High Calcium Conditions Research Articles

Remediation characteristics and effects of electrokinetic-citric acid system on karst soil contaminated by arsenic and cadmium

The features and method of citric acid's remediation on arsenic and cadmium-contaminated soil under electrification were investigated by modeling arsenic and cadmium-contaminated soil in karst areas. According to the experimental findings, arsenic and cadmium in soil dissolve more effectively at higher citric acid concentrations. After being electrified, it can encourage the citric acid system, which breaks down cadmium and arsenic in soil. In soil, oxidizable and acid extractable arsenic are removed at rates of 89.19 % and 48.97 %, respectively, and oxidizable and acid extractable cadmium are removed at rates of 60.88 % and 87.87 %. Alkaline circumstances are also favorable for the desorption and migration of arsenic in karst environments; high calcium conditions, however, will inhibit the dissolution of arsenic while favoring the dissolution of cadmium. The morphological alterations of heavy metals during the electro-enhanced remediation process were examined using Visual MINTEQ software. The findings demonstrated that throughout the process, cadmium predominately existed in the forms of Cd2+ and Cd-Citrate-, whereas arsenic predominately existed in the forms of H2AsO4- and H3AsO4 in acidic environment and HAsO42- and H2AsO4- in alkaline conditions. In summary, electric citric acid plays a crucial role in removing cadmium and arsenic pollution in karst soil, which is of great significance for the remediation of soil contaminated by cadmium and arsenic compounds in karst environments.

Defining the structure and orientation of the N-terminal domain of cardiac troponin T in relationship to actin.

Cardiac troponin T (cTnT) serves as the tropomyosin (Tm) binding subunit of the troponin complex and, when mutated, it often leads to pathologic cardiac remodeling observed in patients with hypertrophic and dilated cardiomyopathies. Part of the N-terminus of cTnT is responsible for positioning Tm along the actin groove and it immediately flanks the Tm overlap domain. Understanding the molecular mechanisms underlying these observations has been hindered by the absence of a high-resolution structure in the highly flexible N-terminal domain. A recently published cryo-electron-microscopy structure shows interactions between the N-terminus of cTnT, around amino acid 87, and the F-actin backbone; however, this interaction was not observed in calcium saturated conditions. Here, we employ single-donor dual-acceptor time-resolved fluorescence resonance energy transfer (TR-FRET) to probe the positioning of the cTnT N-terminal extension in fully reconstituted cardiac thin filaments (CTF). We hypothesize the N-terminal tail interacts with F-actin in the −Ca2+ condition aiding in stabilizing Tm in the blocked position. Conversely, we hypothesize that the that the N-terminal tail is “fly-casting” and not interacting with F-actin in +Ca2+ condition. To test this, a donor probe (IAEDANS) was placed on either the Cys-substituted residues N100C, N73C and M60C of cTnT and an acceptor probe (DABMI) was placed on the endogenous Cys-374 of actin. Preliminary data revealed two measurable discrete distances for all residues, one short and one long, indicating that FRET occurred with two actin monomers. Initial results suggest that the N-terminal tail is interacting with F-actin in high and low calcium conditions. Continuing work aims to probe additional sites on the N-terminus, specifically A20C and A2C. Resolving the structure and orientation of the N-terminal domain of cTnT is critical to understanding the regulatory mechanisms of the CTF.

Characterizations of annexin A1-interacting proteins in apical membrane and cytosolic compartments of renal tubular epithelial cells

Annexin A1 (ANXA1) is a multifunctional calcium-binding protein that can bind to membrane phospholipids. Under high-calcium condition, ANXA1 expression increases on renal epithelial cell surface, leading to enhanced adhesion of calcium oxalate (CaOx) crystal (stone material) onto the cells. To regulate various cellular processes, ANXA1 interacts with many other intracellular protein partners. However, components of the ANXA1-interacting protein complex remain unclear. Herein, we characterized the interacting complexes of apical membrane (ApANXA1) and cytosolic (cyANXA1) forms of ANXA1 in apical membrane and cytosolic compartments, respectively, of renal epithelial cells under high-calcium condition using proteomic and bioinformatic approaches. After fractionation, the ApANXA1- and CyANXA1-interacting partners were identified by immunoprecipitation followed by nanoLC‑ESI‑Qq-TOF tandem mass spectrometry (IP-MS/MS). The ANXA1-interacting partners that were common in both apical membrane and cytosolic compartments and those unique in each compartment were then analyzed for their physico-chemical properties (molecular weight, isoelectric point, amino acid contents, instability index, aliphatic index, and grand average of hydropathicity), secondary structure (α-helix, β-turn, random coil, and extended strand), molecular functions, biological processes, reactome pathways and KEGG pathways. The data demonstrated that each set of these interacting proteins exhibited common and unique characteristics and properties. The knowledge from this study may lead to better understanding of the ApANXA1 and CyAXNA1 biochemistry and functions as well as the pathophysiology of CaOx kidney stone formation induced by high-calcium condition.

Pinoresinol stimulates keratinocyte proliferation and downregulates TNF-α secretion in peripheral blood mononuclear cells: An experimental in vitro study.

Natural coniferous resins are used in traditional medicine for the treatment of skin wounds. Coniferous wood resins ("callus" resin) are a mixture of abietic (resin) acids, lignans such as pinoresinol, and p-coumaric acid. The wound-healing properties of resins are thought to be related to their antimicrobial properties, but also to their effects on cell proliferation and inflammation. The purpose of this study was to identify and investigate the effects of novel aqueous dispersions of resin and its main components in the proliferation of human primary keratinocytes in vitroand in the expression of proinflammatory cytokines in human peripheral blood mononuclear cells. The proliferation studies were performed under low and high calcium conditions with or without added growth stimulators at the time points of 2and 6 days using AlamarBlue Cell Viability Reagent. The cytokine release assay was carried out by incubating the cells with the test articles for 18 h, after which the levels of tumor necrosis factor-α(TNF-α),interleukin-1β (IL-1β), IL-6, and IL-8 were measured in the supernatant by enzyme-linked immunosorbent assay. Resin and the purified lignan PINO, but not p-coumaric acid or abietic acid (industrial tall oil rosin), enhanced the proliferation of human keratinocytes in vitroand inhibited the expression of TNF-α, and to a lesser extent the expression of IL-1β in peripheral blood mononuclear cells. In this study, novel aqueous dispersions of spruce resin were used to investigate the effects of main resin components on keratinocyte proliferation and on the expression of key proinflammatory cytokines known to be associated with chronic wounds. The observations suggest that lignans, such as PINO, but not resin acids, are the components of resins that mediate the proliferative and TNF-α-suppressing effects. Lignans including PINO were identified as novel potential compounds in the treatment of chronic skin ulcers.

Extracellular Matrix Profiling and Disease Modelling in Engineered Vascular Smooth Muscle Cell Tissues

Aortic smooth muscle cells (SMCs) have an intrinsic role in regulating vessel homeostasis and pathological remodelling. In two-dimensional (2D) cell culture formats, however, SMCs are not embedded in their physiological extracellular matrix (ECM) environment. To overcome the limitations of conventional 2D SMC cultures, we established a 3D in vitro model of engineered vascular smooth muscle cell tissues (EVTs). EVTs were casted from primary murine aortic SMCs by suspending a SMC-fibrin master mix between two flexible silicon-posts at day 0 before prolonged culture up to 14 days. Immunohistochemical analysis of EVT longitudinal sections demonstrated that SMCs were aligned, viable and secretory. Mass spectrometry-based proteomics analysis of murine EVT lysates was performed and identified 135 matrisome proteins. Proteoglycans, including the large aggregating proteoglycan versican, accumulated within EVTs by day 7 of culture. This was followed by the deposition of collagens, elastin-binding proteins and matrix regulators up to day 14 of culture. In contrast to 2D SMC controls, accumulation of versican occurred in parallel to an increase in versikine, a cleavage product mediated by proteases of the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family. Next, we tested the response of EVTs to stimulation with transforming growth factor beta-1 (TGFβ-1). EVTs contracted in response to TGFβ-1 stimulation with altered ECM composition. In contrast, treatment with the pharmacological activin-like kinase inhibitor (ALKi) SB 431542 suppressed ECM secretion. As a disease stimulus, we performed calcification assays. The ECM acts as a nidus for calcium phosphate deposition in the arterial wall. We compared the onset and extent of calcification in EVTs and 2D SMCs cultured under high calcium and phosphate conditions for 7 days. Calcified EVTs displayed increased tissue stiffness by up to 30 % compared to non-calcified controls. Unlike the rapid calcification of SMCs in 2D cultures, EVTs sustained expression of the calcification inhibitor matrix Gla protein and allowed for better discrimination of the calcification propensity between independent biological replicates. In summary, EVTs are an intuitive and versatile model to investigate ECM synthesis and turnover by SMCs in a 3D environment. Unlike conventional 2D cultures, EVTs provide a more relevant pathophysiological model for retention of the nascent ECM produced by SMCs.

Emodin alleviates aortic valvular calcification by inhibiting the AKT/FOXO1 pathway

BackgroundValvular calcification commonly occurs in elderly individuals, and is increasingly considered an important economic and health burden. However, no efficient drugs against valvular calcification are available. The present work aimed to examine emodin’s suppressive effect on high-calcium–dependent valve calcification and explore the underpinning mechanisms. MethodsExperiments were carried out in mice receiving vitamin D (Vit D) to induce valvular calcification. Results: Cell viability and apoptosis assays demonstrated celastrol suppressed proliferation and increased apoptosis in porcine aortic valve interstitial cells (PAVICs) at concentrations higher than 10 μM. Emodin (5 μM) attenuated the upregulation of osteogenic genes as well as calcium accumulation in PAVICs under high-calcium conditions. The elevations of calcium content in serum and valve, and calcium accumulation in valve and artery were suppressed by emodin in mice with valvular calcification after joint treatment with adenine and Vit D. In addition, p-AKT and p-FOXO1 were upregulated in PAVICs under high-calcium conditions, and this effect was reversed by emodin treatment. SC79, an AKT activator, reversed emodin’s suppressive effects on increased calcium content, calcium deposition and osteogenic gene expression in PAVICs induced by calcific medium. ConclusionsThese data demonstrated emodin alleviates high-calcium–associated valvular calcification via AKT/FOXO1 signaling suppression, providing new insights into therapeutic strategies for clinical valvular calcification.

Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection

Annexin A11 was identified as autoantigen in IgG4-related cholangitis (IRC), a B-cell driven disease. Annexin A11 modulates calcium-dependent exocytosis, a crucial mechanism for insertion of proteins into their target membranes. Human cholangiocytes form an apical 'biliary bicarbonate umbrella' regarded as defense against harmful hydrophobic bile acid influx. The bicarbonate secretory machinery comprises the chloride/bicarbonate exchanger AE2 and the chloride channel ANO1. We aimed to investigate the expression and function of annexin A11 in human cholangiocytes and a potential role of IgG1/IgG4-mediated autoreactivity against annexin A11 in the pathogenesis of IRC. Expression of annexin A11 in human liver was studied by immunohistochemistry and immunofluorescence. In human control and ANXA11 knockdown H69 cholangiocytes, intracellular pH, AE2 and ANO1 surface expression, and bile acid influx were examined using ratio microspectrofluorometry, cell surface biotinylation, and 22,23-3H-glycochenodeoxycholic acid permeation, respectively. The localization of annexin A11-mEmerald and ANO1-mCherry was investigated by live-cell microscopy in H69 cholangiocytes after incubation with IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies or disease control serum. Annexin A11 was strongly expressed in human cholangiocytes, but not hepatocytes. Knockdown of ANXA11 led to reduced plasma membrane expression of ANO1, but not AE2, alkalization of intracellular pH and uncontrolled bile acid influx. High intracellular calcium conditions led to annexin A11 membrane shift and colocalization with ANO1. Incubation with IRC patient serum inhibited annexin A11 membrane shift and reduced ANO1 surface expression. Cholangiocellular annexin A11 mediates apical membrane abundance of the chloride channel ANO1, thereby supporting biliary bicarbonate secretion. Insertion is inhibited by IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies. Anti-annexin A11 autoantibodies may contribute to the pathogenesis of IRC by weakening the 'biliary bicarbonate umbrella'. We previously identified annexin A11 as a specific autoantigen in immunoglobulin G4-related cholangitis (IRC), a B-cell driven disease affecting the bile ducts. Human cholangiocytes are protected against harmful hydrophobic bile acid influx by a defense mechanism referred to as the 'biliary bicarbonate umbrella'. We found that annexin A11 is required for the formation of a robust bicarbonate umbrella. Binding of patient-derived annexin A11 autoantibodies inhibits annexin A11 function, possibly contributing to bile duct damage by weakening the biliary bicarbonate umbrella in patients with IRC.

Dermal exposure to cobalt studied in vitro in keratinocytes – effects of cobalt exposure on inflammasome activated cytokines, and mRNA response

Background Cobalt is a dermal sensitizer, and keratinocytes respond to cobalt exposure by releasing proinflammatory mediators, regulating the immune response. Objective To determine the effect of cobalt on the inflammasome associated cytokine- and gene expression in cultured human keratinocytes (HaCaT). Cultivation in low- or high calcium conditions model separate differentiation states of keratinocytes in the skin. Method HaCaT cells in two different states of differentiation were exposed to cobalt chloride and caspase-1 activity as well as the production of IL-1β, IL-18 and gene expression of IL1B, IL18, NLRP3, CASP1, and PYCARD was quantified. Results High cobalt chloride exposure mediated significant increase in caspase-1 activity, cytokine levels, and IL1B and NLRP3 expression with a corresponding regulatory decrease for CASP1 and PYCARD expression. No difference between high- and low calcium culturing conditions modelling differentiation states was detected. Conclusions Our data suggest that HaCaT cells respond with inflammmasome associated activity upon cobalt exposure in a concentration-dependent manner. These mechanisms could be of importance for the understanding of the pathophysiology behind allergic sensitization to dermal cobalt exposure.

Abstract 2578: Calcium modulates apoptosis and differentiation in human normal colon 3D organoids irrespective of donor sex and biopsy site

Abstract Background: Observational studies indicate that calcium, a micronutrient involved in numerous cell-signaling cascades, may protect against colon cancer. Stratified analyses suggest the protective effect of calcium may depend on sex and anatomic subsite, but these hypotheses have been difficult to test experimentally. Here, we employ human-derived normal colon 3D organoids as a model of the healthy colon crypt to interrogate the effect of calcium exposure on gene expression and the effect of donor sex and biopsy anatomic subsite on the response to calcium. Methods: A total of 39 3D colon organoid lines derived from colon biopsies (22 proximal, 17 distal) obtained from unrelated subjects (21 female, 18 male) during screening colonoscopy were grown under low (1.7 mM) calcium conditions, and a matching set of the same lines were grown under high (5.0 mM) calcium conditions. After 72 hours, total RNA was extracted from organoid lines, libraries were prepared with poly-A selection, and bulk RNA-sequencing was used to measure gene expression. DNA was extracted from the same lines and genotyped using the OncoArray 500K beadchip. Missing genotypes were imputed to the TOPMed reference panel using the TOPMed Imputation Server. Polygenic risk scores for colorectal cancer were calculated for each organoid line using Plink and 135 of 141 genome-wide significant SNPs associated with colorectal cancer in recent genome-wide association studies. Relative expression between low and high calcium conditions was tested using generalized linear models as implemented in DESeq2. Functional enrichment of gene ontologies among differentially expressed genes was performed with ToppFun. The effects of donor sex, biopsy subsite, and organoid polygenic risk score on response to calcium were tested using single and multiple linear regression as implemented in the base R lm function. Results: Exposure to calcium induced differential expression of 767 genes with a false discovery rate of 5%. Differentially expressed genes were enriched for biological processes including extracellular structure organization (Bonferroni p=1.6E-3) and positive regulation of apoptosis (Bonferroni p=2.2E-2). Although stratified analyses demonstrated more differentially expressed genes in 3D colon organoids derived from male subjects, no statistically significant effect of sex, subsite, or polygenic risk score on response to calcium was observed. Conclusion: We tested the effect of calcium exposure on gene expression in human-derived normal colon 3D organoids and found that calcium modulated processes related to differentiation and apoptosis, but the response to calcium was not affected by sex, anatomic subsite, or polygenic risk score. Citation Format: Christopher H. Dampier, Matthew Devall, Jennifer Bryant, Stephen D. Eaton, Jeroen R. Huyghe, Andre E. Kim, Virginia Diez-Obrero, Franzel J. van Duijnhoven, Duncan C. Thomas, Steven M. Powell, W James Gauderman, Ulrike Peters, Graham Casey. Calcium modulates apoptosis and differentiation in human normal colon 3D organoids irrespective of donor sex and biopsy site [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2578.

Dose- and time-dependent changes in viability and IL-6, CXCL8 and CCL2 production by HaCaT-cells exposed to cobalt. Effects of high and low calcium growth conditions.

Sensitization requires exposure to an allergen with subsequent production of a "danger "signal. In the skin, keratinocytes are the main producers of these signals. To compare dose- and time-effects of cobalt on the viability of and cytokine release from HaCaT cells cultured at low or high calcium. To model two separate states of differentiation of keratinocytes, HaCaT cells were cultured under low or high calcium conditions. HaCaT were exposed to different concentrations of cobalt chloride (10 μm to 5 mM) over time (30 minutes- 48 hours). Cell viability was measured with the Cell-Titer Blue Viability assay. Cytokine production was measured using a bead-based immunoassay and flow cytometry. Gene expression was quantified using qPCR. Data was analyzed by ANOVA and linear mixed model. Viability of the cells was dose- and time-dependent. A linear mixed statistical model showed that cobalt exposure induces increase in IL-6, CXCL8 and CCL2 production over time and whereas increase of IL-6 and a decrease of CCL2 was associated with increasing cobalt chloride concentrations. When comparing the cells incubated under high and low calcium conditions, the more differentiated cells in the high concentration were found to exert a stronger response in terms of IL-6 release. Our data suggest that cobalt chloride triggered an alarm system in HaCaT cells, and proinflammatory cytokines/chemokines were secreted in a dose- and time-dependent manner. When high and low calcium incubations were compared, the difference was seen only for IL-6. These findings indicate that the effect of cobalt chloride on cell toxicity occurs throughout the living epidermis.

SPARC-related modular calcium binding 1 regulates aortic valve calcification by disrupting BMPR-II/p-p38 signalling.

AimsAortic valve calcification is more prevalent in chronic kidney disease accompanied by hypercalcemia. Secreted protein acidic and rich in cysteine (SPARC)-related modular calcium binding 1 (SMOC1) is a regulator of BMP2 signalling, but the role of SMOC1 in aortic valve calcification under different conditions has not been studied. This study aimed to investigate the roles of SMOC1 in aortic valve calcification under normal and high calcium conditions, focusing on the effects on aortic valve interstitial cells (AVICs).Methods and resultsSMOC1 was expressed by aortic valve endothelial cells and secreted into the extracellular matrix in non-calcific valves and downregulated in calcific aortic valves. In vitro studies demonstrated that HUVEC secreted SMOC1 could enter the cytoplasm of AVICs. Overexpression of SMOC1 attenuated warfarin-induced AVIC calcification but promoted high calcium/phosphate or vitamin D-induced AVIC and aortic valve calcification by regulating BMP2 signalling both in vitro and in vivo. Co-immunoprecipitation revealed that SMOC1 binds to BMP receptor II (BMPR-II) and inhibits BMP2-induced phosphorylation of p38 (p-p38) via amino acids 372–383 of its EF-hand calcium-binding domain. Inhibition of p-p38 by the p38 inhibitor SB203580 blocked the effects of SMOC1 on BMP2 signalling and AVIC calcification induced by high calcium/phosphate medium. In high-calcium-treated AVICs, SMOC1 lost its ability to bind to BMPR-II, but not to caveolin-1, promoting p-p38 and cell apoptosis due to increased expression of BMPR-II and enhanced endocytosis.ConclusionsThese observations support that SMOC1 works as a dual-directional modulator of AVIC calcification by regulating p38-dependent BMP2 signalling transduction according to different extracellular calcium concentrations.

Generation of Keratinocytes from Human Induced Pluripotent Stem Cells Under Defined Culture Conditions

Differentiation of keratinocytes from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) has become an important tool for wound healing research and for studying skin diseases in instances where patient cells are not available. Several keratinocyte differentiation protocols using hiPSC colony fragments or embryoid bodies have been published with some requiring prolonged time for differentiation or extended use of reagent cocktails. In this study, we present a simplified method to efficiently generate large numbers of uniformly differentiated keratinocytes in less than 4 weeks from singularized hiPSCs with differentiation factors, retinoic acid and bone morphogenetic protein 4 (BMP4). Low seeding density of singularized iPSCs results in keratinocyte cultures with minimum cell death during differentiation and up to 96% homogeneity for keratin 14-positive cells and low percentage of keratinocyte maturation markers, comparable to early passage primary keratinocytes. hiPSC-derived keratinocytes remain in a proliferative state, can be maintained for prolonged periods of time, and can be terminally differentiated under high calcium conditions in the same way as primary human keratinocytes. Moreover, coculturing hiPSC-derived fibroblasts and keratinocytes consistently formed organotypic 3D skin equivalents. Therefore, keratinocytes generated by this method are a viable source of cells for downstream applications.

Cytotoxic Granule Trafficking and Fusion in Synaptotagmin7-Deficient Cytotoxic T Lymphocytes.

Granules of cytotoxic T lymphocytes (CTL) are derived from the lysosomal compartment. Synaptotagmin7 (Syt7) appears to be the calcium sensor triggering fusion of lysosomes in fibroblasts. Syt7 has been proposed to control cytotoxic granule (CG) fusion in lymphocytes and mice lacking Syt7 have reduced ability to clear infections. However, fusion of CG persists in the absence of Syt7. To clarify the role of Syt7 in CTL function, we have examined the fusion of cytotoxic granules of CD8+ T-lymphocytes from Syt7 knock-out mice. We have recorded granule fusion in living CTL, using total internal reflection microscopy. Since Syt7 is considered a high affinity calcium-sensor specialized for fusion under low calcium conditions, we have compared cytotoxic granule fusion under low and high calcium conditions in the same CTL. There was no difference in latencies or numbers of fusion events per CTL under low-calcium conditions, indicating that Syt7 is not required for cytotoxic granule fusion. A deficit of fusion in Syt7 KO CTL was seen when a high-calcium solution was introduced. Expressing wild type Syt7 in Syt7 KO lymphocytes reversed this deficit, confirming its Syt7-dependence. Mutations of Syt7 which disrupt calcium binding to its C2A domain reduced the efficacy of this rescue. We counted the cytotoxic granules present at the plasma membrane to determine if the lack of fusion events in the Syt7 KO CTL was due to a lack of granules. In low calcium there were no differences in fusion events per CTL, and granule numbers were similar. In high calcium, granule number was similar though wild type CTL exhibited significantly more fusion than Syt7 KO CTL. The modest differences in granule counts do not account for the lack of fusion in high calcium in Syt7 KO CTL. In Syt7 KO CTL expressing wild type Syt7, delivery of cytotoxic granules to the plasma membrane was comparable to that of wild type CTL. Syt7 KO CTL expressing Syt7 with deficient calcium binding in the C2A domain had significantly less fusion and fewer CG at the plasma membrane. These results indicate that Syt7 is involved in trafficking of CG to the plasma membrane.

OR07-04 A Novel Ex Vivo Live-Cell Interrogative Assay of Human Parathyroid Tissue Reveals Distinct Mechanisms of Calcium Sensing Failure in Primary, Secondary, and Tertiary Hyperparathyroidism

Disruption of calcium homeostasis is common to all forms of hyperparathyroidism (HPT), but the underlying biochemical mechanisms that distinguish the various forms of HPT pathology remain poorly characterized. We previously have observed that the kinetics and amplitude of CASR-mediated signaling vary significantly among parathyroid (PT) adenomas and found specific functional and gene expression profiles preferentially associated with increased risk of bone density loss. While these data established a clear connection between CASR activity and clinical phenotype, a direct comparison of the kinetics of PTH secretory behavior between normal and neoplastic intact human PT tissue has yet to be performed. Utilizing eucalcemic normal human organ donor tissues (n=3) as a reference standard, we examined a series of cryopreserved live PT tissue specimens obtained from patients with primary (n=9), secondary (n=12) and tertiary (n=5) HPT. PT tissue fragments matched for viability, mass, and cellular content were placed on permeable membranes and exposed to a series of extracellular calcium concentrations over equivalent time intervals of challenge and normocalcemic recovery to interrogate dynamic PTH secretory induction or suppression. As expected, normal tissue exhibited a sigmoid response curve indicative of allosteric calcium-mediated inhibition, with a mean EC50 of 0.95 mM (95% CI: 0.859–1.254). In contrast, the majority of primary HPT adenomas (n=6) displayed a concave response curve indicative of non-competitive inhibition, consistent with a primary sensing deficit, such as loss of CASR expression. Two distinct PTH secretory behaviors were observed in secondary HPT specimens. One subset (secondary type 1, n=4) retained a sigmoid response curve but with a modest EC50 increase (mean EC50=1.50 mM, 95% CI: 1.41–1.61) and maximal suppression similar to normal tissue, features reflective of competitive inhibition in response to elevated calcium. This pattern could indicate enhanced CASR antagonist activity relative to normal tissue. A second subset, (secondary type 2, n=8) demonstrated a large EC50 shift (mean EC50=2.46 mM; 95% CI: 1.844–2.621), a sigmoid response curve, and an elevated threshold of persistent PTH secretion at high calcium conditions. These parameters are suggestive of non-competitive inhibitory behavior, consistent with loss of a CASR-dependent downstream effector. Three of the primary HPT adenomas shared this response phenotype. Of the tertiary specimens, four matched the primary HPT adenoma pattern, while one exhibited secondary type 2 behavior. These results reveal a series of progressively attenuated dynamic response patterns, where PTH secretion becomes increasingly uncoupled from extracellular calcium sensing. These findings suggest that primary, secondary, and tertiary HPT arise through distinct mechanisms of calcium sensing failure.

Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα.

Progenitor cells at the basal layer of skin epidermis play an essential role in maintaining tissue homeostasis and enhancing wound repair in skin. The proliferation, differentiation, and cell death of epidermal progenitor cells have to be delicately regulated, as deregulation of this process can lead to many skin diseases, including skin cancers. However, the underlying molecular mechanisms involved in skin homeostasis remain poorly defined. In this study, with quantitative proteomics approach, we identified an important interaction between KDF1 (keratinocyte differentiation factor 1) and IKKα (IκB kinase α) in differentiating skin keratinocytes. Ablation of either KDF1 or IKKα in mice leads to similar but striking abnormalities in skin development, particularly in skin epidermal differentiation. With biochemical and mouse genetics approach, we further demonstrate that the interaction of IKKα and KDF1 is essential for epidermal differentiation. To probe deeper into the mechanisms, we find that KDF1 associates with a deubiquitinating protease USP7 (ubiquitin-specific peptidase 7), and KDF1 can regulate skin differentiation through deubiquitination and stabilization of IKKα. Taken together, our study unravels an important molecular mechanism underlying epidermal differentiation and skin tissue homeostasis.

Trafficking of the Prorenin Receptor in Endothelial Cells

Diabetic patients are at higher risk for hypertension and cardiovascular mortality than their normotensive, nondiabetic counterparts. Diabetic patients also have increased circulating prorenin which can bind to its receptor (PRR) to cause activation of the renin‐angiotensin system and fibrosis. We hypothesized that diabetes induces dysregulation of the endothelial PRR to exacerbate cardiovascular damage. Mouse pancreatic endothelial cells (MS1) were cultured and exposed to high glucose or low calcium (Ca2+) conditions for one hour. Cells were fixed for immunocytochemistry or fractionated and immunoblotted for PRR. Treatment with 25 mM glucose or 25 mM mannitol for one hour did not impact full length PRR expression or localization in the total membrane fraction. When Ca2+ was removed from the media, full length PRR increased in the cytosolic fraction by 3.6‐fold (P=0.022) compared to control. Restoration of physiological Ca2+ returned PRR expression to control levels in the cytosolic fraction, while neither sPRR nor full length PRR were altered in the total membrane fraction. Confocal imaging of fixed cells suggested that PRR in the total membrane fraction may be encapsulated in vesicles. These data indicate that Ca2+ may be an important regulator of endothelial PRR trafficking, possibly via a Ca2+‐dependent PRR cleavage pathway. Ongoing studies will establish the relationship between Ca2+ and sPRR/PRR and determine the impact of PRR trafficking on endothelial dysfunction. These studies will determine whether targeting of PRR can lower the risk of cardiovascular disease in patients with diabetes.Support or Funding InformationTulane Center for Engaged Learning and Technology, NIH HL133619, DK104375

Differentiation of human colon tissue in culture: Effects of calcium on trans-epithelial electrical resistance and tissue cohesive properties.

Background and aimsHuman colonoid cultures maintained under low-calcium (0.25 mM) conditions undergo differentiation spontaneously and, concomitantly, express a high level of tight junction proteins, but not desmosomal proteins. When calcium is included to a final concentration of 1.5–3.0 mM (provided either as a single agent or as a combination of calcium and additional minerals), there is little change in tight junction protein expression but a strong up-regulation of desmosomal proteins and an increase in desmosome formation. The aim of this study was to assess the functional consequences of calcium-mediated differences in barrier protein expression.MethodsHuman colonoid-derived epithelial cells were interrogated in transwell culture under low- or high-calcium conditions for monolayer integrity and ion permeability by measuring trans-epithelial electrical resistance (TEER) across the confluent monolayer. Colonoid cohesiveness was assessed in parallel.ResultsTEER values were high in the low-calcium environment but increased in response to calcium. In addition, colonoid cohesiveness increased substantially with calcium supplementation. In both assays, the response to multi-mineral intervention was greater than the response to calcium alone. Consistent with these findings, several components of tight junctions were expressed at 0.25 mM calcium but these did not increase substantially with supplementation. Cadherin-17 and desmoglein-2, in contrast, were weakly-expressed under low calcium conditions but increased with intervention.ConclusionsThese findings indicate that low ambient calcium levels are sufficient to support the formation of a permeability barrier in the colonic epithelium. Higher calcium levels promote tissue cohesion and enhance barrier function. These findings may help explain how an adequate calcium intake contributes to colonic health by improving barrier function, even though there is little change in colonic histological features over a wide range of calcium intake levels.

Physiological Responses of Handeliodendron bodinieri (Levl.) Rehd. to Exogenous Calcium Supply under Drought Stress

The soils in karst regions, with carbonate rocks as the background material, are characterized by two main factors, drought and high calcium levels. Handeliodendron bodinieri (Levl.) Rehd is an endemic plant species in this area of China. However, few studies have been carried out on the adaptation mechanism of H. bodinieri to drought and high calcium soil. To reveal the physiological responses of H. bodinieri to exogenous calcium under drought stress, 10% PEG-6000 was used to simulate drought stress, and the effects of exogenous calcium at different concentrations on the physiology of H. bodinieri seedlings under drought stress were studied. The results showed that drought stress significantly reduced the relative water content and water potential of H. bodinieri seedlings. Malondialdehyde (MDA) content, O2− and H2O2 production rates significantly increased under drought stress. The addition of exogenous calcium significantly reduced MDA content and O2− and H2O2 production rates. Moreover, peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), ascorbic acid peroxidase (APX) and glutathione peroxidase (GPX) activities increased significantly under drought stress. At the same time, the accumulation of osmotic regulators such as soluble sugar, betaine and free proline also increased significantly. The addition of exogenous calcium further increased the activity of antioxidant enzymes and the accumulation of osmotic regulatory substances. Consequently, the oxidative stress and osmotic stress induced by drought decreased. Finally, exogenous calcium enhanced the photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) of H. bodinieri under drought stress and significantly improved water use efficiency (WUE). This study confirmed that the application of exogenous calcium can enhance the water condition, photosynthetic capacity, osmotic regulation and antioxidant capacity of H. bodinieri under drought stress. Of the treatments, the 10 mmol·L−1 CaCl2 treatment is more likely to improve survival of H. bodinieri under drought tolerance. This study provides an important reference for describing the adaptation mechanism and appropriate conservation of H. bodinieri under drought and high calcium conditions.

Extracellular calcium-phosphate nanoparticles activate the NLRP3 inflammasome through CaSR signaling

Abstract Calcium and phosphate are well known as the main minerals in bone ossification. But they also have an immunomodulatory potential as components of calciprotein particles. Such particles precipitate spontaneously in body fluids if the calcium and phosphate homeostasis is disturbed. An abnormally increased mineral load could lead to soft tissue calcification. In our study we investigated the cellular response of freshly isolated peripheral blood monocytes in serum-containing medium under precipitating (high calcium and high phosphate content) conditions. Using dynamic mass redistribution analyses with the EPIC® system and IL-1β measurements as readout, we identified phosphate as a necessary co-modulator of calcium-triggered overall cellular responses and NLRP3 inflammasome activation. Moreover, flow cytometry and ImageStream® analyses revealed the uptake of spontaneously formed nanoparticles under stimulating conditions, which depends on the activation of the G protein-coupled receptor Calcium-sensing receptor (CaSR). Our study unraveled a GPCR-mediated mechanism by which monocytes detect imbalances in calcium phosphate homeostasis in the extracellular space resulting in an enormous pro-inflammatory cellular response.

Cyto/chemokine profile of in vitro scratched keratinocyte model: Implications of significant upregulation of CCL20, CXCL8 and IL36G in Koebner phenomenon

BackgroundScratch injury induces Koebner phenomenon in psoriasis. Smoking is also a risk factor for psoriasis. Keratinocytes can produce various psoriasis-related molecules including TNF, IL1 A, IL1B, IL6, IL12B, IL17C, IL23 A, IL36 A, IL36B, IL36 G, CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL20, IFNB, and CAMP. However, the scratch-induced molecular profiling remains elusive. ObjectiveTo profile the induction pattern of above-mentioned psoriasis-related and keratinocyte-derived molecules by scratch injury in the presence or absence of anti-psoriatic drugs or benzo[a]pyrene, a major environmental pollutant of tobacco smoke. MethodsConfluent normal human keratinocytes were scratched and molecules were assayed by qRT-PCR, ELISA and Western blotting with or without drugs and benzo[a]pyrene. ResultsAmong the 18 molecules, the scratch injury on a confluent keratinocyte sheet significantly and selectively upregulated the mRNA expression of four cyto/chemokines, CXCL8, CCL20, IL36G, and TNF, in a scratch-line-number-dependent manner under either low- or high-calcium condition. However, significant protein secretion was only demonstrated for CXCL8 and CCL20. The IL36 G protein was not secreted, but its intracellular level was significantly upregulated by scratch injury, whereas neither the secretion nor the intracellular level of TNF protein was affected by scratch injury. Dexamethasone, but not maxacalcitol nor the phosphodiesterase 4 inhibitor apremilast, partially inhibited the CXCL8 and CCL20 secretion. Benzo[a]pyrene significantly and synergistically enhanced the scratch-induced CCL20 secretion that may explain why smoking is a risk factor for psoriasis. ConclusionCCL20 and to a less extent CXCL8 may play a key role in triggering the Koebner phenomenon after scratch injury to keratinocytes.