Biology subjects

McClintock, S. D.

Publications and source records attributed to McClintock, S. D..

8 recordsLinked to original sources

bioRxiv2026

Aquamin: A Source of Bioavailable Calcium, Magnesium, and Additional Cationic Trace Elements in Colon Health and Beyond

Introduction: Aquamin is a calcium- and magnesium-rich natural product derived from marine red algae that contains multiple trace elements. Human and mouse studies have demonstrated potential health benefits. Methods: Aquamin and calcium carbonate were compared using a two-stage simulated gastric and intestinal digestion protocol. ICP-OES/ICP-MS was used to quantify cationic elements in soluble and insoluble fractions. Binding of dispersed mineral powders to human colon epithelial cells was assessed by light and scanning electron microscopy, and uptake was assessed by transmission electron microscopy. TMT-based proteomics identified protein changes in colon epithelial organoids following mineral exposure. Serum samples from 90- and 180-day Aquamin intervention trials were analyzed for changes in circulating cationic elements. Results: Both Aquamin and calcium carbonate were solubilized under simulated gastric conditions, although Aquamin was more acid-resistant. After transfer to neutral-pH intestinal conditions, limited precipitation occurred, and most calcium remained soluble. With Aquamin, magnesium and strontium also remained soluble, whereas several trace elements became partially or completely insoluble. Aquamin rapidly bound to colon epithelial cells, with ultrafine Aquamin particles detected between adjacent cells. Calcium carbonate bound more slowly and to fewer cells. Aquamin increased proteins involved in enterocyte cation uptake, intracellular trafficking, and basolateral secretion. Serum strontium levels increased in study participants after 90 or 180 days of Aquamin consumption; most other elements changed minimally, although serum iron increased modestly. Conclusions: Aquamin provides a bioavailable source of several cationic elements. Soluble calcium, magnesium, and strontium may be absorbed primarily through transcellular pathways, while less-soluble elements may also cross the colonic epithelium through paracellular mechanisms.

molecular biology↗

bioRxiv2026

CCN3-derived peptide BLR-200 impairs YAP activation and attenuates bleomycin-induced skin fibrosis through blocking the generation of Sfrp2-positive fibroblasts

An autocrine pro-adhesive/pro-contractile signaling loop, through the mechanosensitive transcriptional cofactor YAP, promotes fibrosis. The CCN family of matricellular proteins modify adhesive signaling. Of these, CCN3 is antifibrotic. We show that BLR-200, a CCN3-derived peptide, has anti-fibrotic properties in the bleomycin-induced model of scleroderma skin fibrosis. In vitro, BLR-200 delayed, but did not abolish, fibroblast adhesion to collagen and nuclear YAP localization. In vivo, BLR-200 prevented/treated bleomycin-induced skin fibrosis, and reduced bleomycin-induced expression of profibrotic genes including -smooth muscle actin, CCN1 and CCN2. Lineage tracing and scRNA-seq analyses revealed that the myofibroblasts in this model were quantitatively derived from collagen-lineage Pi16+/Col15+ve fibroblasts. BLR-200 prevented myofibroblast differentiation in this model and trajectory of fibroblasts toward a Sfrp2-positive subset, a cell type associated with poor clinical outcome. BLR-200 impairs YAP activation in vitro and appearance of translationally-relevant fibroblast subtypes in vivo and is a novel anti-fibrotic agent for SSc skin fibrosis. HIGHLIGHTS-SSc skin fibrosis, a major unmet need, is driven by an activated mechanotransduction/YAP pathway; how to block this pathway clinically is unclear -members of the CCN family of matricellular proteins are adhesive signaling modifiers; herein, we identify BLR-200, a synthetic peptide derived from CCN3, based on its ability to impair, but not ablate, YAP nuclear localization and fibroblast spreading/attachment to collagen -BLR-200 blocks and treats progression of bleomycin-induced skin fibrosis -In the bleomycin model, lineage tracing analysis revealed that collagen-lineage cells are the primary source of myofibroblasts -BLR-200 blocks myofibroblast differentiation concomitant with reduced progression toward Col8a1+ve and Sfrp2+ ve fibroblasts, populations implicated in SSc pathogenesis -BLR-200 represents a novel, translationally relevant drug candidate for SSc skin fibrosis

cell biology↗

bioRxiv2022

Red Algae-Derived Mineral Intervention to Counter Pro-inflammatory Activity in Human Colon Organoids

Human colon organoids were maintained in culture medium alone or exposed to lipopolysaccharide with a combination of three pro-inflammatory cytokines (tumor necrosis factor-, interleukin-1{beta} and interferon-{gamma} [LPS-cytokines]) to mimic the environment in the inflamed colon. Untreated organoids and those exposed to LPS-cytokines were concomitantly treated with a multimineral product that has previously been shown to improve barrier structure/function. The organoids were subjected to proteomic analysis to obtain a broad view of the protein changes induced by these interventions. In parallel, confocal fluorescence microscopy and trans-epithelial electrical resistance measurements were used to assess barrier structure/function. The LPS-cytokines altered expression of multiple proteins that influence innate immunity and promote inflammation. Most of these were unaffected by the multi-mineral intervention, though a subset of inflammation-related proteins including fibrinogen-{beta} and -{gamma} chains, phospholipase A2 and SPARC was down-regulated in the presence of the multi-mineral intervention; another subset of proteins with anti-inflammatory, antioxidant or antimicrobial activity was up-regulated by multi-mineral treatment. When used alone, the multi-mineral intervention strongly up-regulated proteins that contribute to barrier formation and tissue strength. Concomitant treatment with LPS-cytokines did not inhibit barrier formation in response to the multimineral intervention. Altogether, the findings suggest that mineral intervention may provide a novel approach to combating inflammation in the colon by improving barrier structure/function as well as by directly altering expression of pro-inflammatory proteins.

molecular biology↗

bioRxiv2022

Liver Protein Expression in NASH Mice on a High-Fat Diet

Male MS-NASH mice were maintained on a high-fat diet for 16 weeks with and without red algae-derived minerals. Obeticholic acid (OCA) was used as a comparator in the same strain and diet. C57BL/6 mice maintained on a standard (low-fat) rodent chow diet were used as a control. At the end of the in-life portion of the study, body weight, liver weight, liver enzyme levels and liver histology were assessed. Samples obtained from individual livers were subjected to Tandem Mass Tag labeling / mass spectroscopy for protein profile determination. As compared to mice maintained on the low-fat diet, all high-fat-fed mice had increased whole body and liver weight, increased liver enzyme (aminotransferases) levels and widespread steatosis / ballooning hepatocyte degeneration. Histological evidence for liver inflammation and collagen deposition was also present, but changes were to a lesser extent. A moderate reduction in ballooning degeneration and collagen deposition was observed with mineral supplementation. Control mice on the high-fat diet alone demonstrated multiple protein changes associated with dysregulated fat and carbohydrate metabolism, lipotoxicity and oxidative stress. Cholesterol metabolism and bile acid formation were especially sensitive to diet. In mice receiving multi-mineral supplementation along with the high-fat diet, there was reduced liver toxicity as evidenced by a decrease in levels of several cytochrome P450 enzymes and other oxidantgenerating moieties. Additionally, elevated expression of several keratins was also detected in mineral-supplemented mice. The protein changes observed with mineral supplementation were not seen with OCA. Our previous studies have shown that mice maintained on a high-fat diet for up to 18 months develop end-stage liver injury including hepatocellular carcinoma. Mineral-supplemented mice were substantially protected against tumor formation and other end-state consequences of high-fat feeding. The present study identifies early (16-week) protein changes occurring in the livers of the high-fat diet-fed mice, and how the expression of these proteins is influenced by mineral supplementation. These findings help elucidate early protein changes that contribute to end-stage liver injury and potential mechanisms by which dietary minerals may mitigate such damage.

molecular biology↗

bioRxiv2021

Cell-matrix adhesion contributes to permeability control in human colon organoids

Background and aimsCell-cell adhesion structures (desmosomes and, especially, tight junctions) are known to play important roles in control of transepithelial permeability in the colon. The involvement of cell-matrix interactions in permeability control is less clear. The goals of the present study were to: i) determine if disruption of colon epithelial cell interactions with the extracellular matrix alters permeability control and ii) determine if increasing the elaboration of protein components of cell-matrix adhesion complexes improves permeability control and mitigates the effects of cell-matrix disruption. MethodsHuman colon organoids were interrogated for transepithelial electrical resistance (TEER) under control conditions (0.25 mM calcium) and in the presence of Aquamin(R), a multi-mineral product, at a level providing 1.5 mM calcium. The effects of Aquamin(R) on cell-matrix adhesion protein expression were determined in a proteomic screen and by Western blotting. In parallel, TEER was assessed in the presence of a function-blocking antibody directed at an epitope in the C-terminal region of laminin 3 chain. ResultsTreatment of colon organoids with Aquamin(R) increased the expression of multiple basement membrane and hemidesmosomal proteins as well as keratin 8 and 18. TEER values were higher in the presence of Aquamin(R) than they were under control conditions. Anti-laminin antibody reduced TEER values under all conditions but was most effective in the absence of Aquamin(R), where laminin expression was low and TEER values were lower to begin with. ConclusionsThese findings provide evidence that cell-matrix interactions contribute to permeability control in the colon. They suggest that the elaboration of proteins important to cell-matrix interactions can be increased in human colon organoids by exposure to a multi-mineral natural product. Increasing the elaboration of such proteins may help to mitigate the consequences of disrupting cell-matrix interactions on permeability control.

cell biology↗

bioRxiv2019

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 minerals), there is little change in tight junction proteins but a strong up-regulation of desmosomal proteins and an increase in desmosome formation. The aim of this study was to assess functional consequences of the differences in calcium-mediated barrier protein expression.\n\nMethodsHuman colonoid-derived epithelial monolayers were interrogated in transwell culture under low- or high-calcium conditions. Ion permeability and monolayer integrity were assessed by measuring trans-epithelial electrical resistance (TEER) across the confluent monolayer. Colonoid cohesiveness was assessed in parallel.\n\nResultsTEER values were high in the low-calcium environment and increased only modestly in response to calcium. In contrast, colonoid cohesiveness increased substantially with calcium supplementation. In both assays, the response to Aquamin was greater than the response to calcium alone. However, differences between interventions were small and only compared to the 0.25 mM calcium were they statistically significant. Consistent with these findings, occludin expression (a measure of tight junctions) was high at 0.25 mM calcium and did not increase with supplementation. Cadherin-17 and desmoglein-2 were weakly-expressed under low calcium conditions but increased with intervention.\n\nConclusionsThese findings indicate that low ambient calcium levels are sufficient to support formation of a permeability barrier in the colonic epithelium. Higher calcium levels are necessary to promote tissue cohesion and enhance barrier function. These findings may help explain how an adequate daily intake of calcium 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.

physiology↗

bioRxiv2018

CALCIUM-INDUCED DIFFERENTIATION IN NORMAL HUMAN COLONOID CULTURES: Cell-cell / cell-matrix adhesion, barrier formation and tissue integrity

Colonoid cultures were established from histologically-normal human colon tissue and maintained in a low-calcium (0.25 mM) medium or in medium supplemented with an amount of calcium (1.5 - 3.0 mM) that was shown in a previous study to induce differentiation in colonoids derived from large adenomas. Calcium alone was compared to Aquamin, a multi-mineral natural product that contains magnesium and detectable levels of 72 additional trace elements in addition to calcium. Unlike the previously-studied tumor-derived colonoids (which remained un-differentiated in the absence of calcium-supplementation), normal tissue colonoids underwent differentiation as indicated by gross and microscopic appearance, a low proliferative index and high-level expression of cytokeratin 20 (CK20) in the absence of intervention. Only modest additional changes were seen in these parameters with either calcium alone or Aquamin (providing up to 3.0 mM calcium). In spite of this, proteomic analysis and immunohistochemistry revealed that both interventions induced strong up-regulation of proteins that promote cell-cell and cell-matrix adhesive functions, barrier formation and tissue integrity. Transmission electron microscopy revealed an increase in desmosomes in response to intervention. These findings demonstrate that histologically normal human colonoids can undergo differentiation in the presence of a low ambient calcium concentration. However, higher calcium levels can induce elaboration of proteins that promote cell-cell and cell-matrix adhesion. These changes could lead to improved barrier function and improved colon tissue health.

molecular biology↗

bioRxiv2017

Identification, Isolation, and Characterization of Human LGR5-positive Colon Adenoma Cells

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, while less is known about human intestinal stem cells due to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas, and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC) associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from 4 adenoma organoid lines and were subjected to RNA-sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA-seq data from adenoma and normal organoids, we found correlations between LGR5 and CRC- specific genes, including DKK4 (dickkopf WNT signaling pathway inhibitor 4) and SMOC2 (SPARC related modular calcium binding 2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

cancer biology↗