Ca Chelator Research Articles

Optimization of the Preparation Process and Ameliorative Efficacy in Osteoporotic Rats of Peptide-Calcium Chelates from Skipjack Tuna (Katsuwonus pelamis) Meat.

This study aimed to establish the preparation process of peptide-calcium chelates (TMP-Ca) using skipjack tuna meat and investigate the function and mechanism of TMP-Ca in an osteoporosis model of rats. The results indicated that trypsin is more suitable for preparing the Ca-chelating hydrolysates of tuna meat, and the optimal hydrolysis conditions were derived as follows: digestion time 4 h, material-liquid ratio 1:10, and enzyme dose 3%. The conditions for chelating Ca with tuna meat hydrolysate were optimized to be chelation time 50 min, temperature 50 °C, pH 8.0, and a peptide-Ca ratio 1:10. The prepared hydrolysate was subjected to ultrafiltration, and the fraction (TMP) (MW <1 kDa) showed the highest Ca chelation rate (51.27 ± 1.42%) and was made into the peptide-Ca chelates (TMP-Ca). In osteoporotic rats, TMP-Ca significantly improved the decrease in ovarian indexes caused by retinoic acid. It also elevated serum Ca, phosphorus, and bone turnover indexes, increased the number of bone trabeculae, and improved bone microstructure. In addition, we confirmed that TMP-Ca could regulate the OPG/TRAF6 pathway to reduce osteoclast differentiation, inhibit bone resorption, and promote bone formation. Therefore, TMP-Ca could significantly ameliorate osteoporosis, and this study provides a functional component for the preparation of healthcare products using skipjack tuna meat to treat osteoporosis.

NOX2-TRPM2 coupling promotes Zn2+ inhibition of complex III to exacerbate ROS production in a cellular model of Parkinson’s disease

Reactive oxygen species (ROS) serve vital physiological functions, but aberrant ROS production contributes to numerous diseases. Unfortunately, therapeutic progress targeting pathogenic ROS has been hindered by the limited understanding of whether the mechanisms driving pathogenic ROS differ from those governing physiological ROS generation. To address this knowledge gap, we utilised a cellular model of Parkinson’s disease (PD), as an exemplar of ROS-associated diseases. We exposed SH-SY5Y neuroblastoma cells to the PD-toxin, MPP+ (1-methyl-4-phenylpyridinium) and studied ROS upregulation leading to cell death, the primary cause of PD. We demonstrate: (1) MPP+ stimulates ROS production by raising cytoplasmic Ca2+ levels, rather than acting directly on mitochondria. (2) To raise the Ca2+, MPP+ co-stimulates NADPH oxidase-2 (NOX2) and the Transient Receptor Potential Melastatin2 (TRPM2) channel that form a positive feedback loop to support each other’s function. (3) Ca2+ exacerbates mitochondrial ROS (mtROS) production not directly, but via Zn2+. (4) Zn2+ promotes electron escape from respiratory complexes, predominantly from complex III, to generate mtROS. These conclusions are drawn from data, wherein inhibition of TRPM2 and NOX2, chelation of Ca2+ and Zn2+, and prevention of electron escape from complexes -all abolished the ability of MPP+ to induce mtROS production and the associated cell death. Furthermore, calcium ionophore mimicked the effects of MPP+, while Zn2+ ionophore replicated the effects of both MPP+ and Ca2+. Thus, we unveil a previously unrecognized signalling circuit involving NOX2, TRPM2, Ca2+, Zn2+, and complex III that drives cytotoxic ROS production. This circuit lies dormant in healthy cells but is triggered by pathogenic insults and could therefore represent a safe therapeutic target for PD and other ROS-linked diseases.

Purinergic agonists increase [Ca2+]i in rat conjunctival goblet cells through ryanodine receptor type 3.

ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca2+ concentration ([Ca2+]i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca2+i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca2+]i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca2+ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10-6 M), which depletes ER Ca2+ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10-5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca2+]i was significantly lower after chelating Ca2+i with 2 mM EGTA in Ca2+-free buffer. The peak [Ca2+]i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10-5 M and BzATP at 10-4 M. Incubating CGCs with dantrolene alone decreased [Ca2+]i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca2+i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca2+]i. This study contributes to the understanding of diseases arising from defective Ca2+ signaling in nonexcitable cells.NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca2+]i) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca2+]i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca2+]i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca2+]i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca2+i homeostasis.

Effect of extraction method on the calcium binding capacity of faba bean globulin fractions at various pH

Faba bean ingredients are rich in proteins and good sources of calcium (Ca), although containing phytic acid (PA) molecules. PA, a polyphosphate compound, can affect the bioavailability of minerals/proteins through complex formation. This study evaluates the impact of two extraction processes, Alkaline Extraction-IsoElectric Precipitation (AE-IEP) and Sequential Extraction (SE), on the ability of faba bean globulin systems to bind added calcium ions. Increasing concentrations of CaCl2 were introduced into 2.5% (w/v) protein dispersions at pHs 4.5, 5.5, 6.5, and 7.5, and free Ca monitored. Near the isoelectric point of globulin (pH ∼ 4–5), Ca binding capacity was found to be low. At higher pHs, significant Ca chelation occurred, initially attributed to free PA binding sites, resulting in the formation of insoluble complexes and subsequent protein precipitation. The AE-IEP globulin fraction exhibited a higher Ca binding capacity than the SE globulin, attributed to its higher PA and lower initial Ca concentrations.

Targeting LINC00152 activates cAMP/Ca2+/ferroptosis axis and overcomes tamoxifen resistance in ER+ breast cancer

Tamoxifen has been the mainstay therapy to treat early, locally advanced, and metastatic estrogen receptor-positive (ER + ) breast cancer, constituting around 75% of all cases. However, the emergence of resistance is common, necessitating the identification of novel therapeutic targets. Here, we demonstrated that long-noncoding RNA LINC00152 confers tamoxifen resistance by blocking tamoxifen-induced ferroptosis, an iron-mediated cell death. Mechanistically, inhibiting LINC00152 reduces the mRNA stability of phosphodiesterase 4D (PDE4D), leading to activation of the cAMP/PKA/CREB axis and increased expression of the TRPC1 Ca2+ channel. This causes cytosolic Ca2+ overload and generation of reactive oxygen species (ROS) that is, on the one hand, accompanied by downregulation of FTH1, a member of the iron sequestration unit, thus increasing intracellular Fe2+ levels; and on the other hand, inhibition of the peroxidase activity upon reduced GPX4 and xCT levels, in part by cAMP/CREB. These ultimately restore tamoxifen-dependent lipid peroxidation and ferroptotic cell death which are reversed upon chelating Ca2+ or overexpressing GPX4 or xCT. Overexpressing PDE4D reverses LINC00152 inhibition-mediated tamoxifen sensitization by de-activating the cAMP/Ca2+/ferroptosis axis. Importantly, high LINC00152 expression is significantly correlated with high PDE4D/low ferroptosis and worse survival in multiple cohorts of tamoxifen- or tamoxifen-containing endocrine therapy-treated ER+ breast cancer patients. Overall, we identified LINC00152 inhibition as a novel mechanism of tamoxifen sensitization via restoring tamoxifen-dependent ferroptosis upon destabilizing PDE4D, increasing cAMP and Ca2+ levels, thus leading to ROS generation and lipid peroxidation. Our findings reveal LINC00152 and its effectors as actionable therapeutic targets to improve clinical outcome in refractory ER+ breast cancer.

Sodium citrate targeting Ca2+/CAMKK2 pathway exhibits anti-tumor activity through inducing apoptosis and ferroptosis in ovarian cancer

IntroductionOvarian cancer (OC) is known for its high mortality rate. Although sodium citrate has anti-tumor effects in various cancers, its effect and mechanism in OC remain unclear. ObjectivesTo analyze the inhibitory effect of sodium citrate on ovarian cancer cells and the underlying mechanism. MethodsCell apoptosis was examined by TUNEL staining, flow cytometry, and ferroptosis was examined intracellular Fe2+, MDA, LPO assays, respectively. Cell metabolism was examined by OCR and ECAR measurements. Immunoblotting and immunoprecipitation were used to elucidate the mechanism. ResultsThis study suggested that sodium citrate not only promoted ovarian cancer cell apoptosis but also triggeredferroptosis, manifested as elevated levels of Fe2+, LPO, MDA andlipid ROS production. On one hand, sodium citrate treatment led to a decrease of Ca2+ content in the cytosol by chelatingCa2+, which further inhibited the Ca2+/CAMKK2/AKT/mTOR signaling, thereby suppressing HIF1α-dependent glycolysis pathway and inducing cell apoptosis. On the other hand, the chelation of Ca2+ by sodium citrate resulted in inactivation of CAMKK2 and AMPK, leading to increase of NCOA4-mediated ferritinophagy, causing increased intracellular Fe2+ levels. More importantly, the inhibition of Ca2+/CAMKK2/AMPK signaling pathway reduced the activity of the MCU and Ca2+ concentration within the mitochondria, resulting in an increase in mitochondrial ROS. Additionally, metabolomic analysis indicated that sodium citrate treatment significantly increased de novo lipid synthesis. Altogether, these factors contributed to ferroptosis. As expected, Ca2+ supplementation successfully reversed the cell death and decreased tumor growth induced by sodium citrate. Inspiringly, it was found that coadministration of sodium citrate increased the sensitivity of OC cells to chemo-drugs. ConclusionThese results revealed that the sodium citrate exerted its anti-cancer activity by inhibiting Ca2+/CAMKK2-dependent cell apoptosis and ferroptosis. Sodium citrate will hopefully serve as a prospective compound for OC treatment and for improvingthe efficacy of chemo-drugs.

Calcium signaling facilitates chilling- and GA- induced dormancy release in tree peony.

Calcium plays a crucial role in plant growth and development, yet little is known about its function in endodormancy regulation. Tree peony (Paeonia suffruticosa), characterized by compound buds and large flowers, is well-known for its ornamental and medicinal value. To break bud dormancy release is a prerequisite of flowering and forcing culture, particularly during the Spring Festival. In this study, the Ca2+ chelator EGTA and Ca2+ channel blocker LaCl3 were applied, resulting in a significant delay in budburst during both chilling- and gibberellin (GA)- induced dormancy release in a dosage-dependent manner. As expected, the retardation of bud break was recovered by the supplementation of 30 mM CaCl2, indicating a facilitating role of calcium in dormancy release. Accordingly, several calcium-sensor-encoding genes including Calmodulin (CaM) and Ca2+-dependent protein kinases (CDPKs) were significantly up-regulated by prolonged chilling and exogenous GAs. Ultrastructure observations revealed a decline in starch grains and the reopening of transport corridors following prolonged chilling. Calcium deposits were abundant in the cell walls and intercellular spaces at the early dormant stage but were enriched in the cytosol and nucleus before dormancy release. Additionally, several genes associated with dormancy release, including EBB1, EBB3, SVP, GA20ox, RGL1, BG6, and BG9, were differentially expressed after calcium blocking and recovery treatments, indicating that calcium might partially modulate dormancy release through GA and ABA pathways. Our findings provide novel insights into the mechanism of dormancy release and offer potential benefits for improving and perfecting forcing culture technology in tree peonies.

Tumor cell dissociation‐enhanced intravesical chemotherapy of orthotopic bladder cancer

AbstractFrequent intravesical chemotherapy is still the adopted clinical option after bladder cancer surgery with low adhesion, poor selectivity, low permeability, and drug resistance. Herein, we develop an ingenious bladder cancer dissociation method to enhance intravesical chemotherapy and tumor self‐exclusion with urine. Ethylene diamine tetraacetic acid (EDTA), a common Ca2+ chelator, is loaded with the typical clinical bladder instillation drug doxorubicin (Dox) in chitosan‐modified hollow gold nanorods and subsequently coated with cancer cell membranes. After bladder perfusion, the nanoplatform exhibits high affinity toward bladder tumors under homologous targeting, assisting in long‐term retention. Under NIR‐II laser irradiation, the photothermal effect accelerates the unloading of cargo, and the released EDTA then disrupts intratumoral junctions by depriving and chelating Ca2+ from the intercellular calcium‐dependent connexin. The consequential intertumoral dissociation gives access to the deeper penetration of Dox and allows the exclusion of the shed small tumor masses from the body with the urine. This distinctive tumor dissociation concept holds great promise for modern clinical intravesical chemotherapy and perhaps for other gastrointestinal malignancies.

Study on the scale inhibition performance of organic chelating agent aided by surfactants on CaCO3 at high salinity condition

Abstract The salt blockage layer formed in the wellbore during the development of high salinity oil and gas fields has a serious impact on the safety of oil and gas production of the oil and gas fields. CaCO3 is the main component of scale in high salinity oil and gas fields. Herein, focusing on CaCO3 as an example, the precipitation and scaling process as well as the change of crystalline form of CaCO3 under different temperature and salinity conditions were studied. The effects of two addition methods of organic chelating agent on the solubilization and dislodgement of CaCO3 under high salinity conditions were also explored. The optimal concentration of organic chelating agent is determined to be 1 % and 5 % for solubilization and dislodgement, respectively. It was found that the solubilization is achieved by inhibiting the stable and difficult to treat calcite crystal phase to generate more irregular and more dispersed aragonite CaCO3. Organic chelating agent contains a large number of carboxyl groups in its molecules, which can be adsorbed onto the surface of CaCO3 crystals through electrostatic adsorption. By chelating Ca2+ in stable and difficult to treat calcite CaCO3, the further ordered growth of the crystals is prevented, causing lattice distortion or large crystal rupture, thereby achieving dislodgement. This study will provide theoretical and technical support for the solubilization and dislodgement of salt blockage layer in wellbore at high salinity conditions.

Enhanced cytotoxic activity of natural killer cells from increased calcium influx induced by electrical stimulation.

Natural killer (NK) cells play a crucial role in immunosurveillance independent of antigen presentation, which is regulated by signal balance via activating and inhibitory receptors. The anti-tumor activity of NK cells is largely dependent on signaling from target recognition to cytolytic degranulation; however, the underlying mechanism remains unclear, and NK cell cytotoxicity is readily impaired by tumor cells. Understanding the activation mechanism is necessary to overcome the immune evasion mechanism, which remains an obstacle in immunotherapy. Because calcium ions are important activators of NK cells, we hypothesized that electrical stimulation could induce changes in intracellular Ca2+ levels, thereby improving the functional potential of NK cells. In this study, we designed an electrical stimulation system and observed a correlation between elevated Ca2+ flux induced by electrical stimulation and NK cell activation. Breast cancer MCF-7 cells co-cultured with electrically stimulated KHYG-1 cells showed a 1.27-fold (0.5 V/cm) and 1.55-fold (1.0 V/cm) higher cytotoxicity, respectively. Electrically stimulated KHYG-1 cells exhibited a minor increase in Ca2+ level (1.31-fold (0.5 V/cm) and 1.11-fold (1.0 V/cm) higher), which also led to increased gene expression of granzyme B (GZMB) by 1.36-fold (0.5 V/cm) and 1.58-fold (1.0 V/cm) by activating Ca2+-dependent nuclear factor of activated T cell 1 (NFAT1). In addition, chelating Ca2+ influx with 5 μM BAPTA-AM suppressed the gene expression of Ca2+ signaling and lytic granule (granzyme B) proteins by neutralizing the effects of electrical stimulation. This study suggests a promising immunotherapeutic approach without genetic modifications and elucidates the correlation between cytolytic effector function and intracellular Ca2+ levels in electrically stimulated NK cells.

The Effect of Some Different Blood Anticoagulants on Rabbits Plasma Biochemical Parameters under Different Temperatures

The aim of present study was to compare the effect of Ca chelating anticoagulants (EDTA, citrate and oxalate) and antithrombin anticoagulant (heparin) on some rabbits plasma biochemical parameters at room temperature 25℃ and extreme temperatures -18℃and 50℃ for 1hr. Five male rabbits weighting between 800-1000gm, kept in the animal house /College of Veterinary Medicine/University of Diyala, under 12:12 dark light cycle with ad libtum food and water in individual cages (60cm,40cm) during November and December /2022. Four ml of blood samples were collected by cardiac puncture (23G needle) into four tubes contain anticoagulants: (EDTA, citrate, oxalate and heparin)one ml of blood for each tube; then the blood samples were kept at :room temperature 25℃, waterbath50℃ and refrigerator -18 ℃, for 1hr then put in centrifuge at 3000rpm for 15min. From total examined blood samples =24 for each Ca chelating anticoagulants (EDTA, citrate and oxalate) and antithrombin anticoagulant (heparin) after 1hr under in 25c℃ , -18℃ and 50℃. The both groups of anticoagulants (Ca chelating and antithrombin) showed no significant differences P&gt;0.05 between them in level of biochemical parameters (glucose, total protein, cholesterol and urea) after 1 hr at 25C; on the other hand significant increase in plasma glucose, total protein and cholesterol beside significant decrease in plasma urea of Ca chelating anticoagulants after 1hr at -18C and 50C as compare with antithrombin anticoagulant. In conclusion; heparin was more effective than Ca chelating anticoagulant in preserve plasma biochemical parameters within normal values under extreme cool and hot condition.

Adsorption mechanism of green efficient chelating poly-L-aspartic acid in flotation separation of brucite and dolomite

Green and efficient reagents have gained considerable attention in flotation research, where poly-L-aspartic acid (PASP) is an effective reagent to realize the flotation separation of brucite and dolomite. The effect of PASP on the flotation of brucite and dolomite and its mechanism were studied using flotation tests and modern detection methods. Microflotation tests show that PASP can considerably reduce the flotation recovery of dolomite in the Sodium dodecyl sulfonate (‘SDS) system, whereas it only slightly affects the flotation recovery of brucite. Under ‘SDS, PASP can selectively reduce the contact angle of dolomite but only slightly affect that of brucite. Furthermore, atomic force microscopy detection and zeta potential tests demonstrate that PASP can strongly adsorb on the dolomite surface, considerably changing its roughness and potential value but slightly affecting brucite. The infrared spectrum analysis shows that PASP is selectively strongly adsorbed on dolomite, causing strong and weak ‘SDS adsorption on the surfaces of brucite and dolomite, respectively. Moreover, X-ray photoelectron spectroscopy demonstrates that PASP strongly impacts the Ca site of dolomite, and selective chelation of Ca is a key factor in inhibiting dolomite flotation.

Rhythmic calcium ion activity related to glioma growth reveals the mechanism of ion transmission

Rhythmic calcium ion activity related to glioma growth reveals the mechanism of ion transmission

Untargeted Metabolomic Analysis in Endolymphatic Sac Luminal Fluid from Patients with Meniere's Disease.

Dysfunction of the endolymphatic sac (ES) is one of the etiologies of Meniere's disease (MD), the mechanism of which remains unclear. The aim of the present study was to explore the molecular pathological characteristics of ES during the development of MD. Metabolomic profiling of ES luminal fluid from patients with MD and patients with acoustic neuroma (AN) was performed. Diluted ES luminal fluid (ELF) samples were obtained from 10 patients who underwent endolymphatic duct blockage for the treatment of intractable MD and from 6 patients who underwent translabyrinthine surgery for AN. ELF analysis was performed using liquid chromatography-mass spectrometry before the raw data were normalized and subjected to subsequent statistical analysis by MetaboAnalyst. Using thresholds of P ≤ 0.05 and variable important in projection > 1, a total of 111 differential metabolites were screened in the ELF, including 52 metabolites in negative mode and 59 in positive mode. Furthermore, 15 differentially altered metabolites corresponding to 15 compound names were identified using a Student's t-test, including 7 significant increased metabolites and 8 significant decreased metabolites. Moreover, two differentially altered metabolites, hyaluronic acid (HA) and 4-hydroxynonenal (4-HNE), were validated to be upregulated in the epithelial lining of the ES, as well as in the subepithelial connective-tissue in patients with MD comparing with that in patients with AN. Among these differentially altered metabolites, an upregulated expression of HA detected in the ES lumen of the patients with MD was supposed to be associated with the increased endolymph in ES, while an increased level of 4-HNE found in the ELF of the patients with MD provided direct evidence to support that oxidative damage and inflammatory lesions underlie the mechanism of MD. Furthermore, citrate and ethylenediaminetetraacetic acid were detected to be decreased substantially in the ELF of the patients with MD, suggesting the elevated endolymphatic Ca2+ in the ears with chronic endolymphatic hydrops is likely to be associated with the reduction of these two chelators of Ca2+ in ES. The results in the present study indicate metabolomic analysis in the ELF of the patients with MD can potentially improve our understanding on the molecular pathophysiological mechanism in the ES during the development of MD.

Exogenous laminin exhibits a unique vascular pattern in the brain via binding to dystroglycan and integrins

BackgroundUnlike other proteins that exhibit a diffusion pattern after intracerebral injection, laminin displays a vascular pattern. It remains unclear if this unique vascular pattern is caused by laminin-receptor interaction or laminin self-assembly.MethodsWe compared the distribution of various wild-type laminin isoforms in the brain after intracerebral injection. To determine what causes the unique vascular pattern of laminin in the brain, laminin mutants with impaired receptor-binding and/or self-assembly activities and function-blocking antibodies to laminin receptors were used. In addition, the dynamics of laminin distribution and elimination were examined at multiple time points after intracerebral injection.ResultsWe found that β2-containing laminins had higher affinity for the vessels compared to β1-containing laminins. In addition, laminin mutants lacking receptor-binding domains but not that lacking self-assembly capability showed substantially reduced vascular pattern. Consistent with this finding, dystroglycan (DAG1) function-blocking antibody significantly reduced the vascular pattern of wild-type laminin-111. Although failed to affect the vascular pattern when used alone, integrin-β1 function-blocking antibody further decreased the vascular pattern when combined with DAG1 antibody. EDTA, which impaired laminini-DAG1 interaction by chelating Ca2+, also attenuated the vascular pattern. Immunohistochemistry revealed that laminins were predominantly located in the perivascular space in capillaries and venules/veins but not arterioles/arteries. The time-course study showed that laminin mutants with impaired receptor-engaging activity were more efficiently eliminated from the brain compared to their wild-type counterparts. Concordantly, significantly higher levels of mutant laminins were detected in the cerebral-spinal fluid (CSF).ConclusionsThese findings suggest that intracerebrally injected laminins are enriched in the perivascular space in a receptor (DAG1/integrin)-dependent rather than self-assembly-dependent manner and eliminated from the brain mainly via the perivascular clearance system.

The interdependent transport of yeast vacuole Ca2+ and H+ and the role of phosphatidylinositol 3,5-bisphosphate

Yeast vacuoles are acidified by the v-type H+-ATPase (V-ATPase) that is comprised of the membrane embedded VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme on the vacuole is stabilized in part through interactions between the VO a-subunit ortholog Vph1 and the lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). PI(3,5)P2 also affects vacuolar Ca2+ release through the channel Yvc1 and uptake through the Ca2+ pump Pmc1. Here, we asked if H+ and Ca2+ transport activities were connected through PI(3,5)P2. We found that overproduction of PI(3,5)P2 by the hyperactive fab1T2250A mutant augmented vacuole acidification, whereas the kinase-inactive fab1EEE mutant attenuated the formation of a H+ gradient. Separately, we tested the effects of excess Ca2+ on vacuole acidification. Adding micromolar Ca2+ blocked vacuole acidification, whereas chelating Ca2+ accelerated acidification. The effect of adding Ca2+ on acidification was eliminated when the Ca2+/H+ antiporter Vcx1 was absent, indicating that the vacuolar H+ gradient can collapse during Ca2+ stress through Vcx1 activity. This, however, was independent of PI(3,5)P2, suggesting that PI(3,5)P2 plays a role in submicromolar Ca2+ flux but not under Ca2+ shock. To see if the link between Ca2+ and H+ transport was bidirectional, we examined Ca2+ transport when vacuole acidification was inhibited. We found that Ca2+ transport was inhibited by halting V-ATPase activity with Bafilomycin or neutralizing vacuolar pH with chloroquine. Together, these data show that Ca2+ transport and V-ATPase efficacy are connected but not necessarily through PI(3,5)P2.

Study on the effect of SDBS and SDS on deep coal seam water injection

Coal seam water injection, as an important disaster prevention means in the process of coal mining, can effectively suppress coal dust, add water injection additives, can effectively improve the wettability of coal body, improve the permeability of coal body, so as to achieve the prevention of rock burst. To improve the wettability of coal in coal seam water injection, the surfactant is often added to water, but sodium dodecyl benzene sulfonate (SDBS) and sodium dodecyl sulfate (SDS) have limitations in improving wettability of deep coal seam by injecting water. Therefore, it is very important to determine the influencing factors of SDBS and SDS to improve the wettability of coal. In this paper, the effects of oxygen-containing functional groups and minerals in coal on the wettability of coal are revealed, and the wettability mechanism of SDBS and SDS is expounded from the microscopic point of view. SEM was used to characterize the interaction between coal surface and surfactant, and the contact angle experiment was used to verify the influence of minerals in coal on wettability. Inductively coupled plasma atomic emission spectroscopy (ICP) and dynamic light scattering (DLS) tests were used to characterize the interaction of SDBS, SDS with minerals and the size of precipitation generated by the interaction of SDBS, SDS and mineral ions. The results showed that SDBS and SDS interact with Ca2+ to produce precipitation and block the flow of water in coal, which is not conducive to improving the wettability of deep coal seam to a certain extent. The significant chelating effect of chelating agent and Ca2+ provides a feasible solution to this problem.

Hybrid Alginate-Brushite Beads Easily Catalyze the Knoevenagel Condensation On-Water.

An innovative hybrid organic–inorganic material composed of alginate–brushite xerogel beads was successfully applied for the catalysis of the Knoevenagel condensation. The catalyst was derived from phosphated alginate xerogel microspheres formed from the ionotropic gelling effect of phosphated alginate. To this end, alginate was phosphated by the addition of diammonium hydrogen phosphate in a 1% w/w alginate gel. The phosphated alginate was subsequently precipitated by chelation of Ca2+ cations, generating a phosphated alginate hydrogel microsphere, which was washed and dried, forming hybrid organic–inorganic xerogel beads as a crystalline phosphate-rich mineral fraction covered by alginate. X-ray diffraction analysis revealed that the crystalline inorganic matrix of the material was composed predominantly of brushite. SEM analysis revealed plate-like, ribbon-like, or needle-like morphologies in the hybrid alginate–brushite beads. The hybrid material was tested as a catalyst for Knoevenagel condensation, which was performed ″on-water″ under mild conditions with aromatic aldehydes and activated methylene compounds, giving high yields (up to 97%). The reaction rate and product yield increased together with the reaction temperature for all reagents. The recyclable solid catalyst was effective for three runs, revealing the potential of the innovative hybrid catalyst as an eco-friendly heterogeneous catalyst.

An instantly fixable and self-adaptive scaffold for skull regeneration by autologous stem cell recruitment and angiogenesis.

Limited stem cells, poor stretchability and mismatched interface fusion have plagued the reconstruction of cranial defects by cell-free scaffolds. Here, we designed an instantly fixable and self-adaptive scaffold by dopamine-modified hyaluronic acid chelating Ca2+ of the microhydroxyapatite surface and bonding type I collagen to highly simulate the natural bony matrix. It presents a good mechanical match and interface integration by appropriate calcium chelation, and responds to external stress by flexible deformation. Meanwhile, the appropriate matrix microenvironment regulates macrophage M2 polarization and recruits endogenous stem cells. This scaffold promotes the proliferation and osteogenic differentiation of BMSCs in vitro, as well as significant ectopic mineralization and angiogenesis. Transcriptome analysis confirmed the upregulation of relevant genes and signalling pathways was associated with M2 macrophage activation, endogenous stem cell recruitment, angiogenesis and osteogenesis. Together, the scaffold realized 97 and 72% bone cover areas after 12 weeks in cranial defect models of rabbit (Φ = 9 mm) and beagle dog (Φ = 15 mm), respectively.

Precipitating phosphorus as struvite from anaerobically-digested dairy manure

Midwestern US agroecosystems suffer greatly due to excess phosphorus (P) in runoff from land-applied manure. Precipitating P as struvite would remove it from the manure stream, allowing efficient storage, transportation, and use as slow-release fertilizer. To achieve struvite precipitation, organic solids and calcium (Ca) must first be removed from the manure. This study assessed struvite precipitation from dairy manure using a novel processing pipeline: first diluting the manure, then acidifying and treating it with a Ca chelator, followed by simultaneously flocculating Ca and organic solids, and finally raising the pH to precipitate struvite. The pH manipulations were determined through chemical equilibrium modeling in Visual MINTEQ. Oxalic acid was the most efficient chelator tested, reducing soluble Ca from 200 mg/L to below 40 mg/L while leaving magnesium concentrations unaffected. An anionic low-weight flocculant was most effective at removing solids, reducing it from 1.2% to 0.5% in diluted (4:1) manure. This process train comprising oxalic acid and the anionic flocculant, reduced dissolved P by 93% producing an estimated struvite yield equivalent to 72% of total P initially present in manure. Results from this study show that a simple one-step removal of Ca and organic solids can achieve the P removal efficiency comparable to multi-step methods.