A multi-mineral intervention to counter pro-inflammatory activity and to improve the barrier in human colon organoids.

Introduction: Ulcerative colitis is a chronic inflammatory condition, and continuous inflammatory stimulus may lead to barrier dysfunction. The goal of this study was to assess barrier proteomic expression by a red algae-derived multi-mineral intervention in the absence or presence of pro-inflammatory insult. Methods: Human colon organoids were maintained in a control culture medium alone or exposed to lipopolysaccharide with a combination of three pro-inflammatory cytokines [tumor necrosis factor-α, interleukin-1ß and interferon-γ (LPS-cytokines)] to mimic the environment in the inflamed colon. Untreated organoids and those exposed to LPS-cytokines were concomitantly treated for 14 days with a multi-mineral product (Aquamin®) that has previously been shown to improve barrier structure/function. The colon organoids were subjected to proteomic analysis to obtain a broad view of the protein changes induced by the two interventions alone and in combination. In parallel, confocal fluorescence microscopy, tissue cohesion and transepithelial electrical resistance (TEER) measurements were used to assess barrier structure/function. Results: The LPS-cytokine mix altered the expression of multiple proteins that influence innate immunity and promote inflammation. Several of these were significantly decreased with Aquamin® alone but only a modest decrease in a subset of these proteins was detected by Aquamin® in the presence of LPS-cytokines. Among these, a subset of inflammation-related proteins including fibrinogen-ß and -γ chains (FGB and FGG), phospholipase A2 (PLA2G2A) and SPARC was significantly downregulated in the presence of Aquamin® (alone and in combination with LPS-cytokines); another subset of proteins with anti-inflammatory, antioxidant or anti-microbial activity was upregulated by Aquamin® treatment. When provided alone, Aquamin® strongly upregulated proteins that contribute to barrier formation and tissue strength. Concomitant treatment with LPS-cytokines did not inhibit barrier formation in response to Aquamin®. Confocal microscopy also displayed increased expression of desmoglein-2 (DSG2) and cadherin-17 (CDH17) with Aquamin®, either alone or in the presence of the pro-inflammatory stimulus. Increased cohesion and TEER with Aquamin® (alone or in the presence of LPS-cytokines) indicates improved barrier function. Conclusion: Taken together, these findings suggest that multi-mineral intervention (Aquamin®) may provide a novel approach to combating inflammation in the colon by improving barrier structure/function as well as by directly altering the expression of certain pro-inflammatory proteins.

Transferrin Receptor-Mediated Iron Uptake Promotes Colon Tumorigenesis.

Transferrin receptor (TFRC) is the major mediator for iron entry into a cell. Under excessive iron conditions, TFRC is expected to be reduced to lower iron uptake and toxicity. However, the mechanism whereby TFRC expression is maintained at high levels in iron-enriched cancer cells and the contribution of TFRC to cancer development are enigmatic. Here the work shows TFRC is induced by adenomatous polyposis coli (APC) gene loss-driven ß-catenin activation in colorectal cancer, whereas TFRC-mediated intratumoral iron accumulation potentiates ß-catenin signaling by directly enhancing the activity of tankyrase. Disruption of TFRC leads to a reduction of colonic iron levels and iron-dependent tankyrase activity, which caused stabilization of axis inhibition protein 2 (AXIN2) and subsequent repression of the ß-catenin/c-Myc/E2F Transcription Factor 1/DNA polymerase delta1 (POLD1) axis. POLD1 knockdown, iron chelation, and TFRC disruption increase DNA replication stress, DNA damage response, apoptosis, and reduce colon tumor growth. Importantly, a combination of iron chelators and DNA damaging agents increases DNA damage response and reduces colon tumor cell growth. TFRC-mediated iron import is at the center of a novel feed-forward loop that facilitates colonic epithelial cell survival. This discovery may provide novel strategies for colorectal cancer therapy.

Liver Protein Expression in NASH Mice on a High-Fat Diet: Response to Multi-Mineral Intervention.

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 oxidant-generating 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.

Cell-Matrix Interactions Contribute to Barrier Function in Human Colon Organoids.

The importance of cell-matrix adhesion to barrier control in the colon is unclear. The goals of the present study were to: (i) determine if disruption of colon epithelial cell interactions with the extracellular matrix alters permeability control measurement and (ii) determine if increasing the elaboration of protein components of cell-matrix adhesion complexes can mitigate the effects of cell-matrix disruption. Human colon organoids were interrogated for transepithelial electrical resistance (TEER) under control conditions and in the presence of Aquamin®, a multi-mineral product. A function-blocking antibody directed at the C-terminal region of the laminin α chain was used in parallel. The effects of Aquamin® on cell-matrix adhesion protein expression were determined in a proteomic screen and by Western blotting. Aquamin® increased the expression of multiple basement membrane, hemidesmosomal and focal adhesion proteins as well as keratin 8 and 18. TEER values were higher in the presence of Aquamin® than they were under control conditions. The blocking antibody reduced TEER values under both conditions but was most effective in the absence of Aquamin®, where expression of cell-matrix adhesion proteins was lower to begin with. These findings provide evidence that cell-matrix interactions contribute to barrier control in the colon.

A Multi-Mineral Intervention to Modulate Colonic Mucosal Protein Profile: Results from a 90-Day Trial in Human Subjects.

The overall goal of this study was to determine whether Aquamin®, a calcium-, magnesium-, trace element-rich, red algae-derived natural product, would alter the expression of proteins involved in growth-regulation and differentiation in colon. Thirty healthy human subjects (at risk for colorectal cancer) were enrolled in a three-arm, 90-day interventional trial. Aquamin® was compared to calcium alone and placebo. Before and after the interventional period, colonic biopsies were obtained. Biopsies were evaluated by immunohistology for expression of Ki67 (proliferation marker) and for CK20 and p21 (differentiation markers). Tandem mass tag-mass spectrometry-based detection was used to assess levels of multiple proteins. As compared to placebo or calcium, Aquamin® reduced the level of Ki67 expression and slightly increased CK20 expression. Increased p21 expression was observed with both calcium and Aquamin®. In proteomic screen, Aquamin® treatment resulted in many more proteins being upregulated (including pro-apoptotic, cytokeratins, cell-cell adhesion molecules, and components of the basement membrane) or downregulated (proliferation and nucleic acid metabolism) than placebo. Calcium alone also altered the expression of many of the same proteins but not to the same extent as Aquamin®. We conclude that daily Aquamin® ingestion alters protein expression profile in the colon that could be beneficial to colonic health.

Organoid culture to study epithelial cell differentiation and barrier formation in the colon: bridging the gap between monolayer cell culture and human subject research.

Organoid culture provides a powerful technology that can bridge the gap between monolayer cell culture on the one hand and whole animal or human subject research on the other. Tissues from many different organs from multiple species, including human, have already been successfully adapted to organoid growth. While optimal culture conditions have not yet been established for all tissue types, it seems that most tissues will, ultimately, be amenable to this type of culture. The colon is one of the tissues in which organoid culture was first established as a technology and which has been most successfully employed. The ready availability of histologically normal tissue as well as both premalignant and malignant tissue (often from the same individual) makes this possible. While individual tumors are highly variable relative to one another in organoid culture, a high degree of genotypic consistency exists between the tumor tissue and the histologically normal counterpart from a given source. Further, source material and tumor tissue in organoid culture demonstrate a high degree of genotypic consistency. Even after 6-9 mo in continuous culture, drift in the mutational profile has been shown to be minimal. Colon tissue maintained in organoid culture, thus, provides a good surrogate for the tissue of origin-a surrogate, however, that is as amenable to intervention with molecular, pharmacological, and immunological approaches as are more-traditionally studied cell lines.

Ulcerative Colitis-Derived Colonoid Culture: A Multi-Mineral-Approach to Improve Barrier Protein Expression.

BACKGROUND: Recent studies demonstrated that Aquamin®, a calcium-, magnesium-rich, multi-mineral natural product, improves barrier structure and function in colonoids obtained from the tissue of healthy subjects. The goal of the present study was to determine if the colonic barrier could be improved in tissue from subjects with ulcerative colitis (UC). METHODS: Colonoid cultures were established with colon biopsies from 9 individuals with UC. The colonoids were then incubated for a 2-week period under control conditions (in culture medium with a final calcium concentration of 0.25 mM) or in the same medium supplemented with Aquamin® to provide 1.5 - 4.5 mM calcium. Effects on differentiation and barrier protein expression were determined using several approaches: phase-contrast and scanning electron microscopy, quantitative histology and immunohistology, mass spectrometry-based proteome assessment and transmission electron microscopy. RESULTS: Although there were no gross changes in colonoid appearance, there was an increase in lumen diameter and wall thickness on histology and greater expression of cytokeratin 20 (CK20) along with reduced expression of Ki67 by quantitative immunohistology observed with intervention. In parallel, upregulation of several differentiation-related proteins was seen in a proteomic screen with the intervention. Aquamin®-treated colonoids demonstrated a modest up-regulation of tight junctional proteins but stronger induction of adherens junction and desmosomal proteins. Increased desmosomes were seen at the ultrastructural level. Proteomic analysis demonstrated increased expression of several basement membrane proteins and hemidesmosomal components. Proteins expressed at the apical surface (mucins and trefoils) were also increased as were several additional proteins with anti-microbial activity or that modulate inflammation. Finally, several transporter proteins that affect electrolyte balance (and, thereby affect water resorption) were increased. At the same time, growth and cell cycle regulatory proteins (Ki67, nucleophosmin, and stathmin) were significantly down-regulated. Laminin interactions, matrix formation and extracellular matrix organization were the top three up-regulated pathways with the intervention. CONCLUSION: A majority of individuals including patients with UC do not reach the recommended daily intake for calcium and other minerals. To the extent that such deficiencies might contribute to the weakening of the colonic barrier, the findings employing UC tissue-derived colonoids here suggest that adequate mineral intake might improve the colonic barrier.

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

BACKGROUND AND AIMS: Human 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. METHODS: Human 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. RESULTS: TEER 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. CONCLUSIONS: These 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.

A Calcium-Rich Multimineral Intervention to Modulate Colonic Microbial Communities and Metabolomic Profiles in Humans: Results from a 90-Day Trial.

Aquamin is a calcium-, magnesium-, and multiple trace element-rich natural product with colon polyp prevention efficacy based on preclinical studies. The goal of this study was to determine the effects of Aquamin on colonic microbial community and attendant metabolomic profile. Thirty healthy human participants were enrolled in a 90-day trial in which Aquamin (delivering 800 mg of calcium per day) was compared with calcium alone or placebo. Before and after the intervention, colonic biopsies and stool specimens were obtained. All 30 participants completed the study without serious adverse event or change in liver and renal function markers. Compared with pretreatment values, intervention with Aquamin led to a reduction in total bacterial DNA (P = 0.0001) and a shift in the microbial community measured by thetaYC (θYC; P = 0.0087). Treatment with calcium also produced a decline in total bacteria, but smaller than seen with Aquamin, whereas no reduction was observed with placebo in the colon. In parallel with microbial changes, a reduction in total bile acid levels (P = 0.0375) and a slight increase in the level of the short-chain fatty acid (SCFA) acetate in stool specimens (P < 0.0001) from Aquamin-treated participants were noted. No change in bile acids or SCFAs was observed with calcium or placebo. We conclude that Aquamin is safe and tolerable in healthy human participants and may produce beneficial alterations in the colonic microbial community and the attendant metabolomic profile. Because the number of participants was small, the findings should be considered preliminary.

Calcium-induced differentiation in normal human colonoid cultures: Cell-cell / cell-matrix adhesion, barrier formation and tissue integrity.

BACKGROUND AND AIMS: The goal of the study was to assess calcium alone and Aquamin, a multi-mineral natural product that contains magnesium and detectable levels of 72 trace elements in addition to calcium, for capacity to affect growth and differentiation in colonoid cultures derived from histologically-normal human colon tissue. METHODS: Colonoid cultures were maintained in a low-calcium (0.25 mM) medium or in medium supplemented with an amount of calcium (1.5-3.0 mM), either from calcium alone or Aquamin for a period of two weeks. This was shown in a previous study to induce differentiation in colonoids derived from large adenomas. Changes in growth, morphological features and protein expression profile were assessed at the end of the incubation period using a combination of phase-contrast and scanning electron microscopy, histology and immunohistology, proteomic assessment and transmission electron microscopy. RESULTS: 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 in the absence of intervention (i.e., in control condition). 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. CONCLUSIONS: These findings demonstrate that colonoids derived from histologically normal human tissue can undergo differentiation in the presence of a low ambient calcium concentration. However, higher calcium levels 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.

Atrophic and hypertrophic photoaging: Clinical, histologic, and molecular features of 2 distinct phenotypes of photoaged skin.

BACKGROUND: Exposure to the sun causes premature skin aging, known as photoaging. Clinical features of photoaging vary widely among individuals. In one form, skin appears thin with telangiectasia, and in another form, skin appears thickened with coarse wrinkles. Etiologic, clinical, and therapeutic distinctions among different forms of photoaging remain largely unknown. OBJECTIVE: To characterize the clinical, histologic, and molecular features of hypertrophic and atrophic photoaging. METHODS: In total, 53 individuals were clinically classified as having primarily atrophic or hypertrophic photoaging or neither (controls). Participants' demographic and sun exposure-related lifestyle data were captured by questionnaire. Fifteen clinical features of participants were qualitatively or quantitively scored. Facial biopsies were analyzed for gene expression and histologic characteristics. RESULTS: Actinic and seborrheic keratosis, telangiectasia, and prior incidence of skin cancers were statistically significantly greater and photoaging scale severity, coarse wrinkles, thickness, and sallowness were significantly reduced in atrophic versus hypertrophic groups. Histology also revealed significantly less elastotic material in atrophic photoaging. Gene expression of matrix metalloproteinases and collagens did not differ between the 2 forms of photoaging. LIMITATIONS: The study was not designed to identify other possible subtypes of photoaging. CONCLUSION: Systematic, categorical, and quantitative clinical and histologic assessments distinguish atrophic and hypertrophic photoaging.

Pro-inflammatory agents released by pathogens, dying host cells, and neutrophils act synergistically to destroy host tissues: a working hypothesis.

We postulate that the extensive cell and tissue damage inflicted by many infectious, inflammatory and post-inflammatory episodes is an enled result of a synergism among the invading microbial agents, host neutrophils and dead and dying cells in the nidus. Microbial toxins and other metabolites along with the plethora of pro-inflammatory agents released from activated neutrophils massively recruited to the infectious sites and high levels of cationic histones, other cationic peptides, proteinases and Th1 cytokines released from activated polymorphonuclear neutrophils (PMNs) and from necrotized tissues may act in concert (synergism) to bring about cell killing and tissue destruction. Multiple, diverse interactions among the many potential pro-inflammatory moieties have been described in these complex lesions. Such infections are often seen in the skin and aerodigestive tract where the tissue is exposed to the environment, but can occur in any tissue. Commonly, the tissue-destructive infections are caused by group A streptococci, pneumococci, Staphylococcus aureus, meningococci, Escherichia coli and Shigella, although many other microbial species are seen on occasion. All these microbial agents are characterized by their ability to recruit large numbers of PMNs. Given the complex nature of the disease process, it is proposed that, to treat these multifactorial disorders, a "cocktail" of anti-inflammatory agents combined with non-bacteriolytic antibiotics and measures to counteract the critical toxic role of cationic moieties might prove more effective than a strategy based on attacking the bacteria alone.

Calcium-Induced Differentiation of Human Colon Adenomas in Colonoid Culture: Calcium Alone versus Calcium with Additional Trace Elements.

Previous murine studies have demonstrated that dietary Aquamin, a calcium-rich, multi-mineral natural product, suppressed colon polyp formation and transition to invasive tumors more effectively than calcium alone when provided over the lifespan of the animals. In the current study, we compared calcium alone to Aquamin for modulation of growth and differentiation in human colon adenomas in colonoid culture. Colonoids established from normal colonic tissue were examined in parallel. Both calcium alone at 1.5 mmol/L and Aquamin (provided at 1.5 mmol/L calcium) fostered differentiation in the adenoma colonoid cultures as compared with control (calcium at 0.15 mmol/L). When Aquamin was provided at an amount delivering 0.15 mmol/L calcium, adenoma differentiation also occurred, but was not as complete. Characteristic of colonoids undergoing differentiation was a reduction in the number of small, highly proliferative buds and their replacement by fewer but larger buds with smoother surface. Proliferation marker (Ki67) expression was reduced and markers of differentiation (CK20 and occludin) were increased along with E-cadherin translocalization to the cell surface. Additional proteins associated with differentiation/growth control [including histone-1 family members, certain keratins, NF2 (merlin), olfactomedin-4 and metallothioneins] were altered as assessed by proteomics. Immunohistologic expression of NF2 was higher with Aquamin as compared with calcium at either concentration. These findings support the conclusions that (i) calcium (1.5 mmol/L) has the capacity to modulate growth and differentiation in large human colon adenomas and (ii) Aquamin delivering 0.15 mmol/L calcium has effects on proliferation and differentiation not observed when calcium is used alone at this concentration. Cancer Prev Res; 11(7); 413-28. ©2018 AACR.

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, whereas less is known about human intestinal stem cells owing 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 four 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 sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Correction: Calcium Reduces Liver Injury in Mice on a High-Fat Diet: Alterations in Microbial and Bile Acid Profiles.

[This corrects the article DOI: 10.1371/journal.pone.0166178.].

Neuronal Protein 3.1 Deficiency Leads to Reduced Cutaneous Scar Collagen Deposition and Tensile Strength due to Impaired Transforming Growth Factor-ß1 to -ß3 Translation.

Neuronal protein 3.1 (P311), a conserved RNA-binding protein, represents the first documented protein known to stimulate transforming growth factor (TGF)-ß1 to -ß3 translation in vitro and in vivo. Because TGF-ßs play critical roles in fibrogenesis, we initiated efforts to define the role of P311 in skin scar formation. Here, we show that P311 is up-regulated in skin wounds and in normal and hypertrophic scars. Genetic ablation of p311 resulted in a significant decrease in skin scar collagen deposition. Lentiviral transfer of P311 corrected the deficits, whereas down-regulation of P311 levels by lentiviral RNA interference reproduced the deficits seen in P311-/- mice. The decrease in collagen deposition resulted in scars with reduced stiffness but also reduced scar tensile strength. In vitro studies using murine and human dermal fibroblasts showed that P311 stimulated TGF-ß1 to -ß3 translation, a process that involved eukaryotic translation initiation factor 3 subunit b as a P311 binding partner. This resulted in increased TGF-ß levels/activity and increased collagen production. In addition, P311 induced dermal fibroblast activation and proliferation. Finally, exogenous TGF-ß1 to -ß3, each restituted the normal scar phenotype. These studies demonstrate that P311 is required for the production of normal cutaneous scars and place P311 immediately up-stream of TGF-ßs in the process of fibrogenesis. Conditions that decrease P311 levels could result in less tensile scars, which could potentially lead to higher incidence of dehiscence after surgery.

Calcium Reduces Liver Injury in Mice on a High-Fat Diet: Alterations in Microbial and Bile Acid Profiles.

A high-fat "Western-style" diet (HFWD) promotes obesity-related conditions including non-alcoholic steatohepatitis (NASH), the histologic manifestation of non-alcoholic fatty liver disease (NAFLD). In addition to high saturated fat and processed carbohydrates, the typical HFWD is deficient in calcium. Calcium-deficiency is an independent risk factor for many conditions associated with the Western-style diet. However, calcium has not been widely evaluated in the context of NAFLD. The goal of the present study was to determine if dietary calcium supplementation could protect mice fed a HFWD from NAFLD, specifically by decreasing non-alcoholic steatohepatitis (NASH) and its down-stream consequences. Male C57BL/6NCrl mice were maintained for 18-months on a HFWD containing dietary calcium at either 0.41 gm/kg feed (unsupplemented) or 5.25 gm/kg feed (supplemented). Although there was no difference in body weight or steatosis, calcium-supplemented mice were protected against downstream consequences of hepatic steatosis, manifested by lower inflammation, less fibrosis, and by lower overall histologic NAFLD activity scores (NAS). Calcium supplementation correlated with distinctly segregating gut fecal and cecal microbial communities as defined by 16S rRNA gene sequence. Further, calcium supplementation also correlated with decreased hepatic concentration of the major conjugated murine primary bile acid, tauro-ß-muricholic acid (as well as a decrease in the parent unconjugated bile acid). Thus, calcium was protective against progression of diet-induced hepatic steatosis to NASH and end-stage liver disease, suggesting that calcium supplementation may effectively protect against adverse hepatic consequences of HFWD in cases where overall diet modification cannot be sustained. This protective effect occurred in concert with calcium-mediated gut microbial community shifts and alterations of the hepatic bile acid pool.

Nuclear histones: major virulence factors or just additional early sepsis markers? A comment.

In 2009, Xu et al. and Chaput et al. in Nature Medicine had argued that the main cause of death in sepsis is the release from neutrophil nets of nuclear histone, highly toxic to endothelial cells and that these polycations are major and unique virulence factors. Since 2009, numerous researchers have also suggested the involvement of histones in the pathophysiology of many clinical disorders. If histones are indeed major unique virulence toxic agents, then heparin, activated protein C and antibodies to histone should prove excellent antisepsis agents. However, this is provided that these agents are administered to patients early enough before the activation of the cytokine storms, immune responses and the coagulation cascades are irreversibly unleashed. This may not be practical, since a diagnosis of sepsis is usually made much later. Future identifications of novel early markers are therefore needed and a compilation of cocktails of antagonists may replace the faulty single antagonists tried for many years, but in vain, to prevent death in sepsis.

Iron Uptake via DMT1 Integrates Cell Cycle with JAK-STAT3 Signaling to Promote Colorectal Tumorigenesis.

Dietary iron intake and systemic iron balance are implicated in colorectal cancer (CRC) development, but the means by which iron contributes to CRC are unclear. Gene expression and functional studies demonstrated that the cellular iron importer, divalent metal transporter 1 (DMT1), is highly expressed in CRC through hypoxia-inducible factor 2α-dependent transcription. Colon-specific Dmt1 disruption resulted in a tumor-selective inhibitory effect of proliferation in mouse colon tumor models. Proteomic and genomic analyses identified an iron-regulated signaling axis mediated by cyclin-dependent kinase 1 (CDK1), JAK1, and STAT3 in CRC progression. A pharmacological inhibitor of DMT1 antagonized the ability of iron to promote tumor growth in a CRC mouse model and a patient-derived CRC enteroid orthotopic model. Our studies implicate a growth-promoting signaling network instigated by elevated intracellular iron levels in tumorigenesis, offering molecular insights into how a key dietary component may contribute to CRC.