and the general refining behavior. In this work, laboratory-scale experiments have been performed, aiming to emulate the industrial process. Five different alloy compositions, with varying initial Ca and Al concentration, were refined over 11 experiments, with samples taken at 0, 5, 7, 10, and 15 minutes at 1873 K. A FactSage-based model was used to explore the underlying thermodynamic system behavior. The results suggest that OLR of MG-Si occurs in three primary steps, initially by the formation of slag by surface oxidation and purge gas, through the depletion of dissolved Ca in the melt, and secondarily by the formation of silica. Towards the end of the refining process, a critical amount of slag has been formed by the previous steps such that a global equilibrium is attained between the SiO_{2}–CaO–Al_{2}O_{3} slag and the silicon alloy.

P2X4 and P2X7 are essential players in basal T cell activity and Ca2+ signaling milliseconds after T cell activation.

Initial T cell activation is triggered by the formation of highly dynamic, spatiotemporally restricted Ca2+ microdomains. Purinergic signaling is known to be involved in Ca2+ influx in T cells at later stages compared to the initial microdomain formation. Using a high-resolution Ca2+ live-cell imaging system, we show that the two purinergic cation channels P2X4 and P2X7 not only are involved in the global Ca2+ signals but also promote initial Ca2+ microdomains tens of milliseconds after T cell stimulation. These Ca2+ microdomains were significantly decreased in T cells from P2rx4−/− and P2rx7−/− mice or by pharmacological inhibition or blocking. Furthermore, we show a pannexin-1–dependent activation of P2X4 in the absence of T cell receptor/CD3 stimulation. Subsequently, upon T cell receptor/CD3 stimulation, ATP release is increased and autocrine activation of both P2X4 and P2X7 then amplifies initial Ca2+ microdomains already in the first second of T cell activation.

Modulation of carbachol-induced Ca2+ oscillations in airway smooth muscle cells by PGE2

PGE2 is a potent bronchodilator, but the mechanisms underlying this effect have not been fully elucidated. Acetylcholine-induced contractions of airway smooth muscle (ASM) are associated with the generation of repetitive Ca2+ oscillations in airway smooth muscle cells (ASMC) and the force of contraction is positively correlated with the frequency of the underlying Ca2+ oscillations. The purpose of the present study was to examine if carbachol-evoked Ca2+ oscillations in isolated ASMC were inhibited by PGE2.Isolated murine ASMC loaded with fluo4-AM were imaged with a Nipkow spinning disk confocal microscope. Cells responded to application of CCh (1 μM) by generating an initial Ca2+ transient followed by a series of Ca2+ oscillations. This activity was abolished by PGE2 (300 nM) and the EP2R agonist (R)-butaprost (3 μM) and the inhibitory effects of PGE2 were reversed by application of the EP2R antagonist PF-04418948 (100 nM). Activation of adenylate cyclase using forskolin (1 μM) mimicked the effects of PGE2. The PKA activator, 6-MB-cAMP (300 μM) reduced the frequency of CCh-induced Ca2+ oscillations by 33% and the PKA inhibitor Rp-8-CPT-cAMPs partially reversed the inhibitory effects of PGE2. The EPAC activator 007-AM (10 μM) reduced the frequency of the oscillations by 60% and joint application of 007-AM and 6-MB-cAMP reduced oscillation frequency by ∼85%. CCh-induced Ca2+ oscillations were inhibited by 2-APB and tetracaine, but caffeine-evoked Ca2+ transients were resistant to PGE2.These data suggest that PGE2 inhibits CCh-induced Ca2+ oscillations in murine ASMC via stimulation of EP2Rs and a mechanism involving activation of PKA and EPAC.