Pps1, phosphatidylserine synthase, regulates the salt stress response in Schizosaccharomyces pombe.

Phosphatidylserine (PS) is important for maintaining growth, cytoskeleton, and various functions in yeast; however, its role in stress responses is poorly understood. In Schizosaccharomyces pombe, the PS synthase deletion (pps1∆) mutant shows defects in growth, morphology, cytokinesis, actin cytoskeleton, and cell wall integrity, and these phenotypes are rescued by ethanolamine supplementation. Here, we evaluated the role of Pps1 in the salt stress response in S. pombe. We found that pps1∆ cells are sensitive to salt stresses such as KCl and CaCl2 even in the presence of ethanolamine. Loss of the functional cAMP-dependent protein kinase (git3∆ or pka1∆) or phospholipase B Plb1 (plb1∆) enhanced the salt stress-sensitive phenotype in pps1∆ cells. Green fluorescent protein (GFP)-Pps1 was localized at the plasma membrane and endoplasmic reticulum regardless of the stress conditions. In pka1∆ cells, GFP-Pps1 was accumulated around the nucleus under the KCl stress. Pka1 was localized in the nucleus and the cytoplasm under normal conditions and transferred from the nucleus to the cytoplasm under salt-stress conditions. Pka1 translocated from the nucleus to the cytoplasm during CaCl2 stress in the wild-type cells, while it remained localized in the nucleus in pps1∆ cells. Expression and phosphorylation of Pka1-GFP were not changed in pps1∆ cells. Our results demonstrate that Pps1 plays an important role in the salt stress response in S. pombe.

Effect of sheep bone protein hydrolysate on promoting calcium absorption and enhancing bone quality in low-calcium diet fed rats.

Calcium deficiency is prone to fractures, osteoporosis and other symptoms. In this study, sheep bone protein hydrolysates (SBPHs) were obtained by protease hydrolysis. A low-calcium-diet-induced calcium-deficiency rat model was established to investigate the effects of SBPHs on calcium absorption and intestinal flora composition. The results showed that an SBPHs + CaCl2 treatment significantly increased the bone calcium content, bone mineral density, trabecular bone volume, and trabecular thickness, and reduced trabecular separation, and changed the level of bone turnover markers (P < 0.05). Supplementation of SBPHs + CaCl2 can remarkably enhance the bone mechanical strength, and the microstructure of bone was improved, and the trabecular network was more continuous, complete, and thicker. Additionally, SBPHs + CaCl2 dietary increased the abundance of Firmicutes and reduced the abundance of Proteobacteria and Verrucomicrobiota, and promoted the production of short chain fatty acids. This study indicated that SBPHs promoted calcium absorption and could be applied to alleviate osteoporosis.

Effect of experimental boundary conditions and treatment-time on the electro-desalination of soils.

This study investigates the effect of boundary conditions and treatment-time on the electro-desalination of artificially-contaminated soil. The effect of ion exchange membranes (IEM), calcium chloride (CaCl2), and ethylenediaminetetraacetic acid (EDTA) on the removal of salt (i.e., Na+, Cl-, and Ca2+) and metal (i.e., Co2+ and Fe2+) ions from the soil by electrokinetic (EK) was studied. The outcomes demonstrate that an increase in treatment-time decreases the electroosmosis and ion removal rate, which might be attributed to the formation of acid-base fronts in soil, except in the IEM case. Because a high pH jump and electroosmotic flow (EOF) of water were not observed within the soil specimen due to the IEM, the removal of ions was only by diffusion and electromigration. The collision of acid-base fronts produced a large voltage gradient in a narrow soil region with a reduced electric field (EF) in its remaining parts, causing a decrease in EOF and ion transport by electromigration. The results showed that higher electroosmosis was observed by using CaCl2 and EDTA; thus, the removal rate of Co2+, Na+, and Ca2+ was greater than Cl- due to higher EOF. However, for relatively low EOF, the removal of Cl- exceeded that of Co2+, Na+, and Ca2+, possibly due to a lack of EOF. In addition, the adsorption of Fe2+ in soil increased with treatment-time due to the corrosion of the anode during all EK experiments except in the case of IEM, where an anion exchange membrane (AEM) was introduced at the anode-soil interface.

Amelioration of NaCl stress on germination, growth, and nitrogen fixation of Vicia faba at isosmotic Na-Ca combinations and Rhizobium.

MAIN CONCLUSION: The Na+/Ca2+ ratio of 1/5 ameliorated the inhibitory action of NaCl and improved the germination and growth of Vicia faba. Addition of Rhizobium also enhanced nodulation and nitrogen fixation. Casting light upon the impact of salinity stress on growth and nitrogen fixation of Vicia faba supplemented with Rhizobium has been traced in this work. How Ca2+ antagonizes Na+ toxicity and osmotic stress of NaCl was also targeted in isosmotic combinations of NaCl and CaCl2 having various Na+:Ca2+ ratios. Growth of Vicia faba (cultivar Giza 3) was studied at two stages: germination and seedling. At both experiments, seeds or seedlings were exposed to successively increasing salinity levels (0, 50, 100, 150, and 200 mM NaCl) as well as isosmotic combinations of NaCl and CaCl2 (Na+:Ca2+ of 1:1, 1:5, 1:10, 1:15, 1:18, and 1: 20), equivalent to 150 mM NaCl. Inocula of the local nitrogen-fixing bacteria, Rhizobium leguminosarum (OP715892) were supplemented at both stages. NaCl salinity exerted a negative impact on growth and metabolism of Vicia faba; inhibition was proportional with increasing salinity level up to the highest level of 200 mM. Seed germination, shoot and root lengths, fresh and dry weights, chlorophyll content, and nodules (number, weight, leghemoglobin, respiration, and nitrogenase activity) were inhibited by salinity. Ca2+ substitution for Na+, particularly at a Na/Ca ratio of 1:5, was stimulatory to almost all parameters at both stages. Statistical correlations between salinity levels and Na/Ca combinations proved one of the four levels (strong- or weak positive, strong- or weak negative) with most of the investigated parameters, depending on the parameter.

Structural characterization and calcium absorption-promoting effect of sucrose-calcium chelate in Caco-2 monolayer cells and mice.

Sucrose emerges as a chelating agent to form a stable sucrose-metal-ion chelate that can potentially improve metal-ion absorption. This study aimed to analyze the structure of sucrose-calcium chelate and its potential to promote calcium absorption in both Caco-2 monolayer cells and mice. The characterization results showed that calcium ions mainly chelated with hydroxyl groups in sucrose to produce sucrose-calcium chelate, altering the crystal structure of sucrose (forming polymer particles) and improving its thermal stability. Sucrose-calcium chelate dose dependently increased the amount of calcium uptake, retention, and transport in the Caco-2 monolayer cell model. Compared to CaCl2 , there was a significant improvement in the proportion of absorbed calcium utilized for transport but not retention (93.13 ± 1.75% vs. 67.67 ± 7.55%). Further treatment of calcium channel inhibitors demonstrated the active transport of sucrose-calcium chelate through Cav1.3. Cellular thermal shift assay and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays indicated that the ability of sucrose-calcium chelate to promote calcium transport was attributed to its superior ability to bind with PMCA1b, a calcium transporter located on the basement membrane, and stimulate its gene expression compared to CaCl2 . Pharmacokinetic analysis of mice confirmed the calcium absorption-promoting effect of sucrose-calcium chelate, as evident by the higher serum calcium level (44.12 ± 1.90 mg/L vs. 37.42 ± 1.88 mmol/L) and intestinal PMCA1b gene expression than CaCl2 . These findings offer a new understanding of how sucrose-calcium chelate enhances intestinal calcium absorption and could be used as an ingredient in functional foods to treat calcium deficiency. PRACTICAL APPLICATION: The development of high-quality calcium supplements is crucial for addressing the various adverse symptoms associated with calcium deficiency. This study aimed to prepare a sucrose-calcium chelate and analyze its structure, as well as its potential to enhance calcium absorption in Caco-2 monolayer cells and mice. The results demonstrated that the sucrose-calcium chelate effectively promoted calcium absorption. Notably, its ability to enhance calcium transport was linked to its strong binding with PMCA1b, a calcium transporter located on the basement membrane, and its capacity to stimulate PMCA1b gene expression. These findings contribute to a deeper understanding of how the sucrose-calcium chelate enhances intestinal calcium absorption and suggest its potential use as an ingredient in functional foods for treating calcium deficiency.

Strength development and self-desiccation of saline cemented paste backfill.

Given that many mines around the world are located in areas where fresh water is scarce, and companies are being held to increasingly stringent sustainability and environmental responsibility standards, many mines are looking to use locally available saline groundwater or seawater as mixing water in cemented paste backfill (CPB). However, the impacts of this decision on key engineering properties of CPB (e.g. strength and self-desiccation) that affect its mechanical stability need to be better understood to allow confident selection of this practical and more sustainable solution. Thus, the effect of mixing water salinity and binder type on the strength (unconfined compressive strength, UCS) development and self-desiccation (measured by suction and volumetric water content) of CPB is explored in this research. NaCl concentrations from 0 to 300 g/L were used in CPB made with silica tailings and Portland cement type I (PC). Concentrations of 10 and 35 g/L were found to moderately increase UCS, while a concentration of 100 g/L had comparable UCS to non-saline CPB and a concentration of 300 g/L was found to significantly decrease UCS over all curing times. The overall trend is 10 g/L > 35 g/L > 0 g/L > 100 g/L > 300 g/L. The UCS of the 60-day-old CPB with a NaCl of 300 g/L is significantly lower, registering a 26% decrease compared to the UCS of the 60-day-old CPB without salt. In contrast, the UCS of the 60-day-old CPBs containing 10 g/L and 35 g/L of salt exhibits a notable improvement, being 15% and 10% higher, respectively, than the UCS of the 60-day-old CPB without salt. Water content and suction monitoring were conducted up to 28 days of curing time, and it was found that suction only slightly contributed to UCS gain of the saline CPB, and high salt contents (100 and 300 g/L) significantly inhibited the self-desiccation ability of CPB due to inhibition of cement hydration by the excessive amount of salt. The increase in strength of both saline and non-saline samples was attributed primarily to the increase in cement hydration products, while the increased strength of the samples with salinities of 10 and 35 g/L was mainly attributed to the enhancement of the binder hydration due to the low amount of salt and the presence of Friedel's salt in the pores. The effect of PC replacement by 25 to 75% with slag on CPB with 35 g/L mixing water salinity was also studied. Slag replacement of 50% and higher resulted in significantly higher UCS over most curing times. Suction likely moderately contributed to UCS of the saline CPB with slag, in addition to the presence of Friedel's salt in the pores and the acceleration of cement and slag hydration by the presence of NaCl.

Improving liming mode for remediation of Cd-contaminated acidic paddy soils: Identifying the optimal soil pH, model and efficacies.

Liming has been widely taken to remediate Cd-contaminated acidic paddy soils, whereas liming mode involving in the relevant optimal soil pH, model and efficacies remain unclear. Both soil and field liming experiments were conducted to improve liming mode for precise remediation of Cd-contaminated acidic paddy soils. Soil batch liming experiments indicated soil DTPA-Cd and CaCl2-Cd were piecewise linearly correlated to soil pH with nodes of 6.8-8.0, and decreased respectively by 15.3%37.7% and 80.7%93.8% (P < 0.05) when soil pH raised over the nodes, indicating an appropriate target soil pH 7.0 for liming. Stepwise linear regression revealed that liming ratio (LR, kg ha-1) could be estimated from soil basal pH (pH0) and the interval to the target soil pH (ΔpH), as [LR=exp(1.10 ×ΔpH+0.61 ×pH0-4.98), R2 = 0.97, n = 42, P < 0.01]. The model exhibited high prediction accuracy (95.2%), low mean estimation error (-0.02) and root mean square error (0.20). Field liming experiment indicated liming to target pH decreased respectively soil CaCl2-Cd by 95.2-98.0% and rice grain Cd by 59.8-80.6% (P < 0.01), whereas uninfluenced rice grain yield. Correlation analysis and structural equation models (SEM) demonstrated that great reduction in Cd phytoavailability was mainly attributed to the transformation of soil water-soluble and exchangeable Cd to carbonate-bound Cd and Fe/Mn oxides-bound Cd and reduced Cd in iron plaque as increasing soil pH. However, rice grain Cd of 50% samples met national food safety standards limit of China (0.2 mg kg-1) due to the high soil Cd level (0.8 mg kg-1). In conclusion, liming to target soil pH 7.0 could be considered as a precise and effective remediation mode for Cd-contaminated acidic paddy soils and complementary practices should be implemented for severe pollution. Our results could provide novel insights on precise liming remediation of Cd-contaminated acidic paddy soils.

Migration and transformation of phosphorus and toxic metals during sludge incineration with Ca additives.

The recycling and utilization of phosphorus resources in sludge is becoming increasingly important. In this study, we compared the conversion of phosphorus and toxic metal passivation effects of different Ca additives under oxygen-rich combustion conditions and elucidated their specific mechanisms of action. The experimental results indicated that four Ca-based additives improved the recovery rate of total phosphorus, and promoted the generation of stable apatite phosphorus (AP). The effect of CaCl2 and CaO was greater than that of Ca(OH)2 and CaSO4. CaCl2 promoted the formation of Ca3(PO4)2 and Ca2P2O7, and CaSO4 improved the conversion of AlPO4 to Ca(H2PO4)2 with increasing temperature. The conversion capacity of CaO on non-apatite inorganic phosphorus to AP was greater than that of Ca(OH)2, and more CaH2P2O7, Ca(PO3)2, and Ca-Al-P minerals were found. Toxic metal percentages decreased after sludge incineration with CaCl2. Compared with CaO and Ca(OH)2, the toxic metal adsorption effect of CaSO4 was more significant. The influence of Ca additives on the conversion of Zn into stable components was as follows: CaCl2 > Ca(OH)2 > CaO > CaSO4. Ca additives reduced the toxic metal contamination level and ecological risk index values, and the order of toxic metal contamination levels was Ni > Zn > Cr > Cu > Mn. The experiment confirmed the conversion of phosphorus and the toxic metal passivation effect of Ca additives during oxy-fuel combustion of sludge, which is beneficial for its resource utilization.

Calcium regulation of the Arabidopsis Na+/K+ transporter HKT1;1 improves seed germination under salt stress.

Calcium is known to improve seed-germination rates under salt stress. We investigated the involvement of calcium ions (Ca2+) in regulating HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1; 1), which encodes a Na+/K+ transporter, and its post-translational regulator TYPE 2C PROTEIN PHOSPHATASE 49 (PP2C49), in germinating Arabidopsis (Arabidopsis thaliana) seedlings. Germination rates of hkt1 mutant seeds under salt stress remained unchanged by CaCl2 treatment in wild-type Arabidopsis, whereas pp2c49 mutant seeds displayed improved salt-stress tolerance in the absence of CaCl2 supplementation. Analysis of HKT1;1 and PP2C49 promoter activity revealed that CaCl2 treatment results in radicle-focused expression of HKT1;1 and reduction of the native radicle-exclusive expression of PP2C49. Ion-content analysis indicated that CaCl2 treatment improves K+ retention in germinating wild-type seedlings under salt stress, but not in hkt1 seedlings. Transgenic seedlings designed to exclusively express HKT1;1 in the radicle during germination displayed higher germination rates under salt stress than the wild type in the absence of CaCl2 treatment. Transcriptome analysis of germinating seedlings treated with CaCl2, NaCl, or both revealed 118 upregulated and 94 downregulated genes as responsive to the combined treatment. Bioinformatics analysis of the upstream sequences of CaCl2-NaCl-treatment-responsive upregulated genes revealed the abscisic acid response element CACGTGTC, a potential CaM-binding transcription activator-binding motif, as most prominent. Our findings suggest a key role for Ca2+ in mediating salt-stress responses during germination by regulating genes that function to maintain Na+ and K+ homeostasis, which is vital for seed germination under salt stress.

Xylitol biosynthesis enhancement by Candida tropicalis via medium, process parameter optimization, and co-substrate supplementation.

The present study examines the impact of nitrogen sources (yeast extract, ammonium sulfate peptone, ammonium nitrate, urea, and sodium nitrate), salt solution (0.5 g/L MgSO4, 0.5 g/L KH2PO4, 0.3 g/L CaCl2), trace elements solution (0.1 g/L CuSO4, 0.1 g/L FeSO4, 0.02 g/L MnCl2, 0.02 g/L ZnSO4), operational parameters (temperature, aeration, agitation, initial pH and xylose concentration) and co- substrate supplementation (glucose, fructose, maltose, sucrose, and glycerol) on xylitol biosynthesis by Candida tropicalis ATCC 13803 using synthetic xylose. The significant medium components were identified using the Plackett Burman design followed by central composite designs to obtain the optimal concentration for the critical medium components in shaker flasks. Subsequently, the effect of operational parameters was examined using the One Factor At a Time method, followed by the impact of five co-substrates on xylitol biosynthesis in a 1 L bioreactor. The optimal media components and process parameters are as follows: peptone: 12.68 g/L, yeast extract: 6.62 g/L, salt solution (0.5 g/L MgSO4, 0.5 g/L KH2PO4, and 0.3 g/L CaCl2): 1.23 X (0.62 g/L, 0.62 g/L, and 0.37 g/L respectively), temperature: 30 °C, pH: 6, agitation: 400 rpm, aeration: 1 vvm, and xylose: 50 g/L. Optimization studies resulted in xylitol yield and productivity of 0.71 ± 0.004 g/g and 1.48 ± 0.018 g/L/h, respectively. Glycerol supplementation (2 g/L) further improved xylitol yield (0.83 ± 0.009 g/g) and productivity (1.87 ± 0.020 g/L/h) by 1.66 and 3.12 folds, respectively, higher than the unoptimized conditions thus exhibiting the potential of C. tropicalis ATCC 13803 being used for commercial xylitol production.

Texture classification and in situ cation measurements by SEM-EDX provide detailed quantitative insights into cell wall cation interaction changes during ageing, soaking, and cooking of red kidney beans.

Hard-to-cook (HTC) is a textural defect that delays the softening of common bean seeds during cooking. While this defect is commonly associated with conventionally stored beans, soaking/cooking of beans in CaCl2 solutions or sodium acetate buffer can also prolong the cooking time of beans due to formation of Ca2+ crosslinked pectin retarding bean softening during cooking. In this study, the role of the cell wall-bound Mg2+/Ca2+ content and the degree of pectin methyl esterification (DM) was quantified, as important factors for bean texture-related changes stipulated in the pectin-cation-phytate hypothesis, the most plausible hypothesis of HTC development. Evaluation of texture changes during cooking of conventionally aged beans (35 °C and 83% RH for up to 20 weeks), beans soaked/cooked in CaCl2 solutions (0.01 to 0.1 M) or soaked in 0.1 M sodium acetate buffer (pH 4.4) revealed large bean-to-bean variations. Therefore a texture-based classification approach was used to better capture the relation between texture characteristics and cell wall polymer, in particular pectin, related changes. While cell wall-bound Ca2+ and pectin DM did not change/were not related to the texture variation during cooking of fresh beans, increased cell wall-bound Ca2+ and decreased pectin DM were associated with prolonged conventional storage of beans and their texture changes during subsequent cooking (due to pectin cross linking, retarding its solubilization during cooking). Exogenously added Ca2+ from pre-treating beans in CaCl2 solutions promoted to a great extent the cell wall-bound Ca2+ during soaking but even more so during cooking, complementing the harder texture associated with these beans during cooking (compared to conventionally stored and fresh beans). Similarly, free Ca2+ endogenously generated by phytase-catalysed phytate hydrolysis (beans treated by acetate buffer) promoted crosslinking of pectin by Ca2+ (cell wall-bound Ca2+), delaying softening of beans during cooking.

Effect of bio-chemical changes due to conventional ageing or chemical soaking on the texture changes of common beans during cooking.

To establish the HTC defect development, the cooking kinetics of seeds of ten bean accessions (belonging to seven common bean market classes), fresh and conventionally aged (35 °C, 83% RH, 3 months) were compared to those obtained after soaking in specific salt solutions (in 0.1 M sodium acetate buffer at pH 4.4, 41 °C for 12 h, or 0.01 M CaCl2 at pH 6.2, 25 °C for 16 h and subsequently cooking in CaCl2 solution, or deionised water). The extent of phytate (inositol hexaphosphate, IP6) hydrolysis was evaluated to better understand the role of endogenous Ca2+ in the changes of the bean cooking kinetics. A significant decrease in the IP6 content was observed after conventional ageing and after soaking in a sodium acetate solution suggesting phytate hydrolysis (release of endogenous Ca2+). These changes were accompanied by an increase in the cooking time of the beans. Smaller changes in cooking times after soaking in a sodium acetate solution (compared to conventionally aged beans) was attributed to a lower ionisation level of the COOH groups in pectin (pH 4.4, being close to pKa value of pectin) limiting pectin Ca2+ cross-linking. In beans soaked in a CaCl2 solution, the uptake of exogenous cations increased the cooking times (with no IP6 hydrolysis). The change in cooking time of conventionally aged beans was strongly correlated with the extent of IP6 hydrolysis, although two groups of beans with low or high IP6 hydrolysis were distinguished. Comparable trends were observed when soaking in CaCl2 solution (r = 0.67, p = 0.14 or r = 0.97, p = 0.03 for two groups of beans with softer or harder texture during cooking). Therefore a test based on the Ca2+ sensitivity of the cooking times, implemented through a Ca2+ soaking experiment followed by cooking can be used as an accelerated test to predict susceptibility to HTC defect development during conventional ageing. On the other hand, a sodium acetate soaking experiment can be used to predict IP6 hydrolysis of conventionally aged bean accessions and changes of cooking times for these bean accessions (with exception of yellow bean-KATB1).

NPs-Ca promotes Cd accumulation and enhances Cd tolerance of rapeseed shoots by affecting Cd transfer and Cd fixation in pectin.

The use of rapeseed (Brassica napus) as a hyperaccumulator plant has shown great promise for the remediation of cadmium (Cd) contaminated soils. Nanosized materials (NPs) have been shown to mitigate heavy metal toxicity in plants, but it is unknown how l-aspartate nano-calcium (NPs-Ca) affects Cd uptake, transport, and tolerance in rapeseed. A soil pot experiment was conducted with two treatments: a control treatment (CK) with 2.16 g CaCl2 and NPs-Ca treatment with 6.00 g NPs-Ca, to evaluate the effects and mechanisms of NPs-Ca on Cd tolerance in rapeseed. Compared to CaCl2, NPs-Ca promoted Cd transportation from roots to shoots by up-regulating the expression of Cd transport genes (ABCC12, HMA8, NRAM6, ZIP6, CAX4, PCR2, and HIP6). Therefore, NPs-Ca increased Cd accumulation in rapeseed shoots by 39.4%. Interestingly, NPs-Ca also enhanced Cd tolerance in the shoots, resulting in lower hydrogen peroxide (H2O2) accumulation and proline content, as well as higher antioxidant enzyme activities (POD, CAT). Moreover, NPs-Ca reduced the activity of pectin-degrading enzymes (polygalacturonase: PG, ß-galactosidase: ß-GAL), promoted the activity of pectin methyl esterase (PME), and changed transcription levels of related genes (PME, PMEI, PG, PGIP, and ß-GAL). NPs-Ca treatment also significantly increased the Cd content in cell walls by 59.8%, that is, more Cd was immobilized in cell walls, and less Cd entered organelles in shoots of NPs-Ca treatment due to increased pectin content and degree of pectin demethylation. Overall, NPs-Ca increased Cd accumulation in rapeseed shoots by promoting Cd transport from roots to shoots. And meantime, NPs-Ca enhanced Cd tolerance of shoots by inhibiting pectin degradation, promoting pectin demethylation and increasing Cd fixation in pectin. These findings suggest that NPs-Ca can improve the potential of rapeseed as a hyperaccumulator for the remediation of Cd-contaminated soil and the protection of the environment. Furthermore, the study provides a theoretical basis for the application of NPs-Ca in the phytoremediation of Cd-contaminated soils with hyperaccumulating plants.

Renzhu Ointment Regulates L-Type Voltage-Dependent Calcium Channel in Mice Model of Senna-Induced Diarrhea by Transdermal Administration.

Purpose: In China, herbal preparation is commonly administered transdermally for treating pediatric diarrhea. However, few studies have probed into their antidiarrheal mechanisms. This study was designed to investigate the antidiarrheal effect of Renzhu ointment (Renzhuqigao, RZQG) and its underlying mechanisms via transdermal administration. Methods: The main components of RZQG were confirmed by gas chromatography-mass spectrometry (GC-MS). The effect of RZQG on L-type voltage-dependent calcium channel (L-VDCC) was evaluated by CaCl2- and ACh-induced contraction in isolated colon. The antidiarrheal efficacy of RZQG was further investigated by the senna-induced diarrhea mice based on the frequency of loose stools, diarrhea rate and index, fecal moisture content, and the basal tension of the colon. Additionally, the protein expression of CACNA1C, CACNA1D, cAMP, and PKA were detected with Western blot and immunohistochemistry (IHC). Results: GC-MS analysis determined 14 components in RZQG. In vitro, RZQG relaxed the CaCl2- and ACh-induced tension, while nifedipine (a L-VDCC inhibitor) and H-89 (a PKA inhibitor) decreased the relaxation. In vivo, animal model showed that transdermal administration of RZQG exhibited a significant reduction in the frequency of loose stools, diarrhea rate and index, fecal moisture content and the basal tension. Compared to the model group, the colon of mice treated with RZQG showed lower expression of CACNA1C, CACNA1D, cAMP, and PKA. IHC results showed that cAMP was downregulated in colonic smooth muscle after RZQG treatment. Conclusion: RZQG improved diarrhea symptoms and down-regulated the expression of CACNA1C and CACNA1D via transdermal administration, which is closely associated with the cAMP/PKA signaling pathway in colonic smooth muscle.

Oral calcium and vitamin D supplements differentially alter exploratory, anxiety-like behaviors and memory in male rats.

Oral calcium and calcium plus vitamin D supplements are commonly prescribed to several groups of patients, e.g., osteoporosis, fracture, and calcium deficiency. Adequate and steady extracellular calcium levels are essential for neuronal activity, whereas certain forms of calcium supplement (e.g., CaCO3) probably interfere with memory function. However, it was unclear whether a long-term use of ionized calcium (calcium chloride in drinking water ad libitum), vitamin D supplement (oral gavage) or the combination of both affected anxiety and memory, the latter of which was probably dependent on the hippocampal neurogenesis. Here, we aimed to determine the effects of calcium and/or vitamin D supplement on the anxiety- and memory-related behaviors and the expression of doublecortin (DCX), an indirect proxy indicator of hippocampal neurogenesis. Eight-week-old male Wistar rats were divided into 4 groups, i.e., control, calcium chloride-, 400 UI/kg vitamin D3-, and calcium chloride plus vitamin D-treated groups. After 4 weeks of treatment, anxiety-, exploration- and recognition memory-related behaviors were evaluated by elevated pulse-maze (EPM), open field test (OFT), and novel object recognition (NOR), respectively. The hippocampi were investigated for the expression of DCX protein by Western blot analysis. We found that oral calcium supplement increased exploratory behavior as evaluated by OFT and the recognition index in NOR test without any effect on anxiety behavior in EPM. On the other hand, vitamin D supplement was found to reduce anxiety-like behaviors. Significant upregulation of DCX protein expression was observed in the hippocampus of both calcium- and vitamin D-treated rats, suggesting their positive effects on neurogenesis. In conclusion, oral calcium and vitamin D supplements positively affected exploratory, anxiety-like behaviors and/or memory in male rats. Thus, they potentially benefit on mood and memory in osteoporotic patients beyond bone metabolism.

The effect of amorphous calcium carbonate as a culture media supplement on embryonic development of murine sibling embryos.

PURPOSE: The aim of this study was to compare the addition in culture media of stabilized amorphous calcium carbonate (ACC) versus calcium chloride (CaCl2) or calcium carbonate in crystalline form (CCC) on growth rates among sibling mouse embryos. METHODS: We evaluated the effect of different ACC concentrations on the rates of embryo compaction at 60 h, blastocyst rate at 84 h and percentage of fully hatched at 108 h following hCG injection. As ACC is stabilized by tripolyphosphate (TPP), we also evaluated the addition of TPP alone to the culture media. Finally, we compared supplemented ACC culture media to one-step SAGE and Irvine cleavage media. RESULTS: The results revealed that ACC accelerates the compaction and blastocyst rates, as well as the percentage of fully hatched embryos in a dose-dependent manner, with an increased positive effect at 2.5 mM. The magnitude of the effect for ACC-supplemented media on the embryo developmental rate was between 30 to 40% (p < 0.01) faster for each stage, compared to both SAGE and Irvine one-step standard media. Embryos cultured with SAGE or Irvine media with or without supplementation of CaCl2 or CCC, did not produce the same improvements as observed with ACC. CONCLUSION: In conclusion, the ACC demonstrates a rapid modulation effect for restoring media optimal pH. ACC can inhibit cathepsin B activity during in vitro culture of fibroblast cells. The beneficial impact of ACC on cleavage mouse embryos is likely due to an improved buffering effect causing slower pH media variations, which may enhance quality and implantation potential of embryos following in vitro culture.

Qi-Po-Sheng-Mai granule ameliorates Ach-CaCl2 -induced atrial fibrillation by regulating calcium homeostasis in cardiomyocytes.

BACKGROUND: Atrial fibrillation (AF) is one of the most common arrhythmias encountered in clinical settings. Currently, the pathophysiology of AF remains unclear, which severely limits the effectiveness and safety of medical therapies. The Chinese herbal formula Qi-Po-Sheng-Mai Granule (QPSM) has been widely used in China to treat AF. However, its pharmacological and molecular mechanisms remain unknown. PURPOSE: The purpose of this study was to investigate the molecular mechanisms and potential targets of QPSM for AF. STUDY DESIGN AND METHODS: The AF model was induced by Ach (66 µg/ml) and CaCl2 (10 mg/kg), and the dose of 0.1 ml/100 g was injected into the tail vein for 5 weeks. QPSM was administered daily at doses of 4.42 and 8.84 g/kg, and amiodarone (0.18 g/kg) was used as the positive control. The effect of QPSM on AF was assessed by electrocardiogram, echocardiography, and histopathological analysis. Then, we employed network pharmacology with single nucleus RNA sequencing (snRNA-Seq) to investigate the molecular mechanisms and potential targets of QPSM for AF. Furthermore, high performance liquid chromatography (HPLC) method was used for component analysis of QPSM, and molecular docking was used to verify the potential targets. Using the IonOptix single cell contraction and ion synchronization test equipment, single myocyte length and calcium ion variations were observed in real time. The expression levels of calcium Transporter-related proteins were detected by western blot and immunohistochemistry. RESULTS: Based on an Ach-CaCl2-induced AF model, we found that QPSM treatment significantly reduced atrial electrical remodeling-related markers, such as AF inducibility and duration, and attenuated atrial dilation and fibrosis. Network pharmacology identified 52 active ingredients and 119 potential targets for QPSM in the treatment of AF, and 45 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched, among which calcium pathway had the greatest impact. Using single nucleus sequencing (snRNA-seq), we identified cardiomyocytes as the most differentially expressed in response to drug treatment, with nine differentially expressed genes enriched in calcium signaling pathways. High performance liquid chromatography and molecular docking confirmed that the core components of QPSM strongly bind to the key factors in the calcium signaling pathway. Additional experiments have shown that QPSM increases calcium transients (CaT) and contractility in the individual cardiomyocyte. This was accomplished by increasing the expression of CACNA1C and SERCA2a and decreasing the expression of CAMK2B and NCX1. CONCLUSION: The present study has systematically elucidated the role of QPSM in maintaining calcium homeostasis in cardiomyocytes through the regulation of calcium transporters, which could lead to new drug development ideas for AF.

Role of Ca2+ in the pepsin-induced coagulation and in the dynamic in vitro gastric digestion behavior of casein micelles.

The effect of Ca2+ on pepsin-induced hydrolysis of κ-casein and subsequent coagulation of casein micelles was studied in a micellar casein (MC) solution at pH ≈ 6.0 at 37 °C without stirring. An NaCl-supplemented MC solution was used as a positive control to assess the effect of increased ionic strength after CaCl2 addition. Quantitative determination of the released para-κ-casein during the reaction using reverse-phase high-performance liquid chromatography showed that specific hydrolysis of κ-casein by pepsin was little affected by the addition of either CaCl2 or NaCl. However, rheological properties and microstructures of curds induced by pepsin hydrolysis depended markedly on the addition of salts. Addition of CaCl2 up to 17.5 mM facilitated coagulation, with decreases in coagulation time and critical hydrolysis degree, and increases in firming rate and maximum storage modulus (G'max); further addition of CaCl2 (22.5 mM) resulted in a lower G'max. Increased ionic strength to 52.5 mM by adding NaCl retarded the coagulation and resulted in a looser curd structure. In a human gastric simulator, MC, without the addition of CaCl2, did not coagulate until the pH decreased to ≈5.0 after ≈50 min of digestion. Addition of CaCl2 facilitated coagulation of casein micelles and resulted in more cohesive curds with dense structures during digestion, which slowed the emptying rate of caseins. At the same CaCl2 concentration, a sample with higher ionic strength coagulated more slowly. This study provides further understanding on the effect of divalent (Ca2+) ions and ionic strength on the coagulation of casein micelles and the digestion behavior of milk.

Realizing Eco-Friendly Water-Resistant Sodium-Alginate-Based Films Blended with a Polyphenolic Aqueous Extract from Grape Pomace Waste for Potential Food Packaging Applications.

Water-resistant and environmentally friendly sodium-alginate-based films have been investigated to develop functional materials to extend the food's shelf-life. A water-stable alginate-based film was prepared, employing both the internal and external gelation approach in the presence of CaCl2. To apply this film to food packaging and thus preserve food quality, the aim of this work is to perform a chemical and physical characterization of the proposed materials, evidencing the main features and stability under different work conditions. Water contact angle measurements showed a value of 65°, suggesting an important reduced hydrophilic character of the obtained alginate films due to the novel CaCl2-induced compacted polymer network. The film's stability was thus checked through swelling measurements in water after varying pH, temperature, and ionic strength. The film was stable at high temperatures and not pH-responsive. Only highly concentrated salt-based solutions negatively affected the proposed packaging, causing a large swelling. Furthermore, a water-based polyphenolic extract from grape (Vitis vinifera L.) pomace waste was embedded inside the films in different amounts in order to confer additional properties. The extract's polyphenolic content (evaluated from HPLC/MS-MS measurements) endowed the films' UV-light screening and enhanced antioxidant properties. These important findings suggest the additional potential role of these films in protecting food from light deterioration. The stability of these hybrid films was also checked by observation, as the polyphenols' presence did not largely alter the alginate network that occurred yet was water-resistant under the described work conditions.

Utilizing CaCl2 to promote the enrichment and bioavailability of phosphorus in incinerated sludge ash.

Recovering phosphorus from incineration sludge sewage ash (ISSA) is a well-established technology, with a greater recovery potential than that of supernatant or sludge. ISSA can be utilized as a secondary raw material in the fertilizer industry, or as a fertilizer if heavy metal concentrations do not exceed permissible limits, thus reducing the cost of phosphorus recovery. Increasing the temperature to produce ISSA with higher solubility and plant availability of phosphorus is advantageous for both pathways. But a decrease in the extraction of phosphorus is also observed at high temperatures, thereby diminishing the overall economic benefits. In this study, CaCl2 was utilized to mitigate the decrease in the extraction rate and also to promote the bioavailability of phosphorus. The addition of CaCl2 (80 g/kg of dry sludge) effectively promoted the conversion of non-apatite inorganic phosphorus to apatite inorganic phosphorus at a rate of 87.73% at 750 °C. Furthermore, the decrease in the extraction rate of phosphorus at 1,050 °C was comparatively smaller in the presence of CaCl2. If iron flocculants are used to capture P in wastewater management, it may be necessary to pay special attention to the amount of addition and incineration temperature to maximize the economic potential of recycling.