Figure - available from: Nutrients
This content is subject to copyright.
Proteomic expression of colon mucosal biopsies and response to interventions. (A): Upregulated and downregulated proteins in each cohort at 1.5-fold. Each bar represents the number of proteins up-regulated (1.5-fold; ≤1% FDR) or down-regulated (0.67-fold; ≤1% FDR) over the 90-day course of treatment with each of the three interventions. On the right, Venn diagrams show the overlap of these upregulated and downregulated moieties to provide common proteins among 3 groups or between 2 groups or unique to an intervention. The list of these upregulated proteins is presented in Table S3. While downregulated proteins are presented in Table S4. (B): Upregulated and downregulated proteins in each cohort at 1.1-fold. Each bar represents the number of proteins up-regulated (1.1-fold; ≤1% FDR) or down-regulated (0.9-fold; ≤1% FDR) over the 90-day course of treatment with each of the three interventions. On the right, Venn diagrams show the overlap of these upregulated and downregulated moieties to provide common proteins among 3 groups or between 2 groups or unique to an intervention. (C): Differential proteomic expression of upregulated proteins (of all interventions at 1.5-fold; ≤1% FDR) is presented in a heatmap. These proteins are listed in Table S3. (D): Differential proteomic expression of downregulated proteins (of all interventions at 1.5-fold; ≤1% FDR) is presented in a heatmap. These proteins are listed in Table S4.
Source publication
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 interve...
Citations
... Improved barrier function assessed by transepithelial electrical resistance (TEER) and tissue cohesion was also observed with the same mineral supplement under both control conditions and in the presence of the pro-inflammatory stimulus. These data validated previous findings from human colon organoid studies and a biomarker trial in human subjects (Attili et al., 2019;Aslam et al., 2020;McClintock et al., 2020;Aslam et al., 2021). Overall, the data suggest that improving barrier structure and function may have implications regarding inflammatory bowel disease prevention. ...
... Past work in our laboratory has shown that a red algae-derived multi-mineral product (Aquamin®) increases the elaboration of numerous proteins that contribute to barrier structure. These studies have been conducted using human colon tissue in organoid culture (Attili et al., 2019;Aslam et al., 2020a;McClintock et al., 2020) and validated in a small interventional trial with healthy adult subjects (Aslam et al., 2021). In the organoid culture studies (using colon tissue from healthy individuals or lesional tissue from individuals with UC), we demonstrated modest changes in tight junctional protein expression with the multi-mineral intervention but much stronger upregulation of several cadherins and desmosomal proteins. ...
... Increased barrier protein expression was accompanied by improved barrier function (Schmitz et al., 1999;McGuckin et al., 2009;Salim and Söderholm, 2011;Baumgartner, 2013;Antoni et al., 2014;Ungewiß et al., 2017;Chang et al., 2018;Lee et al., 2018;Burkard et al., 2021). Findings in organoid culture were confirmed in a pilot-phase biomarker trial in healthy adults (Aslam et al., 2021). These past results highlight the importance of inorganic minerals to barrier formation in the gastrointestinal tract. ...
... Of course, demonstrating improvement in vivo will require controlled clinical studies. While such trials have not been completed at this point, a recent 90-day interventional trial with the same multi-mineral agent in healthy adults demonstrated a similar increase in barrier protein expression as observed in organoid culture (Aslam et al., 2021). Our ongoing 180-day trial (clinicaltrials.gov/: ...
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-1β 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-β 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.
... This selective process plays a crucial role in preventing the induction of inappropriate inflammatory signals. Mucosal processing of non-immunological antigens and molecules plays a crucial role in how to induce tolerogenic or non-tolerogenic immune responses in maintaining homeostasis [9,10]. ...
Probiotics play a crucial role in immunomodulation by regulating dendritic cell (DC) maturation and inducing tolerogenic DCs. Akkermansia muciniphila affects inflammatory response by elevating inhibitory cytokines. We aimed to evaluate whether Akkermansia muciniphila and its outer membrane vesicles (OMVs) affect microRNA-155, microRNA-146a, microRNA-34a, and let-7i expression of inflammatory and anti-inflammatory pathways. Peripheral blood mononuclear cells (PBMCs) were isolated from the healthy volunteers. To produce DCs, monocytes were cultivated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were allocated into six subgroups: DC + Lipopolysaccharide (LPS), DC + dexamethasone, DC + A. muciniphila (MOI 100, 50), DC + OMVs (50 µg/ml), and DC + PBS. The surface expression of human leukocyte antigen-antigen D related (HLA-DR), CD86, CD80, CD83, CD11c, and CD14 was examined using flow cytometry, and the expression of microRNAs was assessed using qRT-PCR, and the levels of IL-12 and IL-10 were measured using ELISA. A. muciniphila (MOIs 50, 100) could significantly decrease IL-12 levels relative to the LPS group. The IL-10 levels were decreased in the DC + LPS group than the DC + dexamethasone group. Treatment with A. muciniphila (MOI 100) and OMVs could elevate the concentrations of IL-10. DC treatment with LPS led to a significant increment in the expression of microRNA-155, microRNA-34a, and microRNA-146a. The expression of these microRNAs was reversed by A. muciniphilia and its OMVs treatment. Let-7i increased in treatment groups compared to the DC + LPS group. A. muciniphilia (MOI 50) had a substantial effect on the expression of HLA-DR, CD80, and CD83 on DCs. Therefore, DCs treatment with A. muciniphila led to induce tolerogenic DCs and the production of anti-inflammatory IL-10.
... 21 biomarker trial in which thirty healthy adult subjects (10 per arm) were randomized to receive Aquamin ® at a level providing 800 mg of calcium per day, calcium alone at the same level or placebo (clinicaltrials.gov: NCT02647671) (23). Before and after intervention, replicate colonic biopsies and fecal specimens were obtained from each subject. ...
Human colon organoids were maintained in culture medium alone or exposed to lipopolysaccharide with a combination of three pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β and interferon-γ [LPS-cytokines]) to mimic the environment in the inflamed colon. Untreated organoids and those exposed to LPS-cytokines were concomitantly treated with a multi-mineral 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-β and -γ 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 anti-microbial 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 multi-mineral 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.
... The eight studies included in this work were double-blind randomized clinical trials [14,[26][27][28][29][30][31][32]. A total of 5308 individuals were considered, all of them suffering from CRC or being at risk of its development. ...
... Regarding calcium supplementation, Aslam et al. [28] concluded that treatment with both Aquamin ® (composed of 30% calcium plus other minerals) and calcium monotherapy resulted in the upregulation of (i) many proapoptotic proteins, (ii) cytokeratins, (iii) cell-to- cell adhesion molecules and (iv) basement membrane components and in the (v) downregulation of nucleic acid proliferation and metabolism compared to placebo, together with promoting a preventive effect against CRC [28]. In contrast, Crockett et al. [27] suggested that supplementation with calcium carbonate or calcium plus vitamin D increased the risk of sessile serrated adenomas or colorectal polyps 6 to 10 years after starting with this supplementation, especially in women and smokers. ...
... Regarding calcium supplementation, Aslam et al. [28] concluded that treatment with both Aquamin ® (composed of 30% calcium plus other minerals) and calcium monotherapy resulted in the upregulation of (i) many proapoptotic proteins, (ii) cytokeratins, (iii) cell-to-cell adhesion molecules and (iv) basement membrane components and in the (v) downregulation of nucleic acid proliferation and metabolism compared to placebo, together with promoting a preventive effect against CRC [28]. In contrast, Crockett et al. [27] suggested that supplementation with calcium carbonate or calcium plus vitamin D increased the risk of sessile serrated adenomas or colorectal polyps 6 to 10 years after starting with this supplementation, especially in women and smokers. ...
Colorectal cancer (CRC) is currently considered one of the most common and lethal types of tumors. Nutrition is of notorious relevance, given its influence in CRC prevention and treatment. This systematic review aimed to revise and update the state of knowledge regarding the potential role of vitamin D and calcium as key factors involved in the prevention and treatment of CRC. A literature search was performed in PubMed and Web of Science. A total of eight studies were finally included in the present review. Vitamin D showed a protective role by promoting transcriptomic changes associated with antitumor effects. However, no significant effects of vitamin D were noted in the relapse-free survival of patients at 5 years. On the other hand, previous scientific evidence demonstrated that calcium regulates the expression of colonic proteins that decrease cell proliferation and increase cell differentiation. Nevertheless, an increased risk of associated serrated adenomas was found in response to calcium and calcium + vitamin D supplementation. Moreover, supplementation with both nutrients showed positive changes on relevant CRC biomarkers including TGFα, TGFβ1, APC, β-catenin and E-cadherin. In conclusion, vitamin D supplementation seems to have a protective effect in the prevention and treatment of CRC, while calcium intake showed contradictory effects as a prevention or treatment tool; therefore, further studies are necessary to well understand its relevance in patients with CRC.
... For protein mass spectrometry analysis, we employed liver tissue from five mice from each group and assessed each separately. A cryopreserved tissue piece from the left lobe (of each mouse liver) was weighed and homogenized in Radioimmuno-Precipitation Assay (RIPA) -lysis and extraction buffer (Pierce, # 89901; ThermoFisher Scientific) for protein isolation, as described in our previous publications (38)(39)(40). Fifty micrograms of sample protein from each liver were digested separately with trypsin and individual samples labeled with one of 11 isobaric mass tags following the manufacturer's protocol. Tandem Mass Tag (TMT) 11plex Isobaric Label Reagent Set (ThermoFisher Scientific) was utilized for this application. ...
... Whether the mineral supplement used here or some other formulation might provide a way to ensure adequate mineral intake remains to be seen. We have recently completed a 90-day pilot phase trial in which 30 healthy human subjects were randomized to receive Aquamin R formulated to provide 800 mg of calcium per day, calcium carbonate at the same level or placebo (38,85). To summarize, no safety or tolerability issues were seen with Aquamin R . ...
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.
... In an effort to begin addressing this issue, we have recently carried out a pilot phase trial in which 10 healthy subjects were treated with the same multi-mineral product (Aquamin R ) used here. To summarize the results of this pilot study, there were no tolerability issues with daily Aquamin R ingestion over a 90-day period and no safety concerns (55,56). Equally important, when Aquamin R -treated subjects were compared to subjects receiving placebo for the same period, we saw up-regulation of laminin chains along with increased levels of other basement membrane components and hemidesmosome moieties in colonic biopsies (56). ...
... To summarize the results of this pilot study, there were no tolerability issues with daily Aquamin R ingestion over a 90-day period and no safety concerns (55,56). Equally important, when Aquamin R -treated subjects were compared to subjects receiving placebo for the same period, we saw up-regulation of laminin chains along with increased levels of other basement membrane components and hemidesmosome moieties in colonic biopsies (56). Subjects receiving calcium alone (i.e., the most abundant mineral in Aquamin R ) also demonstrated increases in several of the same molecules, but the degree of up-regulation with calcium alone was lower than that seen with Aquamin R (56). ...
... Equally important, when Aquamin R -treated subjects were compared to subjects receiving placebo for the same period, we saw up-regulation of laminin chains along with increased levels of other basement membrane components and hemidesmosome moieties in colonic biopsies (56). Subjects receiving calcium alone (i.e., the most abundant mineral in Aquamin R ) also demonstrated increases in several of the same molecules, but the degree of up-regulation with calcium alone was lower than that seen with Aquamin R (56). ...
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.
... ThermoFisher Scientific) for protein isolation, as described in our previous publications (37)(38)(39). Fifty micrograms of sample protein from each liver were digested separately with trypsin and individual samples labeled with one of 11 isobaric mass tags following the manufacturer's protocol. Tandem Mass Tag (TMT)11plex Isobaric Label Reagent Set (ThermoFisher Scientific) was utilized for this application. ...
... We have recently completed a 90-day pilot phase trial in which 30 healthy human subjects were randomized to receive Aquamin ® formulated to provide 800 mg of calcium per day, calcium carbonate at the same level or placebo (37,77). To summarize, no safety or tolerability issues were seen with Aquamin ® . ...
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.
... And of course, the decrease in death rate is also because of improved treatment options and targeted therapies [12]. Similarly, dietary and chemopreventive measures also play a role in decreasing the overall incidence of CRC [13,14]. ...
Colonoscopy procedure has been the key screening method to detect colorectal cancer (CRC). As a fatal disease, CRC needs early detection. The COVID-19 pandemic caused screening tests (colonoscopy) to be halted and delayed. As a result, there could be dire consequences such as later-stage or missed diagnosis or greater mortality. This report will analyze scientific literature pertaining to interrupted CRC screenings due to COVID-19 while drawing historical parallels from the 1918 flu pandemic. We conducted literature searches in the PubMed database as well as in Google Scholar. One of the main lessons learned from the 1918 flu pandemic was to employ social distancing to stop the spread of the virus. So, the global response at the start and peak of the COVID-19 pandemic was decreased hospital visits for any non-emergency cases. That led to a halt and delays in cancer (including CRC) screenings. The Medical community predicted this lag will cause more CRC cases and deaths in the future. However, reorganizing and changing screening method strategies were helpful during the ongoing pandemic. In conclusion, COVID-19 greatly affected CRC screening, including how we view the future of CRC screening. We can learn from this prospect to better prepare for future pandemics or other public health crises.
