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Accurately predicting phosphorous (P) and calcium (Ca) dietary requirements is critical for optimizing dairy cattle performance, and minimizing mineral excretions and ecosystems eutrophication. This study provides a new factorial system to determine net and dietary P and Ca requirements for maintenance and lactation, derived from a meta-regression of mineral trials involving lactating dairy cows. A comprehensive global database was constructed from 57 peer-reviewed articles of mineral balance trials, with a wide range of dietary and animal performance data. We estimated the net requirements for maintenance from the intercept of a nonlinear equation between mineral intake and the sum of total fecal and urinary excretions, which is an estimate of endogenous mineral loss. Mineral secreted in milk was used to obtain net requirements for lactation. The mineral metabolizable coefficient was quantified through observed (treatment means) mineral intake and total fecal and urinary excretions, discounting the estimated endogenous excretions from our proposed models. The nonlinear models of total fecal and urinary mineral excretion were evaluated (observed versus predicted values) using a 5-fold cross validation approach. The models to estimate the sum of endogenous fecal and urinary excretions of P (0.135±0.043 g P/kg BW0.75) and Ca (0.360±0.144 g Ca/kg BW0.75) exhibited suitable precision and accuracy; r = 0.89 and 0.79, concordance correlation coefficient = 0.85 and 0.77, and root mean square prediction error = 24.1 and 20.5% observed means, respectively. Dietary variables (forage level, fiber, starch, crude protein, and ether extract) did not affect the metabolizable coefficient (MC) of P and Ca; therefore, an overall dietary MC of P (0.69±0.01) and Ca (0.65±0.02) were proposed. Our new system estimates lower net and dietary P requirements for lactating dairy cows compared to the NASEM-2021 and NRC-2001 models, but slightly higher Ca requirements than NASEM-2021.This proposed system holds potential to reduce the use of phosphorus in diets for dairy cows, and thus to enhance economic efficiency and environmental sustainability of the dairy industry., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Oliveira, Soares. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Skeleton bones, distinguished by trabecular and cortical bone tissue content, exhibit varied growth and composition, in response to modified dietary calcium and phosphorus levels. The study investigated how gilts adapt their individual bone and bone region mineralisation kinetics in response to changing intake of Ca and P. A total of 24 gilts were fed according to a two-phase (Depletion (D) 60-95 and Repletion (R) 95-140 kg BW, respectively). During the D phase, gilts were fed either 60% (D60) or 100% (D100) of the estimated P requirement. Subsequently, during the R phase, half of the gilts from each D diet were fed either 100% (R100) or 160% (R160) of the estimated P requirement according to a 2 × 2 factorial arrangement. Bone mineral content (BMC) was assessed in the whole body, individual bones (femur and lumbar spine L2-L4), and bone regions (head, front legs, trunk, pelvis, femur, and hind legs) every 2 weeks using dual-energy X-ray absorptiometry (DXA). At 95 kg BW, gilts fed D60 showed reduced BMC and BMC/BW ratio in all studied sites compared to those fed D100 (P < 0.001). During the depletion phase, the allometric BW-dependent regressions slopes for BMC of D100 gilts remained close to 1 for all sites and did not differ from each other. In contrast, the slopes were lower in D60 gilts (P < 0.05), with an 18% reduction in the whole body, except for the front and hind legs, femur, and pelvis, which exhibited higher reductions (P < 0.05). At 140 kg BW, BMC and BMC/BW ratio of all studied sites were similar in gilts previously fed D60 and D100, but higher in R160 than in R100 gilts (P < 0.05), except for front and hind legs. During the repletion phase, the allometric BW dependent regressions slopes for BMC were lower (P < 0.05) in R100 than in R160 gilts (for whole body -10%; P < 0.01) except for front and hind legs, femur, and pelvis. In conclusion, bone demineralisation and recovery followed similar trends for all measured body sites. However, the lumbar spine region was most sensitive whereas the hind legs were least sensitive. These data suggest that using bone regions such as the head and forelegs that can be collected easily at the slaughterhouse may be a viable alternative to whole body DXA measurement., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Insect Malpighian tubules contribute to Ca 2+ homeostasis via Ca 2+ storage in intracellular compartments, Ca 2+ secretion into the tubule lumen, and Ca 2+ reabsorption into the hemolymph. A plasma membrane Ca 2+ -ATPase (PMCA) is hypothesized to be a Ca 2+ -transporter involved in renal Ca 2+ transport of insects, however few studies have investigated its immunochemical expression in Malpighian tubules. Here we characterized the abundance and localization of PMCA-like immunoreactivity in Malpighian tubules of adult female mosquitoes Aedes aegypti using an antibody against Drosophila melanogaster PMCA. Western blotting revealed expression of a relatively abundant 109 kDa isoform and a relatively sparse 115 kDa isoform. Feeding mosquitoes 10% sucrose with 50 mM CaCl 2 for 7 days did not affect PMCA immunoreactivity. However, at 24, 48, and 96 h post-blood feeding (PBF), the relative abundance of the 109 kDa isoform decreased while that of the 115 kDa isoform increased. Immunolabeling of Malpighian tubules revealed PMCA-like immunoreactivity in both principal and stellate cells; principal cell labeling was intracellular, whereas stellate cell labeling was along the basal membrane. Blood feeding enhanced immunolabeling of PMCA in stellate cells but weakened that in principal cells. Moreover, a unique apicolateral pattern of PMCA-like immunolabeling occurred in principal cells of the proximal segment at 24 h PBF, suggesting potential trafficking to septate junctions. Our results suggest PMCA isoforms are differentially expressed and localized in mosquito Malpighian tubules where they contribute to redistributing tubule Ca 2+ during blood meal processing., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Intramuscular fat (IMF) in pork holds significant importance for economic performance within the pig industry and dietary calcium supplementation enhances the accumulation of intramuscular fat. Additionally, calcium ions inhibit translation and reduce protein synthesis. However, the mechanism by which calcium regulates IMF deposition in muscle through translation remains largely unknown. In this study, we compared the ribosome profiles of the longissimus dorsi muscles of Duroc × Landrace × Large white pigs from the normal calcium (NC) group or calcium supplement (HC) group by Ribo-seq, and RNA-seq. By integrating multiple-omics analysis, we further discovered 437 genes that were transcriptionally unchanged but translationally altered and these genes were significantly enriched in the oxidative phosphorylation signaling pathway. Furthermore, experimental data showed that inhibiting the expression of COX10 and mtND4L increased triglyceride accumulation in C2C12 cells, providing new targets for intramuscular fat deposition. Finally, this work links dietary calcium, translation regulation and IMF deposition, providing a new strategy for both meat quality and economic performance within the pig industry., (© 2024. The Author(s).)

Environmental factors and genetic variation individually impact bone. However, it is not clear how these factors interact to influence peak bone mass accrual. Here we tested whether genetically programmed high bone formation driven by missense mutations in the Lrp5 gene (Lrp5A214V) altered the sensitivity of mice to an environment of inadequate dietary calcium (Ca) intake. Weanling male Lrp5A214V mice and wildtype littermates (control) were fed AIN-93G diets with 0.125%, 0.25%, 0.5% (reference, basal), or 1% Ca from weaning until 12 weeks of age (ie, during bone growth). Urinary Ca, serum Ca, Ca regulatory hormones (PTH, 1,25 dihydroxyvitamin D3 (1,25(OH)2D3)), bone parameters (μCT, ash), and renal/intestinal gene expression were analyzed. As expected, low dietary Ca intake negatively impacted bones and Lrp5A214V mice had higher bone mass and ash content. Although bones of Lrp5A214V mice have more matrix to mineralize, their bones were not more susceptible to low dietary Ca intake. In control mice, low dietary Ca intake exerted expected effects on serum Ca (decreased), PTH (increased), and 1,25(OH)2D3 (increased) as well as their downstream actions (ie, reducing urinary Ca, increasing markers of intestinal Ca absorption). In contrast, Lrp5A214V mice had elevated serum Ca with a normal PTH response but a blunted 1,25(OH)2D3 response to low dietary Ca that was reflected in the renal 1,25(OH)2D3 producing/degrading enzymes, Cyp27b1 and Cyp24a1. Despite elevated serum Ca in Lrp5A214V mice, urinary Ca was not elevated. Despite an abnormal serum 1,25(OH)2D3 response to low dietary Ca, intestinal markers of Ca absorption (Trpv6, S100g mRNA) were elevated in Lrp5A214V mice and responded to low Ca intake. Collectively, our data indicate that the Lrp5A214V mutation induces changes in Ca homeostasis that permit mice to retain more Ca and support their high bone mass phenotype., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The purpose of this study was to examine transient plasma membrane disruptions (TPMDs) and TPMD-induced Ca++ waves (TPMD Ca++ Wvs) in human and mouse corneal epithelium (HCEC and MCEC). A multi-photon microscope was used to create laser-induced TPMDs in single cultured cells and in intact ex vivo and in vivo MCECs and ex vivo human cornea rim HCECs. Eye rubbing-induced TPMDs were studied by gentle rubbing with a cotton tipped applicator over a closed eyelid in ex vivo and in vivo MCECs. Ca++ sources for TPMD-induced Ca++ waves were explored using Ca++ channel inhibitors and Ca++-free media. TPMDs and TPMD Ca++ Wvs were observed in all cornea epithelial models examined, often times showing oscillating Ca++ levels. The sarcoplasmic reticulum Ca++ ATPase inhibitors thapsigargin and CPA reduced TPMD Ca++ Wvs. TRP V1 antagonists reduced TPMD Ca++ Wvs in MCECs but not HCECs. Ca++-free medium, 18α-GA (gap junction inhibitor), apyrase (hydrolyzes ATP), and AMTB (TRPM8 inhibitor) did not affect TPMD Ca++ Wvs. These results provide a direct demonstration of corneal epithelial cell TPMDs and TPMDs in in vivo cells from a live animal. TPMDs were observed following gentle eye rubbing, a routine corneal epithelial cell mechanical stress, indicating TPMDs and TPMD Ca++ Wvs are common features in corneal epithelial cells that likely play a role in corneal homeostasis and possibly pathophysiological conditions. Intracellular Ca++ stores are the primary Ca++ source for corneal epithelial cell TPMD Ca++ Wvs, with TRPV1 Ca++ channels providing Ca++ in MCECs but not HCECs. Corneal epithelial cell TPMD Ca++ Wv propagation is not influenced by gap junctions or ATP., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rice protein peptides (RPP) are a potentially valuable source of high-quality calcium chelating properties. However, there is a lack of information regarding the calcium-absorption-promoting effect of RPP and its underlying mechanism. The present study adopted molecular docking methodologies to analyze the 10 most potent peptide segments from RPP. Results revealed that the peptide AHVGMSGEEPE (AHV) displayed optimal calcium binding properties (calcium-chelating capacity 55.69 ± 0.66 mg/g). Quantum chemistry analysis revealed that the AHV peptide effectively binds and forms stable complexes with calcium via the carbonyl oxygen atoms in valine at position 3 and the carbonyl of the C-terminal carboxyl group of glutamate at position 11. The spectral analysis results indicated that AHV may bind to calcium through carboxyl oxygen atoms, resulting in a transition from a smooth surface block-like structure to a dense granular structure. Furthermore, this study demonstrated that the 4 mmol/L AHV-Ca chelate (61.75 ± 13.23 μg/well) significantly increases calcium absorption compared to 1 mM CaCl 2 (28.57 ± 8.59 μg/well) in the Caco-2 cell monolayer. In terms of mechanisms, the novel peptide-calcium chelate AHV-Ca derived from RPP exerts a cell-level effect by upregulating the expression of TRPV6 calcium-ion-channel-related genes and proteins (TRPV6 and Calbindin-D9k). This study provides a theoretical basis for developing functional foods with the AHV peptide as ingredients to improve calcium absorption.

GCaMP is a genetically encoded calcium indicator (GECI) widely used in neuroscience research. It measures intracellular Ca 2+ level by fluorescence changes as it directly binds to Ca 2+ . In this process, the effect of this calcium buffer on the intracellular calcium signaling and cell physiology is often not taken into consideration. However, growing evidence from calcium imaging studies shows GCaMP expression under certain conditions can generate aberrant activity, such as seizures. In this study, we examined the effect of GCaMP6 expression in the dentate gyrus (DG) on epileptogenesis. We found that viral expression of GCaMP6s but not GCaMP6f in the DG induces tonic-clonic seizures several weeks after viral injection. Cell-type specific expression of GCaMP6s revealed the granule cells (GCs) as the key player in GCaMP6s-induced epilepsy. Finally, by using slice electrophysiology, we demonstrated that GCaMP6s expression increases neuronal excitability in the GCs. Together, this study highlights the ability of GCaMP6s in DG-associated epileptogenesis., (© 2024. The Author(s).)

Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (w enzyme /w meat ) for papain with a hydrolysis time of 3.5 h and 0.18% (w enzyme /w meat ) for flavourzyme with a hydrolysis time of 2.8 h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8 ± 1.8 mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6 ± 0.6 mg calcium/g protein with a high association constant of 420 M -1 (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of ─COOH and ─NH 2 groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat., (© 2024 Institute of Food Technologists.)

An experiment was conducted to evaluate the effects of phytase in calcium (Ca) and available phosphorous (avP)-reduced diet on growth performance, body composition, bone health, and intestinal integrity of broilers challenged with Eimeria maxima and Eimeria acervulina. A total of 672 14-day-old male broilers were allocated to a 2 × 4 factorial arrangement with 6 replicates per treatment and 14 birds per replicate. Two factors were Eimeria challenge and 4 dietary treatments: 1) a positive control (PC; 0.84% Ca and 0.42% avP); 2) a negative control (NC; 0.74% Ca and 0.27% avP); 3) NC + 500 FTU/Kg of phytase (NC + 500PHY); and 4) NC + 1,500 FTU/Kg of phytase (NC + 1500PHY). On d 14, birds in the Eimeria-challenged groups received a solution containing 15,000 sporulated oocysts of E. maxima and 75,000 sporulated oocysts of E. acervulina via oral gavage. At 5 d postinoculation (DPI), the challenged birds showed a higher (P < 0.01) FITC-d level than the unchallenged birds. While the permeability of the NC group did not differ from the PC group, the phytase supplementation groups (NC + 500PHY and NC + 1500PHY) showed lower (P < 0.05) serum FITC-d levels compared to the NC group. Interaction effects (P < 0.05) of Eimeria challenge and dietary treatments on feed intake (FI), mucin-2 (MUC2) gene expression, bone ash concentration, and mineral apposition rate (MAR) were observed. On 0 to 6 and 0 to 9 DPI, Eimeria challenge decreased (P < 0.01) body weight (BW), body weight gain (BWG), FI, bone mineral density (BMD), bone mineral content (BMC), bone area, fat free bone weight (FFBW), bone ash weight, bone ash percentage and bone ash concentration; and it showed a higher FCR (P < 0.01) compared to the unchallenged group. The reduction Ca and avP in the diet (NC) did not exert adverse effects on all parameters in birds, and supplementing phytase at levels of 500 or 1,500 FTU/Kg improved body composition, bone mineralization, and intestinal permeability, with the higher dose of 1,500 FTU/Kg showing more pronounced enhancements. There was an observed increase in FI (P < 0.01) when phytase was supplemented at 1,500 FTU/Kg during 0 to 6 DPI. In conclusion, results from the current study suggest that dietary nutrients, such as Ca and avP, can be moderately reduced with the supplementation of phytase, particularly in birds infected with Eimeria spp., which has the potential to save feed cost without compromising growth performance, bone health, and intestinal integrity of broilers., (Copyright © 2024. Published by Elsevier Inc.)

The calcium-binding capacity and osteoblast proliferation and differentiation were studied in Alaska pollock surimi hydrolysate (APSH) using a system that mimics the gastrointestinal digestive system. Evaluation of the calcium absorption-promoting ability of APSH revealed that the best calcium-binding ability was achieved after hydrolysis with a combination of pepsin, α-chymotrypsin, and trypsin, and separation into <3 kDa (APSH-I), 3-5 kDa (APSH-II), 5-10 kDa (APSH-III), and <10 kDa (APSH-IV) fractions. Scanning electron microscopy with energy-dispersive X-ray spectroscopy analysis confirmed that the hydrolysate and calcium ions formed a complex. Comparison of the calcium absorption capacity using Caco-2 cells showed that calcium absorption was promoted by these hydrolysates. Measurement of the osteoblast activation revealed higher alkaline phosphatase activity, collagen synthesis, and mineralization effect for the low-molecular-weight hydrolysate (LMH) than for the other hydrolysates. In addition, LMH promoted the expression of osteocalcin, osteopontin, and bone morphogenetic protein-2 and -4, which are hormones related to bone formation. Expression of the Runx2 transcription factor, which regulates the expression of these hormones, also increased. These results suggest that Alaska pollock surimi protein hydrolysates prepared using a system that mimics gastrointestinal hydrolysis may result in better osteoblast proliferation and bone health than those prepared using other proteases., (© 2024 The Authors. Journal of Food Science published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

The type III secretion system (T3SS) is a key factor for the symbiosis between rhizobia and legumes. In this study, we investigated the effect of calcium on the expression and secretion of T3SS effectors (T3Es) in Sinorhizobium fredii NGR234, a broad host range rhizobial strain. We performed RNA-Seq analysis of NGR234 grown in the presence of apigenin, calcium, and apigenin plus calcium and compared it with NGR234 grown in the absence of calcium and apigenin. Calcium treatment resulted in a differential expression of 65 genes, most of which are involved in the transport or metabolism of amino acids and carbohydrates. Calcium had a pronounced effect on the transcription of a gene (NGR&#95;b22780) that encodes a putative transmembrane protein, exhibiting a 17-fold change when compared to NGR234 cells grown in the absence of calcium. Calcium upregulated the expression of several sugar transporters, permeases, aminotransferases, and oxidoreductases. Interestingly, calcium downregulated the expression of nodABC , genes that are required for the synthesis of nod factors. A gene encoding a putative outer membrane protein (OmpW) implicated in antibiotic resistance and membrane integrity was also repressed by calcium. We also observed that calcium reduced the production of nodulation outer proteins (T3Es), especially NopA, the main subunit of the T3SS pilus. Additionally, calcium mediated the cleavage of NopA into two smaller isoforms, which might affect the secretion of other T3Es and the symbiotic establishment. Our findings suggest that calcium regulates the T3SS at a post-transcriptional level and provides new insights into the role of calcium in rhizobia-legume interactions.

The effect of sound waves (SWs) on plant cells can be considered as important as other mechanical stimuli like touch, wind, rain, and gravity, causing certain responses associated with the downstream signaling pathways on the whole plant. The objective of the present study was to elucidate the response of suspension-cultured tobacco cells (Nicotiana tabacum L. cv Burley 21) to SW at different intensities. The sinusoidal SW (1,000 Hz) was produced through a signal generator, amplified, and beamed to the one layer floating tobacco cells inside a soundproof chamber at intensities of 60, 75, and 90 dB at the plate level for 15, 30, 45, and 60 min. Calibration of the applied SW intensities, accuracy, and uniformity of SW was performed by a sound level meter, and the cells were treated. The effect of SW on tobacco cells was monitored by quantitation of cytosolic calcium, redox status, membrane integrity, wall components, and the activity of wall modifying enzymes. Cytosolic calcium ions increased as a function of sound intensity with a maximum level of 90 dB. Exposure to 90 dB was also accompanied by a significant increase of H2O2 and membrane lipid peroxidation rate but the reduction of total antioxidant and radical scavenging capacities. The increase of wall rigidity in these cells was attributed to an increase in wall-bound phenolic acids and lignin and the activities of phenylalanine ammonia-lyase and covalently bound peroxidase. In comparison, in 60- and 75 dB, radical scavenging capacity increased, and the activity of wall stiffening enzymes reduced, but cell viability showed no changes. The outcome of the current study reveals that the impact of SW on plant cells is started by an increase in cytosolic calcium. However, upon calcium signaling, downstream events, including alteration of H2O2 and cell redox status and the activities of wall modifying enzymes, determined the extent of SW effects on tobacco cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sardari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Deer sinew as a by-product has high collagen and nutritional value. This study focuses on its hydrolysate being used as a calcium carrier to develop functional foods. The chelation mechanism was analyzed by SEM, EDS, UV-vis, FTIR, and fluorescence spectroscopy and zeta potential analysis after using peptide-sequenced deer sinew peptides for chelation with calcium ions. The results showed that the chelation of deer sinew peptides with calcium ions occurs mainly at the O and N atoms of carboxyl, amino and amide bonds. In vitro and in vivo studies revealed that deer sinew peptide-calcium chelate (DSPs-Ca) promoted the proliferation of MC3T3-E1 cells without toxic side effects and increased the alkaline phosphatase activity. The DSPs-Ca group improved the bone microstructure induced by low calcium, as well as up-regulated the expression of genes responsible for calcium uptake in the kidneys, as evidenced by serum markers, bone sections, bone parameters, and gene expression analyses in low-calcium-fed mice. From the above, it can be concluded that DSPs-Ca is expected to be a calcium supplement food for promoting bone health.

As an important functional protein molecule in the human body, human annexin A5 (hAnxA5) is widely found in human cells and body fluids. hAnxA5, the smallest type of annexin, performs a variety of biological functions by reversibly and specifically binding phosphatidylserine (PS) in a calcium-dependent manner and plays an important role in many human physiological and pathological processes. The free state hAnxA5 exists in the form of monomers and usually forms a polymer in a specific self-assembly manner when exerting biological activity. This review systematically discusses the current knowledge and understanding of hAnxA5 from three perspectives: physiopathological relevance, diagnostic value, and therapeutic utility. h AnxA5 affects the occurrence and development of many physiopathological processes. Moreover, hAnxA5 can be used independently or in combination as a biomarker of physiopathological phenomena for the diagnosis of certain diseases. Importantly, based on the properties of hAnxA5, many novel drug candidates have been designed and prepared for application in actual medical practice. However, there are also some gaps and shortcomings in h AnxA5 research. This in-depth study will not only expand the understanding of structural and functional relationships but also promote the application of hAnxA5 in the field of biomedicine.

Colostrum basic protein (CBP) is a trace protein extracted from bovine colostrum. Previous studies have shown that CBP can promote bone cell differentiation and increase bone density. However, the mechanism by which CBP promotes bone activity remains unclear. This study investigated the mechanism of the effect of CBP on bone growth in mice following dietary supplementation of CBP at doses that included 0.015%, 0.15%, 1.5%, and 5%. Compared with mice fed a normal diet, feeding 5% CBP significantly enhanced bone rigidity and improved the microstructure of bone trabeculae. Five-percent CBP intake triggered significant positive regulation of calcium metabolism in the direction of bone calcium accumulation. The expression levels of paracellular calcium transport proteins CLDN2 and CLDN12 were upregulated nearly 1.5-fold by 5% CBP. We conclude that CBP promotes calcium absorption in mice by upregulating the expression of the calcium-transporting paracellular proteins CLND2 and CLND12, thereby increasing bone density and promoting bone growth. Overall, CBP contributes to bone growth by affecting calcium metabolism.

Our study aimed to investigate the effects of different dietary vitamin D (VD) combinations during the grower (1-32 d of age) and feed restriction (33-52 d of age) phases on growth performance. We also evaluated sternal morphology, mineralization, and related genes expression of bone metabolism as well as absorption of calcium and phosphorous in duodenal mucosa and kidney in Pekin ducks. During the grower phase, we used 2 VD regimes (Group A: 3,160 IU/kg VD 3 ; Group B: 400 IU/kg VD 3 + 69 μg/kg 25-OH-D 3 ). Each dietary treatment had 50 replicate pens of 10 ducks per pen. During the feed restriction phase, 30 replicate pens selected from Group A and Group B, repetitively, were redivided into 5 different dietary VD regimes to form a 2 × 5 experimental design. Each group consisted of 6 replicates, each with 10 ducks. During the feed restriction phase, we evaluated 5 different dietary VD combinations were as follows: T1: 2,000 IU/kg VD 3 ; T2: 5,000 IU/kg VD 3 ; T3: 3,620 IU/kg VD 3 + 34.5 μg/kg 25-OH-D 3 ; T4: 2,240 IU/kg VD 3 + 69 μg/kg 25-OH-D 3 ; T5: 1,800 IU/kg VD 3 + 80 μg/kg 25-OH-D 3 ). Results showed that Group B combinations with T5 had a better growth performance and breast meat deposition (P < 0.1). Regardless of 5 dietary VD regimes during the feed restriction phase, Group B significantly increased (P < 0.05) 52 d sternal depth and tended to increase (P < 0.1) 52 d sternal defatted weight, ash content, and phosphate (P) content of ducks. A significant interactive effect (P < 0.05) was observed on the mRNA abundance of DMP1 and Sost1 as well as RANKL/OPG in sternum and of VDR in duodenal mucosa of ducks at 52 d of age between dietary VD combinations during 2 phases. These results indicated that dietary VD regimes during the grower phase could affect the effectiveness of dietary VD regimes during the feed restriction phases; Dietary VD combinations of both phases could affect the genes expression of bone formation and the absorption as well as reabsorption of calcium and phosphorus in duodenum and kidney., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A total of 360 Ross 708 male broiler chicks were used in an 8 treatment and 9 replicate cage study to explore the influence of day-old chick weight on the efficacy of exogenous phytase. Treatments were arranged as a 2 × 4 factorial with the factors being diet (a positive (PC) and negative control (NC) varying in nutrient density fed without or with 2 concentrations of exogenous phytase) and chick weight (light; <38.5 g or heavy; >42 g). Chicks were sourced from the same breeder flock, with light and heavy chicks being selected from the naturally occurring heterogeneity in the population. The diets were corn-soybean meal based and the PC was formulated to meet the nutrient requirements of male broiler chicks. The NC was formulated to contain 120 kcal/kg, 0.5, 0.18, and 0.18% less apparent metabolizable energy (AME), crude protein (CP), calcium (Ca), and phosphorus (P), respectively, than the PC. Amino acid (AA) density in the NC was also reduced in line with the reduction in CP and the manufacturers' recommendations for the effect of phytase on amino acid digestibility. Phytase at either 1,000 FYT/kg or 3,000 FYT/kg was added only to the NC to create a total of 4 diets. Water and mash feed were available ad libitum and were offered to 8 replicate cages, each containing 5 chicks. The experiment was conducted over a period of 3 wk and diets were offered in 2 phases (starter from d 1 to 10 and grower from d 11 to 21). Growth performance was monitored at the end of each diet phase and on d 21 ileal digesta were collected for estimation of apparent digestibility of energy (DE), nitrogen (N), Ca, P, dry matter (DM), and AA. There were no statistically significant interactions between diet and day-old chick weight for any of the measured parameters. Light chicks had significantly lower weight gain (approx. 5%) at both d 10 and d 21 compared with heavy chicks. This effect was principally associated with reduced feed intake and there was no significant effect of chick weight on feed conversion ratio (FCR). Chick weight had no effect on ileal nutrient digestibility. The reduction in nutrient density from the PC to the NC generated a significant reduction in weight gain (around 12%) and a significant increase in FCR (1.68 vs. 1.83). This effect was associated with a significant reduction in ileal DE (approximately 150 kcal/kg) and in the digestibility of several AA. Exogenous phytase significantly increased weight gain, reduced FCR and generated a significant increase in the ileal digestibility of energy, N, P, and several AA. Although chick weight and diet did not interact statistically, heavy chicks benefited more than light chicks from high doses of exogenous phytase across almost all measured end points which was confirmed by regression analysis. In conclusion, light chicks have inferior performance outcomes than heavy chicks principally because of reduced feed intake, but putatively not in digestive capacity per se. Exogenous phytase is effective in improving performance and nutrient digestibility in nutrient deficient diets. The effect of chick weight per se, and also breeder flock age, on the utility of supra-nutritional inclusion concentrations of exogenous phytase warrants further study., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Maintenance of calcium and phosphorus homeostasis in laying hens is crucial for preservation of skeletal integrity and eggshell quality, though physiological regulation of these systems is incompletely defined. To investigate changes in mineral and vitamin D 3 homeostasis during the 24-h egg formation cycle, 32-wk-old commercial laying hens were sampled at 1, 3, 4, 6, 7, 8, 12, 15, 18, 21, 23, and 24 h post-oviposition (HPOP; n ≥ 4). Ovum location and egg calcification stage were recorded, and blood chemistry, plasma vitamin D 3 metabolites, circulating parathyroid hormone (PTH), and expression of genes mediating uptake and utilization of calcium and phosphorus were evaluated. Elevated levels of renal 25-hydroxylase from 12 to 23 HPOP suggest this tissue might play a role in vitamin D 3 25-hydroxylation during eggshell calcification. In shell gland, retinoid-x-receptor gamma upregulation between 6 and 8 HPOP followed by subsequently increased vitamin D receptor indicate that vitamin D 3 signaling is important for eggshell calcification. Increased expression of PTH, calcitonin, and fibroblast growth factor 23 (FGF23) receptors in the shell gland between 18 and 24 HPOP suggest elevated sensitivity to these hormones toward the end of eggshell calcification. Shell gland sodium-calcium exchanger 1 was upregulated between 4 and 7 HPOP and plasma membrane calcium ATPase 1 increased throughout eggshell calcification, suggesting the primary calcium transporter may differ according to eggshell calcification stage. Expression in shell gland further indicated that bicarbonate synthesis precedes transport, where genes peaked at 6 to 7 and 12 to 18 HPOP, respectively. Inorganic phosphorus transporter 1 (P i T-1) expression peaked in kidney between 12 and 15 HPOP, likely to excrete excess circulating phosphorus, and in shell gland between 18 and 21 HPOP. Upregulation of FGF23 receptors and PiT-1 during late eggshell calcification suggest shell gland phosphorus uptake is important at this time. Together, these findings identified potentially novel hormonal pathways involved in calcium and phosphorus homeostasis along with associated circadian patterns in gene expression that can be used to devise strategies aimed at improving eggshell and skeletal strength in laying hens., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The choice of the calcium (Ca) source in pig diets and the addition of formic acid may affect the gastrointestinal inositol phosphate (InsP) degradation and thereby, phosphorus (P) digestibility in pigs. This study assessed the effects of different Ca sources (Ca carbonate, Ca formate), exogenous phytase, and chemical acidification on InsP degradation, nutrient digestion and retention, blood metabolites, and microbiota composition in growing pigs. In a randomized design, 8 ileal-cannulated barrows (24 kg initial BW) were fed 5 diets containing Ca formate or Ca carbonate as the only mineral Ca addition, with or without 1,500 FTU/kg of an exogenous hybrid 6-phytase. A fifth diet was composed of Ca carbonate with phytase but with 8 g formic acid/kg diet. No mineral P was added to the diets. Prececal InsP6 disappearance and P digestibility were lower (P ≤ 0.032) in pigs fed diets containing Ca formate. In the presence of exogenous phytase, InsP5 and InsP4 concentrations in the ileal digesta were lower (P ≤ 0.019) with Ca carbonate than Ca formate. The addition of formic acid to Ca carbonate with phytase diet resulted in greater (P = 0.027) prececal InsP6 disappearance (87% vs. 80%), lower (P = 0.001) InsP5 concentration, and greater (P ≤ 0.031) InsP2 and myo-inositol concentrations in the ileal digesta. Prececal P digestibility was greater (P = 0.004) with the addition of formic acid compared to Ca carbonate with phytase alone. Prececal amino acid (AA) digestibility of some AA was greater with Ca formate compared to Ca carbonate but only in diets with phytase (P ≤ 0.048). The addition of formic acid to the diet with Ca carbonate and phytase increased (P ≤ 0.006) the prececal AA digestibility of most indispensable AA. Exogenous phytase affected more microbial genera in the feces when Ca formate was used compared to Ca carbonate. In the ileal digesta, the Ca carbonate diet supplemented with formic acid and phytase led to a similar microbial community as the Ca formate diets. In conclusion, Ca formate reduced prececal InsP6 degradation and P digestibility, but might be of advantage in regard to prececal AA digestibility in pigs compared to Ca carbonate when exogenous phytase is added. The addition of formic acid to Ca carbonate with phytase, however, resulted in greater InsP6 disappearance, P and AA digestibility values, and changed ileal microbiota composition compared to Ca carbonate with phytase alone., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

This study evaluated the effect of dietary calcium (Ca) levels and deoxynivalenol (DON) contamination on Ca and phosphorus (P) utilization and bone mineralization in piglets. During an initial 13-d depletion phase, 64 piglets (15.7 ± 0.7 kg) received a control (DON-) or DON-contaminated treatment (DON+, 2.7 mg DON/kg) with either a low Ca (Ca-, 0.39%) or normal Ca level (Ca+, 0.65%) with a constant digestible P level (0.40%). A second group of 16 piglets received DON- or DON+ treatments for 9 d for gene expression analysis. During the subsequent 14-d repletion phase, all piglets were fed a Ca+ DON- diet containing 0.65% Ca and 0.35% digestible P without DON. After 5 d of the depletion phase, the absorption of P (DON × Ca; P < 0.05) and Ca was increased by the Ca- (P < 0.01) and DON+ (P < 0.01) diet. After 13 d, feed conversion ratio (P < 0.01) and average daily feed intake (P = 0.06) tended to decrease with the Ca- diet. The bone mineral content (BMC) gain was decreased by Ca, especially with Ca- DON + (DON × Ca, P < 0.05). The P absorption was increased by Ca- DON + (DON × Ca, P < 0.01), although the P retention efficiency was only increased by Ca+ DON + (DON × Ca, P < 0.001). The absorption of Ca was increased by DON+ (P < 0.001), and the Ca efficiency was increased by Ca- DON- (DON × Ca, P < 0.01). After 9 d, the gene expression of intestinal claudin 12 (P < 0.01) and CYP24A1 (P < 0.05), femur cortical RANKL (P < 0.05) and OPG (P = 0.06), and renal calbindin D9K (P < 0.05) and Klotho (P = 0.07) were decreased by DON+. The Ca (P = 0.06) and magnesium (P < 0.01) concentrations were decreased by DON+, and the Ca (P = 0.06) and P digestibility (P < 0.01) were increased. After the repletion phase, Ca- piglets recovered their BMC deficit, but not those receiving DON+ (DON × Ca; P = 0.06). The Ca (P < 0.05) and P (P = 0.06) retention efficiency tended to increase with Ca-. The absorption of Ca and P was increased by Ca- and DON+ (DON × Ca, P < 0.05). The results show that piglets increased their Ca and P utilization efficiency, allowing them to recover the BMC deficit caused by Ca-, but not when the piglets were exposed to DON. Pigs previously receiving Ca-deficient diet with DON still have lower body Ca and P, leading to elevated calcitriol concentrations and enhanced Ca and P intestinal absorption. The fact that DON decreased the expression of genes implicated in Ca intestinal and renal transport and P excretion after 9 d can potentially explain the reduced plasma Ca concentration., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Periparturient hypocalcemia is a complex metabolic disorder that occurs at the onset of lactation because of a sudden irreversible loss of Ca incorporated into colostrum and milk. Some cows are unable to quickly adapt to this demand and succumb to clinical hypocalcemia, commonly known as milk fever, whereas a larger proportion of cows develop subclinical hypocalcemia. The main goal of this study was to identify causative mutations and candidate genes affecting postpartum blood calcium concentration in Holstein cows. Data consisted of blood calcium concentration measured in 2513 Holstein cows on the first three days after parturition. All cows had genotypic information for 79 k SNP markers. Two consecutive rounds of imputation were performed: first, the 2513 Holstein cows were imputed from 79 k to 312 k SNP markers. This imputation was performed using a reference set of 17,131 proven Holstein bulls with 312 k SNP markers. Then, the 2513 Holstein cows were imputed from 312 k markers to whole-genome sequence data. This second round of imputation used 179 Holstein animals from the 1000 Bulls Genome Project as a reference set. Three alternative phenotypes were evaluated: (1) total calcium concentration in the first 24 h postpartum, (2) total calcium concentration in the first 72 h postpartum calculated as the area under the curve; and (3) the recovery of total calcium concentration calculated as the difference in total calcium concentration between 72 and 24 h. The identification of genetic variants associated with these traits was performed using a two-step mixed model-based approach implemented in the R package MixABEL. The most significant variants were located within or near genes involved in calcium homeostasis and vitamin D transport (GC), calcium and potassium channels (JPH3 and KCNK13), energy and lipid metabolism (CA5A, PRORP, and SREBP1), and immune response (IL12RB2 and CXCL8), among other functions. This work provides the foundation for the development of novel breeding and management tools for reducing the incidence of periparturient hypocalcemia in dairy cattle., (© 2023. The Author(s).)

The preservation of liquid semen is pivotal for both industrial livestock production and genetic management/conservation of species with sperm that are not highly cryo-tolerant. Nevertheless, with regard to poultry semen, even brief in vitro storage periods can lead to a notable decline in fertility, despite the in vivo capacity to maintain fertility for several weeks when within the hen's sperm storage tubules. For fertility in sperm, intracellular calcium ions ([Ca 2+ ]i) play a key role in signaling towards modifying energy metabolism. While reducing [Ca 2+ ]i has been found to enhance the preservation of sperm fertility in some mammals, the connection between semen fertility and calcium availability in avian sperm has received limited attention. In this study, we demonstrate that the use of extracellular and intracellular calcium chelators in liquid semen extenders, specifically EGTA and EGTA-AM, has distinct effects on prolonging the fertility of chicken sperm. These results were validated through in vivo fertility tests. Mechanistically, the effects observed were linked to coordination of mitochondrial metabolism and ATP catabolism. Despite both calcium chelators inducing hypoxia, they differentially regulated mitochondrial respiration and ATP accumulation. This regulation was closely linked to a bimodal control of dynein ATPase activity; a direct initial activation with reduction in [Ca 2+ ]i, and subsequent suppression by cytoplasmic acidification caused by lactic acid. These findings not only contribute to advancing poultry liquid semen preservation techniques, but also elucidates biologically relevant mechanisms that may underlie storage within the female reproductive tract in birds., (© 2023. The Author(s).)

Vanadium (V) is an essential mineral element in animals, but excessive V can lead to many diseases, affecting the health of humans and animals. However, the molecular crosstalk between mitochondria-associated endoplasmic reticulum membranes (MAMs) and inflammation under V exposure is still at the exploratory stage. This study was conducted to determine the molecular crosstalk between MAMs and inflammation under V exposure in ducks. In this study, duck hepatocytes were treated with NaVO 3 (0 μM, 100 μM, and 200 μM) and 2-aminoethyl diphenyl borate (2-APB) (IP 3 R inhibitor) alone or in combination for 24 h. The data showed that V exposure-induced cell vacuolization, enlarged intercellular space, and decreased density and viability. Meanwhile, hydrogen peroxide (H 2 O 2 ), malonaldehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and reactive oxygen species (ROS) levels were upregulated under V treatment. In addition, excessive V could lead to a marked reduction in the MAMs structure, destruction of the membrane structure and overload of intracellular Ca 2+ and mitochondrial Ca 2+ . Moreover, V treatment resulted in notable upregulation of the levels of MAMs-relevant factors (IP 3 R, Mfn2, Grp75, MCU, VDAC1) but downregulated the levels of IL-18, IL-1β, and lactate dehydrogenase (LDH) in the cell supernatant. Additionally, it also significantly elevated the levels of inflammation-relevant factors (NLRP3, ASC, caspase-1, MAVS, IL-18, IL-1β, and TXNIP). However, the inhibition of IP 3 R expression attenuated the V-induced variations in the above indicators. Collectively, our results revealed that the maintenance of calcium homeostasis could protect duck hepatocytes from V-induced inflammation injury via MAMs., Competing Interests: DISCLOSURES The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in the present study., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

To reduce P excretion and increase the sustainability of poultry farms, one needs to understand the mechanisms surrounding P metabolism and its close link with Ca metabolism to precisely predict the fate of dietary P and Ca and related requirements for birds. This study describes and evaluates a model developed to estimate the fate of Ca and P consumed by broilers. The Ca and P model relies on three modules: (1) digestion of Ca and P; (2) dynamics of Ca and P in soft tissue and feathers; and (3) dynamics of body ash. Exogenous phytase affects the availability of Ca and P; thus, to predict the absorption of those minerals, the model also accounts for the effect of phytase on Ca and P digestibility. We used a database to estimate the consequences of dietary Ca, P, and phytase over feed intake response. This study followed a four-step process: (1) Ca and P model development and its coupling with a growth broiler model; (2) model behavior assessment; (3) sensitivity analysis to identify the most influential parameters; and (4) external evaluation based on three databases. The proportion of P in body protein and the Ca to P ratio in bone are the most sensitive parameters of P deposition in soft tissue and bone, representing 91 and 99% of the total variation. The external evaluation results indicated that body water and protein had an overall mean square prediction error (rMSPE) of 7.22 and 12.3%, respectively. The prediction of body ash, Ca, and P had an rMSPE of 7.74, 11.0, and 6.56%, respectively, mostly errors of disturbances (72.5, 51.6, and 90.7%, respectively). The rMSPE for P balance was 13.3, 18.4, and 22.8%, respectively, for P retention, excretion, and retention coefficient, with respective errors due to disturbances of 69.1, 99.9, and 51.3%. We demonstrated a mechanistic model approach to predict the dietary effects of Ca and P on broiler chicken responses with low error, including detailed simulations to show the confidence level expected from the model outputs. Overall, this model predicts broilers' response to dietary Ca and P. The model could aid calculations to minimize P excretion and reduce the impact of broiler production on the environment. A model inversion is ongoing that will enable the calculation of Ca and P dietary quantities for a specific objective. This will simplify the use of the model and the feed formulation process., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Carbohydrates increase kidney stone risk and increase urine calcium and magnesium. We hypothesize that the effects of glucose as an allosteric modulator of calcium-sensing receptors may mediate this effect. Six healthy subjects were on a low-sodium diet before consuming 100 g of glucose beverage. Timed fasting (3) and postglucose (6) urine and blood samples were collected every 30 min. Urine composition and serum markers were measured and microvesicular abundance of tubular transport proteins (NHE3, NKCC2, NCC, and TRPV5) were quantified. Postglucose, serum glucose, and insulin rose rapidly with a parallel increase in calcium and magnesium excretion and no change in fractional excretion of sodium. Both serum parathyroid hormone (PTH) and urine TRPV5 fell in the postglucose periods. The rise in the calcium and magnesium excretion likely occurred primarily in the thick ascending limb where they are both under control of the calcium-sensing receptor. The fall in PTH and TRPV5 support the role of glucose as an allosteric modulator of calcium-sensing receptor. NEW & NOTEWORTHY Sugar increases urine calcium and magnesium as well as kidney stone and bone disease risk. Our study provided new insights into the underlying mechanism as we gave healthy subjects an oral glucose load and used newer tools such as fractional excretion of lithium, serum parathyroid hormone, and microvesicular abundance of tubular transport proteins to characterize the mechanism and identify the thick ascending limb with possible calcium-sensing receptor mediation as a likely contributor to this mechanism.

Orai1 is the pore-forming subunit of the Ca 2+ -release activated Ca 2+ channels that mediate store-operated Ca 2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1β, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca 2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1β, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1β support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1β in spheroid forming efficiency and self-renewal in breast cancer stem cells., (© 2023. The Author(s).)

There are only a few studies devoted to the comparative and simultaneous study of the mechanisms of the length-dependent regulation of atrial and ventricular contractility. Therefore, an isometric force-length protocol was applied to isolated guinea pig right atrial (RA) strips and ventricular (RV) trabeculae, with a simultaneous measurement of force (Frank-Starling mechanism) and Ca 2+ transients (CaT) or transmembrane action potentials (AP). Over the entire length-range studied, the duration of isometric contraction, CaT and AP, were shorter in the RA myocardium than in the RV myocardium. The RA myocardium was stiffer than the RV myocardium. With the increasing length of the RA and RV myocardium, the amplitude and duration of isometric contraction and CaT increased, as well as the amplitude and area of the "CaT difference curves" (shown for the first time). However, the rates of the tension development and relaxation decreased. No contribution of AP duration to the heterometric regulation of isometric tension was found in either the RA or RV myocardium of the guinea pig. Changes in the degree of overlap of the contractile proteins of the guinea pig RA and RV myocardium mainly affect CaT kinetics but not AP duration.

Prokineticin 2 (Prok2) is a small protein expressed in a subpopulation of neurons in the suprachiasmatic nucleus (SCN), the primary circadian pacemaker in mammals. Prok2 has been implicated as a candidate output molecule from the SCN to control multiple circadian rhythms. Genetic manipulation specific to Prok2-producing neurons would be a powerful approach to understanding their function. Here, we report the generation of Prok2-tTA knock-in mice expressing the tetracycline transactivator (tTA) specifically in Prok2 neurons and an application of these mice to in vivo recording of Ca 2+ rhythms in these neurons. First, the specific and efficient expression of tTA in Prok2 neurons was verified by crossing the mice with EGFP reporter mice. Prok2-tTA mice were then used to express a fluorescent Ca 2+ sensor protein to record the circadian Ca 2+ rhythm in SCN Prok2 neurons in vivo. Ca 2+ in these cells showed clear circadian rhythms in both light-dark and constant dark conditions, with their peaks around midday. Notably, the hours of high Ca 2+ nearly coincided with the rest period of the behavioral rhythm. These observations fit well with the predicted function of Prok2 neurons as a candidate output pathway of the SCN by suppressing locomotor activity during both daytime and subjective daytime., (© 2023. Springer Nature Limited.)

Competing Interests: The authors disclose no relevant conflicts of interest.

The endoplasmic reticulum (ER) is the largest organelle of the cell, composed of a continuous network of sheets and tubules, and is involved in protein, calcium (Ca 2+ ), and lipid homeostasis. In neurons, the ER extends throughout the cell, both somal and axodendritic compartments, and is highly important for neuronal functions. A third of the proteome of a cell, secreted and membrane-bound proteins, are processed within the ER lumen and most of these proteins are vital for neuronal activity. The brain itself is high in lipid content, and many structural lipids are produced, in part, by the ER. Cholesterol and steroid synthesis are strictly regulated in the ER of the blood-brain barrier protected brain cells. The high Ca 2+ level in the ER lumen and low cytosolic concentration is needed for Ca 2+ -based intracellular signaling, for synaptic signaling and Ca 2+ waves, and for preparing proteins for correct folding in the presence of high Ca 2+ concentrations to cope with the high concentrations of extracellular milieu. Particularly, ER Ca 2+ is controlled in axodendritic areas for proper neurito- and synaptogenesis and synaptic plasticity and remodeling. In this review, we cover the physiologic functions of the neuronal ER and discuss it in context of common neurodegenerative diseases, focusing on pharmacological regulation of ER Ca 2+ Furthermore, we postulate that heterogeneity of the ER, its protein folding capacity, and ensuring Ca 2+ regulation are crucial factors for the aging and selective vulnerability of neurons in various neurodegenerative diseases. SIGNIFICANCE STATEMENT: Endoplasmic reticulum (ER) Ca 2+ regulators are promising therapeutic targets for degenerative diseases for which efficacious drug therapies do not exist. The use of pharmacological probes targeting maintenance and restoration of ER Ca 2+ can provide restoration of protein homeostasis (e.g., folding of complex plasma membrane signaling receptors) and slow down the degeneration process of neurons., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lapmanee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A novel calcium-binding peptide was purified from peanut protein hydrolysate using gel filtration chromatography and identified using HPLC-MS/MS. Its amino acid sequence was determined as Phe-Pro-Pro-Asp-Val-Ala (FPPDVA, named as FA6) with the calcium-binding capacity of 15.67 ± 0.39 mg/g. Then, the calcium chelating characteristics of FPPDVA were investigated using ultraviolet-visible absorption spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, particle size, and zeta potential. The results showed that FPPDVA interacted with calcium ions, the chelation of calcium ions induced FPPDVA to fold and form a denser structure, the calcium-binding sites may mainly involve oxygen atoms from the carboxyl residues of Asp and Ala, and Phe possessed contact energy and carbonyl residues of Val. Microstructure analysis showed that FPPDVA-calcium chelate exhibited a regularly ordered and tightly aggregated sheets or block structures. Additionally, FPPDVA-calcium chelate had good gastrointestinal digestive stability and thermal stability. The results of everted rat intestinal sac and Caco-2 cell monolayer experiments showed that FPPDVA-calcium chelate could promote calcium absorption and transport through the Cav1.3 and TRPV6 calcium channels. These data suggest that FPPDVA-calcium chelate possesses the potential to be developed and applied as calcium supplement.

Aerobic exercise training (AET) has been used to manage heart disease. AET may totally or partially restore the activity and/or expression of proteins that regulate calcium (Ca 2+ ) handling, optimize intracellular Ca 2+ flow, and attenuate cardiac functional impairment in failing hearts. However, the literature presents conflicting data regarding the effects of AET on Ca 2+ transit and cardiac function in rats with heart failure resulting from aortic stenosis (AoS). This study aimed to evaluate the impact of AET on Ca 2+ handling and cardiac function in rats with heart failure due to AoS. Wistar rats were distributed into two groups: control (Sham; n = 61) and aortic stenosis (AoS; n = 44). After 18 weeks, the groups were redistributed into: non-exposed to exercise training (Sham, n = 28 and AoS, n = 22) and trained (Sham-ET, n = 33 and AoS-ET, n = 22) for 10 weeks. Treadmill exercise training was performed with a velocity equivalent to the lactate threshold. The cardiac function was analyzed by echocardiogram, isolated papillary muscles, and isolated cardiomyocytes. During assays of isolated papillary muscles and isolated cardiomyocytes, the Ca 2+ concentrations were evaluated. The expression of regulatory proteins for diastolic Ca 2+ was assessed via Western Blot. AET attenuated the diastolic dysfunction and improved the systolic function. AoS-ET animals presented an enhanced response to post-rest contraction and SERCA2a and L-type Ca 2+ channel blockage compared to the AoS. Furthermore, AET was able to improve aspects of the mechanical function and the responsiveness of the myofilaments to the Ca 2+ of the AoS-ET animals. AoS animals presented an alteration in the protein expression of SERCA2a and NCX, and AET restored SERCA2a and NCX levels near normal values. Therefore, AET increased SERCA2a activity and myofilament responsiveness to Ca 2+ and improved the cellular Ca 2+ influx mechanism, attenuating cardiac dysfunction at cellular, tissue, and chamber levels in animals with AoS and heart failure.

White Roman goose (12-wk-old male, N = 30) carcasses were obtained from a local government-inspected slaughter plant at approximately ∼10-min postmortem. Each carcass was individually sealed in a zip-lock bag and chilled immediately in a water bath at 15°C for 1 h. Both sides of Pectoralis major muscles were excised from each carcass and incubated in 30 mM CaCl 2 or 30 mM EDTA at 15°C for 5 h. After incubation, calcium-incubated and EDTA-incubated breast muscles were vacuum-packaged individually and stored at 5°C for 72 h. Control samples (without CaCl 2 or EDTA incubation) were directly vacuum-packaged and chilled in a water bath at 15°C for 5 h and stored at 5°C for 72 h. Muscle specimens were taken from the left side of breast muscles at 1 h of chilling (∼1-h postmortem) and at 5 h of incubation at 15°C (∼6-h postmortem), as well as 24, 48, and 72 h of aging at 5°C for measuring the activities of calpain-1 and calpain-11 as well as the contents of 80 kDa calpain-1 subunit and desmin. The samples of shear force value and myofibril fragmentation index (MFI) were taken from the right side of breast muscle at 24 h and 72 h of 5°C storage. Our results showed that the decrease of the activities of calpain-1 and calpain-11 and the contents of 80 kDa calpain-1 subunit and desmin was more rapid (P < 0.05) in calcium-incubated samples than in control and EDTA-incubated samples. The shear force was lower, but the MFI was higher in calcium-incubated samples than in control and EDTA-incubated samples (P < 0.05). Therefore, our results suggest that the calpain-mediated proteolysis and tenderization in postmortem goose muscle could be greatly enhanced by combine effects of stepwise chilling with calcium incubation at 15°C and thereafter aging at 5°C. With applying this procedure, commercial slaughter plants may have an alternative way to improve the tenderness of goose meat., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The dietary needs of calcium (Ca) and phosphorus (P) are interdependent, thus accurate evaluation of Ca and P requirements of broilers to support skeleton health and optimal growth is critical. The present study was carried out to investigate the effects of dietary Ca and nonphytate P (NPP) levels and their interactions on growth performance, tibiotarsus characteristics, tibiotarsus metabolism-related enzyme and proteins, and their gene expression of broilers, so as to provide a rational recommendation for Ca and NPP levels in diet. A total of 540 one-day-old Arbor Acres male broilers were randomly allotted to 1 of 15 treatments with 6 replicate cages of 6 birds per cage for each treatment in a completely randomized design involving a 5 × 3 factorial arrangement of treatments (5 levels of Ca × 3 levels of NPP). The birds were fed the corn-soybean meal diet containing 0.60%, 0.70%, 0.80%, 0.90%, or 1.00% Ca and 0.35%, 0.40%, or 0.45% NPP for 21 d. Dietary Ca level affected (P < 0.03) the bone mineral density, bone mineral content (BMC), breaking strength, ash percentage and ash Ca contents in tibia, which showed linear (P < 0.006) responses to dietary Ca levels. Dietary NPP level affected (P < 0.05) tibia BMC, ash percentage, and FGF23 mRNA level. Broilers that received 0.40% and 0.45% NPP had higher (P < 0.04) tibia BMC and ash percentage than those that received 0.35% NPP, but no differences (P > 0.05) were found between 0.40% and 0.45% NPP. Broilers that received 0.40% NPP had higher (P = 0.02) tibia FGF23 mRNA level than those that received 0.35% NPP, but no differences (P > 0.05) were detected between 0.40% and 0.45% NPP or 0.45% and 0.35% NPP. The interactions between dietary Ca and NPP affected (P < 0.05) ADG, ALP activity, bone gal protein, FGF23 contents, and the mRNA expression levels ALP and bone gal protein in tibia of broilers. Results from the present study indicate that dietary Ca and NPP interaction influences growth, tibiotarsus development, and related gene expression of broiler chickens. Considering all the criteria, the dietary levels of 0.90% Ca and 0.45% NPP would be optimal for both growth and tibiotarsus development of broilers fed a conventional corn-soybean meal diet from 1 to 21 d of age., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cadmium (Cd) contamination in rice grains is posing a significant threat to global food security. To restrict the transport of Cd in the soil-rice system, an efficient way is to use the ionomics strategy. Since calcium (Ca) and Cd have similar ionic radii, their uptake and translocation may be linked in multiple aspects in rice. However, the underlying antagonistic mechanisms are still not fully understood. Therefore, we first summarized the current knowledge on the physiological and molecular footprints of Cd translocation in plants and then explored the potential antagonistic points between Ca and Cd in rice, including exchange adsorption on roots, plant cell-wall composition, co-transporter gene expression, and transpiration inhibition. This review provides suggestions for Ca/Cd interaction studies on rice and introduces ionomics research as a means of better controlling the accumulation of Cd in plants.

Ninjurin 1 (NINJ1) is a double-transmembrane cell-surface protein that might mediate plasma membrane rupture (PMR) and the diffusion of inflammatory factors. PMR is a characteristic of acinar cell injury in severe acute pancreatitis (SAP). However, the involvement of NINJ1 in mediating the PMR of acinar cells in SAP is currently unclear. Our study has shown that NINJ1 is expressed in acinar cells, and the expression is significantly upregulated in sodium-taurocholate-induced SAP. The knockout of NINJ1 delays PMR in acinar cells and alleviates SAP. Moreover, we observed that NINJ1 expression is mediated by Ca 2+ concentration in acinar cells. Importantly, we found that Ca 2+ overload drives mitochondrial stress to upregulate the P53/NINJ1 pathway, inducing PMR in acinar cells, and amlodipine, a Ca 2+ channel inhibitor, can reduce the occurrence of PMR by decreasing the concentration of Ca 2+ . Our results demonstrate the mechanism by which NINJ1 induces PMR in SAP acinar cells and provide a potential new target for treatment of SAP.

Background: Sporadic multiple parathyroid gland disease is ¼ cases of primary hyperparathyroidism (PHPT). However, a single tactic for diagnosing and operating volume in patients with this variant of PHPT has not yet been developed. One of the possible directions in the search for pathogenetically substantiated methods of diagnosis and treatment is the study of the molecular genetic features of the disease and associated clinical and laboratory factors., Aim: To study the features of the expression of calcium sensitive (CaSR) and vitamin D (VDR) receptors on the surface of parathyroid cells in primary hyperparathyroidism with solitary and multiple lesions of the parathyroid glands, as well as its changes under the influence of a decrease in the filtration function of the kidneys., Materials and Methods: In a single center observational prospective study with retrospective data collection, there were patients who during 2019-2021. operated on for PHPT, secondary hyperparathyroidism (SHPT) and all cases of tertiary hyperparathyroidism (THPT) operated during 2014-2021. The expression of CaSR, VDR and its relationship with the main laboratory parameters, the clinical variant of hyperparathyroidism, and the morphological substrate were studied., Results: The study included 69 patients: 19 with multiple and 25 with solitary PTG near PHPT, 15 with SHPT, 10 with THPT. A statistically significant decrease in the frequency of detection of normal expression of CaSR and VDR receptors occurs in any morphological variant of hyperparathyroidism and is observed in 93-60% of drugs. A decrease in the normal expression of CaSR in hyperplasia is detected statistically significantly less frequently than in adenoma (p≤0.01). The median expression intensity in adenoma was 2.5 (2:3), in hyperplasia 3.5 (3-4) (p≤0.01). The difference in the molecular mechanisms of the development of hyperparathyroidism with a predominance of a morphological substrate in the form of adenoma (PHPT with solitary adenoma) or hyperplasia (SHPT and PHPT with multiple PTG lesions) is realized in the frequency of maintaining normal CaSR expression in the PTG tissue. These mechanisms are implemented at the local level, their variability does not change under the influence of RRT. A common molecular genetic mechanism for the development of hyperparathyroidism with a predominance of a morphological substrate in the form of adenoma or hyperplasia has been found to reduce the frequency of maintaining normal VDR expression in PTG (up to 7-13%), p&lt;0.01. This mechanism is implemented at the local level, its variability changes under the influence of RRT, reaching statistically significant differences in patients with THPT., Conclusion: The study demonstrates the features of changes in the expression of CaSR and VDR in PHPT with multiple lesions of the parathyroid glands. The relationship between the expression of these receptors and the clinical variant of hyperparathyroidism, the morphological substrate, the main laboratory parameters, and renal function was shown.

A higher concentration of calcium in breast milk than blood favors paracellular calcium absorption enabling growth during postnatal development. We aimed to determine whether suckling animals have greater intestinal calcium permeability to maximize absorption and to identify the underlying molecular mechanism. We examined intestinal claudin expression at different ages in mice and in human intestinal epithelial (Caco-2) cells in response to hormones or human milk. We also measured intestinal calcium permeability in wildtype, Cldn2 and Cldn12 KO mice and Caco-2 cells in response to hormones or human milk. Bone mineralization in mice was assessed by μCT. Calcium permeability across the jejunum and ileum of mice were 2-fold greater at 2 wk than 2 mo postnatal age. At 2 wk, Cldn2 and Cldn12 expression were greater, but only Cldn2 KO mice had decreased calcium permeability compared to wildtype. This translated to decreased bone volume, cross-sectional thickness, and tissue mineral density of femurs. Weaning from breast milk led to a 50% decrease in Cldn2 expression in the jejunum and ileum. Epidermal growth factor (EGF) in breast milk specifically increased only CLDN2 expression and calcium permeability in Caco-2 cells. These data support intestinal permeability to calcium, conferred by claudin-2, being greater in suckling mice and being driven by EGF in breast milk. Loss of the CLDN2 pathway leads to suboptimal bone mineralization at 2 wk of life. Overall, EGF-mediated control of intestinal claudin-2 expression contributes to maximal intestinal calcium absorption in suckling animals., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Physiological Society.)

Genetically encoded biosensors based on fluorescent proteins (FPs) are widely used to monitor dynamics and sub-cellular spatial distribution of calcium ion (Ca2+) fluxes and their role in intracellular signaling pathways. The development of different mutations in the Ca2+-sensitive elements of the cameleon probes has allowed sensitive range of Ca2+ measurements in almost all cellular compartments. Region of the endoplasmic reticulum (ER) tethered to mitochondria, named as the mitochondrial-associated membranes (MAMs), has received an extended attention since the last 5 years. Indeed, as MAMs are essential for calcium homeostasis and mitochondrial function, molecular tools have been developed to assess quantitatively Ca2+ levels in the MAMs. However, sensitivity of the first generation Ca2+ biosensors on the surface of the outer-mitochondrial membrane (OMM) do not allow to measure μM or sub-μM changes in Ca2+ concentration which prevents to measure the native activity (unstimulated exogenously) of endogenous channels. In this study, we assembled a new ratiometric highly sensitive Ca2+ biosensor expressed on the surface of the outer-mitochondrial membrane (OMM). It allows the detection of smaller differences than the previous biosensor in or at proximity of the MAMs. Noteworthy, we demonstrated that IP3-receptors have an endogenous activity which participate to the Ca2+ leak channel on the surface of the OMM during hypoxia or when SERCA activity is blocked., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gouriou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Calcium oxalate (CaOx) crystals in plants act as a sink for excess Ca and play an essential role in detoxifying heavy metals (HMs). However, the mechanism and related influencing factors remain unclear. Amaranth (Amaranthus tricolor L.) is a common edible vegetable rich in CaOx and a potential Cd hyperaccumulation species. In this study, the hydroponic experiment was carried out to investigate the effect of exogenous Ca concentrations on Cd uptake by amaranth. The results showed that either insufficient or excess Ca supply inhibited amaranth growth, while the Cd bioconcentration factor (BCF) increased with Ca concentration. Meanwhile, the sequence extraction results demonstrated that Cd mainly accumulated as pectate and protein-bound species (NaCl extracted) in the root and stem, compared to pectate, protein, and phosphate-bound (acetic acid extractable) species in the leaf. Correlation analysis showed that the concentration of exogenous Ca was positively correlated with amaranth-produced CaOx crystals but negatively correlated with insoluble oxalate-bound Cd in the leaf. However, since the accumulated insoluble oxalate-bound Cd was relatively low, Cd detoxification via the CaOx pathway in amaranth is limited., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A novel calcium-binding peptide from bovine bone collagen hydrolysate was screened based on a new target-the calcium-sensing receptor (CaSR), and its chelation mechanism and calcium absorption activity were investigated. Glu-Tyr-Gly exhibited superior binding affinities to CaSR because of its interaction with the active sites of the CaSR Venus Flytrap (VFT) domain. Glu-Tyr-Gly-Ca may exist in five potential chelation modes and its potential chelation mechanism was that calcium ions were located in the center and surrounded by ionic bonds (carboxyl group) and coordination bonds (carbonyl, amino, and carboxyl group). Glu-Tyr-Gly-Ca was slightly damaged in the intestinal fluid and at different temperatures, whereas it was severely damaged in the gastric fluid and acidic conditions. The results of the calcium dialysis percentage and Caco-2 cells experiments showed that Glu-Tyr-Gly-Ca possessed good calcium transport activity and bioavailability. The findings provided theoretical basis for Glu-Tyr-Gly-Ca as potential calcium supplement to improve intestinal calcium absorption., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Twelve multiparous Holstein cows (42.2 ± 5.6 kg of milk/d; 83 ± 27 d in milk) were used in a split-plot design testing the effects of mineral and vitamin supplementation on the time course of animal performance, metabolism, and inflammation markers during heat stress. The main plot was the average concentrations of dietary vitamin E and Se (adequate: 11.1 IU/kg of vitamin E and 0.55 mg/kg of Se, and high: 223 IU/kg of vitamin E and 1.8 mg/kg of Se, respectively). Within each plot, cows were randomly assigned to (1) heat stress (HS) with adequate concentrations of vitamin D 3 and Ca (1,012 IU/kg and 0.73%, respectively), (2) HS with high concentrations of vitamin D 3 and Ca (HS+D 3 /Ca; 3,764 IU/kg and 0.97%, respectively), or (3) pair-feeding (PF) in thermoneutrality with adequate concentrations of vitamin D 3 and Ca (1,012 IU/kg and 0.73% Ca) in a Latin square design with 14-d periods and 7-d washouts. The highest rectal temperature was recorded at 1700 h for HS (39.4°C; mean of d 1 to 14), being 1.2 and 0.8°C greater than for PF and HS+D 3 /Ca, respectively. Respiratory rate and water intake were higher in HS (73 breaths/min and 115 L/d, respectively) relative to PF (28 breaths/min and 76 L/d). Heat stress decreased dry matter intake progressively, reaching a nadir on d 5 to 7 (33% reduction) and was not different between treatments. Milk yield decreased progressively in all treatments, but remained greater in PF relative to HS from d 3 to 14 (10%), whereas HS and HS+D 3 /Ca were not different. Milk fat, protein, and lactose concentrations and yields were lower in HS relative to PF from d 3 to 14, but not different between HS and HS+D 3 /Ca. Relative to PF, preprandial insulin concentrations were increased in HS, whereas plasma nonesterified fatty acids were decreased on d 7 and 14. Plasma lipopolysaccharide-binding protein concentrations increased in HS cows on d 7 and 14, respectively, relative to PF, whereas they were reduced in HS + D 3 /Ca on d 14. Plasma C-reactive protein, tumor necrosis factor-α, and fecal calprotectin were increased in HS relative to both PF and HS+D 3 /Ca on d 7 and 14. Rectal temperature was positively associated with plasma lipopolysaccharide-binding protein (r = 0.72), tumor necrosis factor-α (r = 0.74), C-reactive protein (r = 0.87), and with milk somatic cells (r = 0.75). Plasma 8-hydroxy-2-deoxyguanosine concentrations presented a 3-way interaction, where 8-hydroxy-2-deoxyguanosine was lower in HS than in PF on d 7 and 14, and lower in HS+D 3 /Ca relative to HS on d 14 in the adequate vitamin E and Se treatment, but no effects were observed in the high vitamin E and Se group. Plasma superoxide dismutase concentrations increased over time, and were higher in HS relative to PF on d 14, whereas HS+D 3 /Ca was similar to HS. Heat stress markedly reduced milk production and milk components while increasing markers of leaky gut and inflammation. In contrast, vitamin D 3 and Ca supplementation reduced hyperthermia (d 7-14), markers of leaky gut, and inflammation independent of dietary concentrations of vitamin E and Se., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Calpain-3 (CAPN3) is a muscle-specific member of the calpain family of Ca 2+ -dependent proteases. It has been reported that CAPN3 can also be autolytically activated by Na + ions in the absence of Ca 2+ , although this was only shown under non-physiological ionic conditions. Here we confirm that CAPN3 does undergo autolysis in the presence of high [Na + ], but this only occurred if all K + normally present in a muscle cell was absent, and it did not occur even in 36 mM Na + , higher than what would ever be reached in exercising muscle if normal [K + ] was present. CAPN3 in human muscle homogenates was autolytically activated by Ca 2+ , with ~50% CAPN3 autolysing in 60 min in the presence of 2 µM Ca 2+ . In comparison, autolytic activation of CAPN1 required about 5-fold higher [Ca 2+ ] in the same conditions and tissue. After it was autolysed, CAPN3 unbound from its tight binding on titin and became diffusible, but only if the autolysis led to complete removal of the IS1 inhibitory peptide within CAPN3, reducing the C-terminal fragment to 55 kDa. Contrary to a previous report, activation of CAPN3, either by raised [Ca 2+ ] or Na + treatment, did not cause proteolysis of the skeletal muscle Ca 2+ release channel-ryanodine receptor, RyR1, in physiological ionic conditions. Treatment of human muscle homogenates with high [Ca 2+ ] caused autolytic activation of CAPN1, accompanied by proteolysis of some titin and complete proteolysis of junctophilin (JP1, full length ~95 kDa), generating an equimolar amount of a diffusible ~75 kDa N-terminal JP1 fragment, but without any proteolysis of RyR1.

Strontium (Sr) belongs to the same group in the periodic table as calcium (Ca). Sr level can serve as an index of rumen Ca absorption capacity; however, the effects of Sr on Ca 2+ metabolism are unclear. This study aims to investigate the effect of Sr on Ca 2+ metabolism in bovine rumen epithelial cells. The bovine rumen epithelial cells were isolated from the rumen of newborn Holstein male calves ( n = 3, 1 day old, 38.0 ± 2.8 kg, fasting). The half maximal inhibitory concentration (IC50) of Sr-treated bovine rumen epithelial cells and cell cycle were used to establish the Sr treatment model. Transcriptomics, proteomics, and network pharmacology were conducted to investigate the core targets of Sr-mediated regulation of Ca 2+ metabolism in bovine rumen epithelial cells. The data of transcriptomics and proteomics were analyzed using bioinformatic analysis (Gene Ontology and Kyoto Encyclopedia of genes/protein). Quantitative data were analyzed using one-way ANOVA in GraphPad Prism 8.4.3 and the Shapiro-Wilk test was used for the normality test. Results presented that the IC50 of Sr treatment bovine rumen epithelial cells for 24 h was 43.21 mmol/L, and Sr increased intracellular Ca 2+ levels. Multi-omics results demonstrated the differential expression of 770 mRNAs and 2436 proteins after Sr treatment; network pharmacology and reverse transcriptase polymerase chain reaction (RT-PCR) revealed Adenosylhomocysteine hydrolase-like protein 2 (AHCYL2) , Semaphoring 3A (SEMA3A) , Parathyroid hormone-related protein (PTHLH) , Transforming growth factor β 2 ( TGF-β2) , and Cholesterol side-chain cleavage enzyme (CYP11A1) as potential targets for Sr-mediated Ca 2+ metabolism regulation. Together these results will improve the current comprehension of the regulatory effect of Sr on Ca 2+ metabolism and pave a theoretical basis for Sr application in bovine hypocalcemia.

In a recent study, visual signals were recorded for the first time in starburst amacrine cells of the macaque retina, and, as for mouse and rabbit, a directional bias observed in calcium signals was recorded from near the dendritic tips. Stimulus motion from the soma toward the tip generated a larger calcium signal than motion from the tip toward the soma. Two mechanisms affecting the spatiotemporal summation of excitatory postsynaptic currents have been proposed to contribute to directional signaling at the dendritic tips of starbursts: (1) a "morphological" mechanism in which electrotonic propagation of excitatory synaptic currents along a dendrite sums bipolar cell inputs at the dendritic tip preferentially for stimulus motion in the centrifugal direction; (2) a "space-time" mechanism that relies on differences in the time-courses of proximal and distal bipolar cell inputs to favor centrifugal stimulus motion. To explore the contributions of these two mechanisms in the primate, we developed a realistic computational model based on connectomic reconstruction of a macaque starburst cell and the distribution of its synaptic inputs from sustained and transient bipolar cell types. Our model suggests that both mechanisms can initiate direction selectivity in starburst dendrites, but their contributions differ depending on the spatiotemporal properties of the stimulus. Specifically, the morphological mechanism dominates when small visual objects are moving at high velocities, and the space-time mechanism contributes most for large visual objects moving at low velocities.

Locomotion triggers a coordinated response of both neurons and astrocytes in the brain. Here we performed calcium (Ca 2+ ) imaging of these two cell types in the somatosensory cortex in head-fixed mice moving on the airlifted platform. Ca 2+ activity in astrocytes significantly increased during locomotion from a low quiescence level. Ca 2+ signals first appeared in the distal processes and then propagated to astrocytic somata, where it became significantly larger and exhibited oscillatory behaviour. Thus, astrocytic soma operates as both integrator and amplifier of Ca 2+ signal. In neurons, Ca 2+ activity was pronounced in quiescent periods and further increased during locomotion. Neuronal Ca 2+ concentration ([Ca 2+ ] i ) rose almost immediately following the onset of locomotion, whereas astrocytic Ca 2+ signals lagged by several seconds. Such a long lag suggests that astrocytic [Ca 2+ ] i elevations are unlikely to be triggered by the activity of synapses among local neurons. Ca 2+ responses to pairs of consecutive episodes of locomotion did not significantly differ in neurons, while were significantly diminished in response to the second locomotion in astrocytes. Such astrocytic refractoriness may arise from distinct mechanisms underlying Ca 2+ signal generation. In neurons, the bulk of Ca 2+ enters through the Ca 2+ channels in the plasma membrane allowing for steady-level Ca 2+ elevations in repetitive runs. Astrocytic Ca 2+ responses originate from the intracellular stores, the depletion of which affects subsequent Ca 2+ signals. Functionally, neuronal Ca 2+ response reflects sensory input processed by neurons. Astrocytic Ca 2+ dynamics is likely to provide metabolic and homeostatic support within the brain active milieu., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. A.V. holds the position of Editorial Board Member for Function and was blinded from reviewing or making decisions for the manuscript., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Physiological Society.)

Functional magnetic resonance imaging relies on the coupling between neuronal and vascular activity, but the mechanisms behind this coupling are still under discussion. Recent experimental evidence suggests that calcium signaling may play a significant role in neurovascular coupling. However, it is still controversial where this calcium signal is located (in neurons or elsewhere), how it operates and how relevant is its role. In this paper we introduce a biologically plausible model of the neurovascular coupling and we show that calcium signaling in astrocytes can explain main aspects of the dynamics of the coupling. We find that calcium signaling can explain so-far unrelated features such as the linear and non-linear regimes, the negative vascular response (undershoot) and the emergence of a (calcium-driven) Hemodynamic Response Function. These features are reproduced here for the first time by a single model of the detailed neuronal-astrocyte-vascular pathway. Furthermore, we analyze how information is coded and transmitted from the neuronal to the vascular system and we predict that frequency modulation of astrocytic calcium dynamics plays a key role in this process. Finally, our work provides a framework to link neuronal activity to the BOLD signal, and vice-versa, where neuronal activity can be inferred from the BOLD signal. This opens new ways to link known alterations of astrocytic calcium signaling in neurodegenerative diseases (e.g. Alzheimer's and Parkinson's diseases) with detectable changes in the neurovascular coupling., (© 2023. The Author(s).)

Musculoskeletal disorders represent one of the main causes of disability worldwide, and their prevalence is predicted to increase in the coming decades. Stem cell therapy may be a promising option for the treatment of some of the musculoskeletal diseases. Although significant progress has been made in musculoskeletal stem cell research, osteoarthritis, the most-common musculoskeletal disorder, still lacks curative treatment. To fine-tune stem-cell-based therapy, it is necessary to focus on the underlying biological mechanisms. Ion channels and the bioelectric signals they generate control the proliferation, differentiation, and migration of musculoskeletal progenitor cells. Calcium- and voltage-activated potassium (K Ca ) channels are key players in cell physiology in cells of the musculoskeletal system. This review article focused on the big conductance (BK) K Ca channels. The regulatory function of BK channels requires interactions with diverse sets of proteins that have different functions in tissue-resident stem cells. In this narrative review article, we discuss the main ion channels of musculoskeletal stem cells, with a focus on calcium-dependent potassium channels, especially on the large conductance BK channel. We review their expression and function in progenitor cell proliferation, differentiation, and migration and highlight gaps in current knowledge on their involvement in musculoskeletal diseases.