MACC1 Regulates LGR5 to Promote Cancer Stem Cell Properties in Colorectal Cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease, which is thought to be initiated by colon cancer stem cells, according to the cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcomes. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our MACC1 transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and the CSC-associated invasive phenotype based on patient-derived tumor organoids (PDOs), patient-derived xenografts (PDXs) and human CRC cell lines. We showed that CD44-enriched CSCs from PDO models express significantly higher levels of MACC1 and LGR5 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PDO and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with the CSC markers CD44, NANOG and LGR5 in PDO models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter, uncovering the long-known CSC marker LGR5 as a novel essential signaling mediator employed by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by a significant positive correlation of MACC1 with LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis inducer MACC1 acts as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs and prevent cancer recurrence and distant metastasis formation.

Impact of social distancing from the COVID-19 pandemic on the immuno-inflammatory response of older adults.

BACKGROUND: Older adults, as the population considered at increased risk for severe COVID-19, were the most impacted by social isolation. Thus, this study aimed to assess the salivary immune/inflammatory response of older adults before and during the COVID-19 pandemic. METHODS: A cohort of 11 older adults (mean age 66.8 ± 6.1) was followed at three different time points: before (S1) and after 6 (S2) and 20 months (S3) of the beginning of the COVID-19 pandemic in Brazil. Unstimulated saliva samples were obtained to assess the levels of antibodies (secretory IgA, IgG and IgM) by ELISA and cytokines (IL-2, IL-5, IL-6, IL-8 and IL-10, TSLP, IFN-γ, TNF-α) by multiplex analysis. Significant differences were evaluated using the Kruskal-Wallis test with Dunn's post-test. RESULTS: None volunteer presented periodontal disease or caries. All volunteers received at least two doses of the COVID-19 vaccines after S2 and before S3. A tendency to increase salivary levels of SIgA and IgM at S2 and of IgG at S3 were observed compared to the values found at S1 and S2. Significantly decreased levels of IL-2 and IL-5 were found at S2 and S3 (p < 0.001) time points. Lower levels of IFN-γ were found at S2 as compared to the values observed at S1 (p < 0.01). A significant decrease in the IFN-γ/IL-10 ratio was found at S2 (p < 0.01). When assessing the Th1/Th2 ratios, a significant decrease was found in the IFN-γ/TSLP ratio at S2 (p < 0.001) and S3 (p < 0.001) when compared to the values at S1. In addition, a significant increase was observed in the TNF-α/IL-5 ratio at S2 (p < 0.001) and S3 (p < 0.001) in comparison to the values at S1. In a similar way, an increase in the TNF-α/IL-6 ratio (Fig. 5E) was observed at S3 (p < 0.001) when compared to the values at S1. CONCLUSIONS: Overall, this study provides valuable insights into the impact of COVID-19-induced social isolation on immune/inflammatory responses in the upper airway mucosa, particularly those present in oral cavity, of older adults. It demonstrates that a controlled shift in Th1 and Th2 immune responses, both during infection and post-vaccination, can create favorable conditions to combat viral infections without exacerbating the immune response or worsening the pathology.

Organic Matrix Derived from Host-Microbe Interplay Contributes to Pathological Renal Biomineralization.

Matrix stones are a rare form of kidney stones. They feature a high percentage of hydrogel-like organic matter, and their formation is closely associated with urinary tract infections. Herein, comprehensive materials and biochemical approaches were taken to map the organic-inorganic interface and gather insights into the host-microbe interplay in pathological renal biomineralization. Surgically extracted soft and slimy matrix stones were examined using micro-X-ray computed tomography and various microspectroscopy techniques. Higher-mineral-density laminae were positive for calcium-bound Alizarin red. Lower-mineral-density laminae revealed periodic acid-Schiff-positive organic filamentous networks of varied thickness. These organic filamentous networks, which featured a high polysaccharide content, were enriched with zinc, carbon, and sulfur elements. Neutrophil extracellular traps (NETs) along with immune response-related proteins, including calprotectin, myeloperoxidase, CD63, and CD86, also were identified in the filamentous networks. Expressions of NETs and upregulation of polysaccharide-rich mucin secretion are proposed as a part of the host immune defense to "trap" pathogens. These host-microbe derived organic matrices can facilitate heterogeneous nucleation and precipitation of inorganic particulates, resulting in macroscale aggregates known as "matrix stones". These insights into the plausible aggregation of constituents through host-microbe interplay underscore the unique "double-edged sword" effect of the host immune response to pathogens and the resulting renal biominerals.

Specific host metabolite and gut microbiome alterations are associated with bone loss during spaceflight.

Understanding the axis of the human microbiome and physiological homeostasis is an essential task in managing deep-space-travel-associated health risks. The NASA-led Rodent Research 5 mission enabled an ancillary investigation of the gut microbiome, varying exposure to microgravity (flight) relative to ground controls in the context of previously shown bone mineral density (BMD) loss that was observed in these flight groups. We demonstrate elevated abundance of Lactobacillus murinus and Dorea sp. during microgravity exposure relative to ground control through whole-genome sequencing and 16S rRNA analyses. Specific functionally assigned gene clusters of L. murinus and Dorea sp. capable of producing metabolites, lactic acid, leucine/isoleucine, and glutathione are enriched. These metabolites are elevated in the microgravity-exposed host serum as shown by liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomic analysis. Along with BMD loss, ELISA reveals increases in osteocalcin and reductions in tartrate-resistant acid phosphatase 5b signifying additional loss of bone homeostasis in flight.

Transcriptome of Epibiont Saccharibacteria Nanosynbacter lyticus Strain TM7x During the Establishment of Symbiosis.

Saccharibacteria Nanosynbacter lyticus strain TM7x is a member of the broadly distributed candidate phylum radiation. These bacteria have ultrasmall cell sizes, have reduced genomes, and live as epibionts on the surfaces of other bacteria. The mechanisms by which they establish and maintain this relationship are not yet fully understood. The transcriptomes of the epibiont TM7x and its host bacteria Schaalia odontolytica strain XH001 were captured across the establishment of symbiosis during both the initial interaction and stable symbiosis. The results showed a dynamic interaction with large shifts in gene expression for both species between the initial encounter and stable symbiosis, notably in transporter genes. During stable symbiosis, the host XH001 showed higher gene expression for peptidoglycan biosynthesis, mannosylation, cell cycle and stress-related genes, whereas it showed lower expression of chromosomal partitioning genes. This was consistent with the elongated cell shape seen in XH001 infected with TM7x and our discovery that infection resulted in thickened cell walls. Within TM7x, increased pili, type IV effector genes, and arginine catabolism/biosynthesis gene expression during stable symbiosis implied a key role for these functions in the interaction. Consistent with its survival and persistence in the human microbiome as an obligate epibiont with reduced de novo biosynthetic capacities, TM7x also showed higher levels of energy production and peptidoglycan biosynthesis, but lower expression of stress-related genes, during stable symbiosis. These results imply that TM7x and its host bacteria keep a delicate balance in order to sustain an episymbiotic lifestyle. IMPORTANCE Nanosynbacter lyticus type strain TM7x is the first cultivated member of the Saccharibacteria and the candidate phyla radiation (CPR). It was discovered to be ultrasmall in cell size with a highly reduced genome that establishes an obligate epibiotic relationship with its host bacterium. The CPR is a large, monophyletic radiation of bacteria with reduced genomes that includes Saccharibacteria. The vast majority of the CPR have yet to be cultivated, and our insights into these unique organisms to date have been derived from only a few Saccharibacteria species. Being obligate parasites, it is unknown how these ultrasmall Saccharibacteria, which are missing many de novo biosynthetic pathways, are maintained at a high prevalence within the human microbiome as well as in the environment.

Episymbiotic Saccharibacteria suppresses gingival inflammation and bone loss in mice through host bacterial modulation.

Saccharibacteria (TM7) are obligate epibionts living on the surface of their host bacteria and are strongly correlated with dysbiotic microbiomes during periodontitis and other inflammatory diseases, suggesting they are putative pathogens. However, due to the recalcitrance of TM7 cultivation, causal research to investigate their role in inflammatory diseases is lacking. Here, we isolated multiple TM7 species on their host bacteria from periodontitis patients. These TM7 species reduce inflammation and consequential bone loss by modulating host bacterial pathogenicity in a mouse ligature-induced periodontitis model. Two host bacterial functions involved in collagen binding and utilization of eukaryotic sialic acid are required for inducing bone loss and are altered by TM7 association. This TM7-mediated downregulation of host bacterial pathogenicity is shown for multiple TM7/host bacteria pairs, suggesting that, in contrast to their suspected pathogenic role, TM7 could protect mammalian hosts from inflammatory damage induced by their host bacteria.

Safety and Preliminary Efficacy of a Novel Host-Modulatory Therapy for Reducing Gingival Inflammation.

Background: Periodontal disease is among the sixth most common inflammatory diseases worldwide with high risk to promote complications from other inflammatory diseases including diabetes, cardiovascular disease and Alzheimer's Disease. Failure of active resolution of inflammation pathways is implicated in pathogenesis of periodontal diseases, including gingivitis. Lipoxin A4 (LXA4), a member of the specialized pro-resolving lipid mediators (SPMs) that drive resolution of inflammation via GPC-receptor mediated pathways, offered therapeutic advantages in preclinical models of periodontitis. Methods: We conducted a randomized, placebo-controlled, parallel-group Phase 1 clinical trial to determine the safety and preliminary efficacy of an LXA4 analog in patients with gingival inflammation. One hundred twenty-seven (127) individuals were randomized to daily use of an oral rinse containing a LXA4 mimetic, methyl ester-benzo-lipoxin A4 (BLXA4), placebo rinse or a no-rinse control group for 28 days. Treatment emergent adverse events (TEAEs) were assessed for safety, the primary outcome. Secondary outcomes included the change in the level of gingival inflammation and periodontal pocket depth (PD). Serum SPMs were monitored using targeted lipid mediator lipidomics to assess potential systemic impact of BLXA4. Results: The frequency of TEAEs was similar in BLXA4 and placebo-treated groups with no study-related SAEs. Once-daily rinsing with BLXA4 for 28-days resulted in a greater decrease in gingival inflammation compared to placebo rinse and no-rinse control groups (mean change: 0.26 GI unit vs 0.21 and 0.17, respectively). PD reduction was also greater with BLXA4 oral rinse compared to placebo and no-rinse groups (mean reduction: 1.23 mm vs. 0.71 mm and 0.46 mm, respectively). Topical application of BLXA4 increased serum levels of SPMs. Conclusion: Treatment with BLXA4 reduces local inflammation, and increases abundance of pro-resolution molecules systemically, which may dampen inflammation that can mediate progression and course of inflammatory diseases beyond periodontitis. Clinical Trial Registration: ClinicalTrials.gov, identifier (NCT02342691).

Regression of human coronary artery plaque is associated with a high ratio of (18-hydroxy-eicosapentaenoic acid + resolvin E1) to leukotriene B4.

Inflammation in arterial walls leads to coronary artery disease (CAD). We previously reported that a high omega-3 fatty index was associated with prevention of progression of coronary atherosclerosis, a disease of chronic inflammation in the arterial wall. However, the mechanism of such benefit is unclear. The two main omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are precursors of specialized pro-resolving lipid mediators (SPMs)-resolvins and maresins-which actively resolve chronic inflammation. To explore whether SPMs are associated with coronary plaque progression, levels of SPMs and proinflammatory mediators (leukotriene B4 [LTB4 ] and prostaglandins) were measured using liquid chromatography-tandem mass spectrometry in 31 statin-treated patients with stable CAD randomized to either EPA and DHA, 3.36 g daily, or no EPA/DHA (control). Coronary plaque volume was measured by coronary computed tomographic angiography at baseline and at 30-month follow-up. Higher plasma levels of EPA+DHA were associated with significantly increased levels of two SPMs-resolvin E1 and maresin 1-and 18-hydroxy-eicosapentaenoic acid (HEPE), the precursor of resolvin E1. Those with low plasma EPA+DHA levels had a low (18-HEPE+resolvin E1)/LTB4 ratio and significant plaque progression. Those with high plasma EPA+DHA levels had either low (18-HEPE+resolvin E1)/LTB4 ratios with significant plaque progression or high (18-HEPE+resolvin E1)/LTB4 ratios with significant plaque regression. These findings suggest that an imbalance between pro-resolving and proinflammatory lipid mediators is associated with plaque progression and potentially mediates the beneficial effects of EPA and DHA in CAD patients.

Spatial survey of non-collagenous proteins in mineralizing and non-mineralizing vertebrate tissues ex vivo.

Bone biomineralization is a complex process in which type I collagen and associated non-collagenous proteins (NCPs), including glycoproteins and proteoglycans, interact closely with inorganic calcium and phosphate ions to control the precipitation of nanosized, non-stoichiometric hydroxyapatite (HAP, idealized stoichiometry Ca10(PO4)6(OH)2) within the organic matrix of a tissue. The ability of certain vertebrate tissues to mineralize is critically related to several aspects of their function. The goal of this study was to identify specific NCPs in mineralizing and non-mineralizing tissues of two animal models, rat and turkey, and to determine whether some NCPs are unique to each type of tissue. The tissues investigated were rat femur (mineralizing) and tail tendon (non-mineralizing) and turkey leg tendon (having both mineralizing and non-mineralizing regions in the same individual specimen). An experimental approach ex vivo was designed for this investigation by combining sequential protein extraction with comprehensive protein mapping using proteomics and Western blotting. The extraction method enabled separation of various NCPs based on their association with either the extracellular organic collagenous matrix phases or the inorganic mineral phases of the tissues. The proteomics work generated a complete picture of NCPs in different tissues and animal species. Subsequently, Western blotting provided validation for some of the proteomics findings. The survey then yielded generalized results relevant to various protein families, rather than only individual NCPs. This study focused primarily on the NCPs belonging to the small leucine-rich proteoglycan (SLRP) family and the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). SLRPs were found to be associated only with the collagenous matrix, a result suggesting that they are mainly involved in structural matrix organization and not in mineralization. SIBLINGs as well as matrix Gla (γ-carboxyglutamate) protein were strictly localized within the inorganic mineral phase of mineralizing tissues, a finding suggesting that their roles are limited to mineralization. The results from this study indicated that osteocalcin was closely involved in mineralization but did not preclude possible additional roles as a hormone. This report provides for the first time a spatial survey and comparison of NCPs from mineralizing and non-mineralizing tissues ex vivo and defines the proteome of turkey leg tendons as a model for vertebrate mineralization.

The relationship between specialized pro-resolving lipid mediators, morbid obesity and weight loss after bariatric surgery.

Obesity and diabetes are associated with chronic inflammation. Specialized pro-resolving lipid mediators (SPMs)-resolvins (Rv), protectins (PD) and maresins (MaR)-actively resolve inflammation. Bariatric surgery achieves remission of diabetes, but mechanisms are unclear. We measured SPMs and proinflammatory eicosanoid levels using liquid chromatography-tandem mass spectrometry in 29 morbidly obese subjects (13 with diabetes) and 15 nondiabetic, mildly obese subjects. Compared to the mildly obese, the morbidly obese had higher levels of SPMs-RvD3, RvD4 and PD1-and white blood cells (WBC) and platelets. Post-surgery, SPM and platelet levels decreased in morbidly obese nondiabetic subjects but not in diabetic subjects, suggesting continued inflammation. Despite similar weight reductions 1 year after surgery (44.6% vs. 46.6%), 8 diabetes remitters had significant reductions in WBC and platelet counts whereas five non-remitters did not. Remitters had a 58.2% decrease (p = 0.03) in 14-HDHA, a maresin pathway marker; non-remitters had an 875.7% increase in 14-HDHA but a 36.9% decrease in MaR1 to a median of 0. In conclusion, higher levels of RvD3, PD1 and their pathway marker, 17-HDHA, are markers of leukocyte activation and inflammation in morbid obesity and diabetes and diminish with weight loss in nondiabetic but not diabetic subjects, possibly representing sustained inflammation in the latter. Lack of diabetes remission after surgically-induced weight loss may be associated with reduced ability to produce MaR1 and sustained inflammation.

Maresin-1 and Resolvin E1 Promote Regenerative Properties of Periodontal Ligament Stem Cells Under Inflammatory Conditions.

Maresin-1 (MaR1) and Resolvin E1 (RvE1) are specialized pro-resolving lipid mediators (SPMs) that regulate inflammatory processes. We have previously demonstrated the hard and soft tissue regenerative capacity of RvE1 in an in vivo model of the periodontal disease characterized by inflammatory tissue destruction. Regeneration of periodontal tissues requires a well-orchestrated process mediated by periodontal ligament stem cells. However, limited data are available on how SPMs can regulate the regenerative properties of human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions. Thus, we measured the impact of MaR1 and RvE1 in an in vitro model of hPDLSC under stimulation with IL-1ß and TNF-α by evaluating pluripotency, migration, viability/cell death, periodontal ligament markers (α-smooth muscle actin, tenomodulin, and periostin), cementogenic-osteogenic differentiation, and phosphoproteomic perturbations. The data showed that the pro-inflammatory milieu suppresses pluripotency, viability, and migration of hPDLSCs; MaR1 and RvE1 both restored regenerative capacity by increasing hPDLSC viability, accelerating wound healing/migration, and up-regulating periodontal ligament markers and cementogenic-osteogenic differentiation. Protein phosphorylation perturbations were associated with the SPM-induced regenerative capacity of hPDLSCs. Together, these results demonstrate that MaR1 and RvE1 restore or improve the regenerative properties of highly specialized stem cells when inflammation is present and offer opportunities for direct pharmacologic treatment of lost tissue integrity.

Salivary metabolite levels in perinatally HIV-infected youth with periodontal disease.

INTRODUCTION: Salivary metabolite profiles are altered in adults with HIV compared to their uninfected counterparts. Less is known about youth with HIV and how oral disorders that commonly accompany HIV infection impact salivary metabolite levels. OBJECTIVE: As part of the Adolescent Master Protocol multi-site cohort study of the Pediatric HIV/AIDS Cohort Study (PHACS) network we compared the salivary metabolome of youth with perinatally-acquired HIV (PHIV) and youth HIV-exposed, but uninfected (PHEU) and determined whether metabolites differ in PHIV versus PHEU. METHODS: We used three complementary targeted and discovery-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflows to characterize salivary metabolite levels in 20 PHIV and 20 PHEU youth with and without moderate periodontitis. We examined main effects associated with PHIV and periodontal disease, and the interaction between them. RESULTS: We did not identify differences in salivary metabolite profiles that remained significant under stringent control for both multiple between-group comparisons and multiple metabolites. Levels of cadaverine, a known periodontitis-associated metabolite, were more abundant in individuals with periodontal disease with the difference being more pronounced in PHEU than PHIV. In the discovery-based dataset, we identified a total of 564 endogenous peptides in the metabolite extracts, showing that proteolytic processing and amino acid metabolism are important to consider in the context of HIV infection. CONCLUSION: The salivary metabolite profiles of PHIV and PHEU youth were overall very similar. Individuals with periodontitis particularly among the PHEU youth had higher levels of cadaverine, suggesting that HIV infection, or its treatment, may influence the metabolism of oral bacteria.

Sex-mediated elevation of the specialized pro-resolving lipid mediator levels in a Sjögren's syndrome mouse model.

Our previous results showed that the specialized pro-resolving mediator (SPM) Resolvin D1 (RvD1) promotes resolution of inflammation in salivary glands in non-obese diabetic (NOD)/ShiLtJ, a mouse model for Sjögren's syndrome (SS). Additionally, mice lacking the RvD1 receptor ALX/FPR2 show defective innate and adaptive immune responses in salivary glands. Particularly, ALX/FPR2 KO mice exhibit exacerbated inflammation in their salivary glands in response to systemic LPS treatment. Moreover, female ALX/FPR2 KO mice show increased autoantibody production and loss of salivary gland function with age. Together, these studies suggest that an underlying SPM dysregulation could be contributing to SS progression. Therefore, we investigated whether SPM production is altered in NOD/ShiLtJ using metabololipidomics and enzyme-linked immunosorbent assay (ELISA). Our results demonstrate that SPM levels were broadly elevated in plasma collected from NOD/ShiLtJ female mice after disease onset, whereas these drastic changes did not occur in male mice. Moreover, gene expression of enzymes involved in SPM biosynthesis were altered in submandibular glands (SMG) from NOD/ShiLtJ female mice after disease onset, with 5-LOX and 12/15-LOX being downregulated and upregulated, respectively. Despite this dysregulation, the abundances of the SPM products of these enzymes (ie, RvD1 and RvD2) were unaltered in freshly isolated SMG cells suggesting that other cell populations (eg, lymphocytes) may be responsible for the overabundance of SPMs that we observed. The elevation of SPMs noted here appeared to be sex mediated, meaning that it was observed only in one sex (females). Given that SS primarily affects females (roughly 90% of diagnosed cases), these results may provide some insights into the mechanisms underlying the observed sexual dimorphism.

Human and Nonhuman Primate Lineage-Specific Footprints in the Salivary Proteome.

Proteins in saliva are needed for preprocessing food in the mouth, maintenance of tooth mineralization, and protection from microbial pathogens. Novel insights into human lineage-specific functions of salivary proteins and clues to their involvement in human disease can be gained through evolutionary studies, as recently shown for salivary amylase AMY1 and salivary agglutinin DMBT1/gp340. However, the entirety of proteins in saliva, the salivary proteome, has not yet been investigated from an evolutionary perspective. Here, we compared the proteomes of human saliva and the saliva of our closest extant evolutionary relatives, chimpanzees and gorillas, using macaques as an outgroup, with the aim to uncover features in saliva protein composition that are unique to each species. We found that humans produce a waterier saliva, containing less than half total protein than great apes and Old World monkeys. For all major salivary proteins in humans, we could identify counterparts in chimpanzee and gorilla saliva. However, we discovered unique protein profiles in saliva of humans that were distinct from those of nonhuman primates. These findings open up the possibility that dietary differences and pathogenic pressures may have shaped a distinct salivary proteome in the human lineage.

Mapping Relative Differences in Human Salivary Gland Secretions by Dried Saliva Spot Sampling and nanoLC-MS/MS.

Dried saliva spot sampling is a minimally invasive technique for the spatial mapping of salivary protein distribution in the oral cavity. In conjunction with untargeted nano-flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) analysis, DSS is used to compare the proteomes secreted by unstimulated parotid and submandibular/sublingual salivary glands. Two hundred and twenty proteins show a statistically significant association with parotid gland secretion, while 30 proteins are at least tenfold more abundant in the submandibular/sublingual glands. Protein identifications and label-free quantifications are highly reproducible across the paired glands on three consecutive days, enabling to establish the core proteome of glandular secretions categorized into eight salivary protein groups according to their biological functions. The data suggest that the relative contributions of the salivary glands fine-tune the biological activity of human saliva via medium-abundant proteins. A number of biomarker candidates for Sjögren's syndrome are observed among the gland-specifically expressed proteins, which indicates that glandular origin is an important factor to consider in salivary biomarker discovery.

Mapping the Tooth Enamel Proteome and Amelogenin Phosphorylation Onto Mineralizing Porcine Tooth Crowns.

Tooth enamel forms in an ephemeral protein matrix where changes in protein abundance, composition and posttranslational modifications are critical to achieve healthy enamel properties. Amelogenin (AMELX) with its splice variants is the most abundant enamel matrix protein, with only one known phosphorylation site at serine 16 shown in vitro to be critical for regulating mineralization. The phosphorylated form of AMELX stabilizes amorphous calcium phosphate, while crystalline hydroxyapatite forms in the presence of the unphosphorylated protein. While AMELX regulates mineral transitions over space and time, it is unknown whether and when un-phosphorylated amelogenin occurs during enamel mineralization. This study aims to reveal the spatiotemporal distribution of the cleavage products of the most abundant AMLEX splice variants including the full length P173, the shorter leucine-rich amelogenin protein (LRAP), and the exon 4-containing P190 in forming enamel, all within the context of the changing enamel matrix proteome during mineralization. We microsampled permanent pig molars, capturing known stages of enamel formation from both crown surface and inner enamel. Nano-LC-MS/MS proteomic analyses after tryptic digestion rendered more than 500 unique protein identifications in enamel, dentin, and bone. We mapped collagens, keratins, and proteolytic enzymes (CTSL, MMP2, MMP10) and determined distributions of P173, LRAP, and P190 products, the enamel proteins enamelin (ENAM) and ameloblastin (AMBN), and matrix-metalloprotease-20 (MMP20) and kallikrein-4 (KLK4). All enamel proteins and KLK4 were near-exclusive to enamel and in excellent agreement with published abundance levels. Phosphorylated P173 and LRAP products decreased in abundance from recently deposited matrix toward older enamel, mirrored by increasing abundances of testicular acid phosphatase (ACPT). Our results showed that hierarchical clustering analysis of secretory enamel links closely matching distributions of unphosphorylated P173 and LRAP products with ACPT and non-traditional amelogenesis proteins, many associated with enamel defects. We report higher protein diversity than previously published and Gene Ontology (GO)-defined protein functions related to the regulation of mineral formation in secretory enamel (e.g., casein α-S1, CSN1S1), immune response in erupted enamel (e.g., peptidoglycan recognition protein, PGRP), and phosphorylation. This study presents a novel approach to characterize and study functional relationships through spatiotemporal mapping of the ephemeral extracellular matrix proteome.

Salivary N1-Methyl-2-Pyridone-5-Carboxamide, a Biomarker for Uranium Uptake, in Kuwaiti Children Exhibiting Exceptional Weight Gain.

In a longitudinal study of 6,158 Kuwaiti children, we selected 94 for salivary metabolomic analysis who were neither obese (by waist circumference) nor metabolic syndrome (MetS) positive (<3 diagnostic features). Half (43) remained healthy for 2 years. The other half (51) were selected because they became obese and MetS positive 2 years later. In the half becoming obese, metabolomic analysis revealed that the level of salivary N1-Methyl-2-pyridone-5-carboxamide (2PY) had the highest positive association with obesity (p = 0.0003, AUC = 0.72) of 441 salivary biochemicals detected. 2PY is a recognized uremic toxin. Also, 2PY has been identified as a biomarker for uranium uptake. Considering that a relatively recent military conflict with documented uranium contamination of the area suggests that this weight gain could be a toxicological effect of long-time, low-level uranium ingestion. Comparison of salivary 2PY in samples from the USA and Kuwait found that only Kuwait samples were significantly related to obesity. Also, the geographic distribution of both reported soil radioactivity from 238U and measured salivary 2PY was highest in the area where military activity was highest. The prevalence pattern of adult diabetes in Kuwait suggests that a transient diabetogenic factor has been introduced into the Kuwaiti population. Although we did not measure uranium in our study, the presence of a salivary biomarker for uranium consumption suggests potential toxicity related to obesity in children.

The double-edged role of copper in the fate of amyloid beta in the presence of anti-oxidants.

The biological fate of amyloid beta (Aß) species is a fundamental question in Alzheimer's disease (AD) pathogenesis. The competition between clearance and aggregation of Aßs is critical for the onset of AD. Copper has been widely considered to be an inducer of harmful crosslinking of Aßs, and an important triggering factor for the onset of AD. In this report, however, we present data to show that copper can also be an inducer of Aß degradation in the presence of a large excess of well-known intrinsic (such as dopamine) or extrinsic (such as vitamin C) anti-oxidants. The degraded fragments were identified using SDS-Page gels, and validated via nanoLC-MS/MS. A tentative mechanism for the degradation was proposed and validated with model peptides. In addition, we performed electrophysiological analysis to investigate the synaptic functions in brain slices, and found that in the presence of a significant excess of vitamin C, Cu(ii) could prevent an Aß-induced deficit in synaptic transmission in the hippocampus. Collectively, our evidence strongly indicated that a proper combination of copper and anti-oxidants might have a positive effect on the prevention of AD. This double-edged function of copper in AD has been largely overlooked in the past. We believe that our report is very important for fully understanding the function of copper in AD pathology.

A robust protocol for the isolation of cellular proteins from Xanthomonas campestris to analyze the methionine effect in 2D-gel experiments.

Two-dimensional PAGE (2D-PAGE) is a key technique for the separation of complex protein samples to survey the protein inventory of prokaryotic microorganisms. Although the preparation of proteins is a critical step in 2D gel experiments, its effect on the outcome of proteome data is often underestimated. In this work, we show that the choice of protein isolation method can have a profound impact on the quality of 2D gels of protein extracts from prokaryotes. Based on Xanthomonas campestris, a commercially relevant producer of the thickening agent xanthan, we validated a phenol extraction protocol for the purification of bacterial proteins that provides excellent 2D gel separation. As a proof of concept, this method was used to study the effect of methionine-a medium compound that reduces the xanthan output of industrial fermentations-on the cellular proteome of Xanthomonas. The detection of nine regulated proteins associated with sulfur metabolism (Cgl, CysI, CysJ, CysK, MetH1, MetY) and sugar nucleotide biosynthesis (Pgi, Ugd, XanA) proved the efficiency of phenol extraction for the screening of statistically significant abundance changes in 2D gel spots and MALDI-TOF-MS based identification in bacteria. Since this method is very robust, it may be useful for the study of other prokaryotes that are relevant in industrial biotechnology.

MACC1 Induces Tumor Progression in Transgenic Mice and Colorectal Cancer Patients via Increased Pluripotency Markers Nanog and Oct4.

PURPOSE: We have previously identified the gene MACC1 as a strong prognostic biomarker for colorectal cancer metastasis and patient survival. Here, we report for the first time the generation of transgenic mouse models for MACC1. EXPERIMENTAL DESIGN: We generated mice with transgenic overexpression of MACC1 in the intestine driven by the villin promoter (vil-MACC1) and crossed them with Apc(Min) mice (vil-MACC1/Apc(Min)). RESULTS: vil-MACC1/Apc(Min) mice significantly increased the total number of tumors (P = 0.0056). This was particularly apparent in large tumors (≥3-mm diameter; P = 0.0024). A detailed histopathologic analysis of these lesions demonstrated that the tumors from the vil-MACC1/Apc(Min) mice had a more invasive phenotype and, consequently, showed a significantly reduced survival time than Apc(Min) mice (P = 0.03). Molecular analysis revealed an increased Wnt and pluripotency signaling in the tumors of vil-MACC1/Apc(Min) mice. Specifically, we observed a prominent upregulation of the pluripotency markers Oct4 and Nanog in these tumors compared with Apc(Min) controls. Finally, we could also validate that Oct4 and Nanog are regulated by MACC1 in vitro and strongly correlate with MACC1 levels in a cohort of 60 tumors of colorectal cancer patients (r = 0.7005 and r = 0.6808, respectively; P > 0.0001 and P > 0.0002, respectively). CONCLUSIONS: We provide proof of principle that MACC1-induced tumor progression in colorectal cancer acts, at least in part, via the newly discovered MACC1/Nanog/Oct4 axis. These findings might have important implications for the design of novel therapeutic intervention strategies to restrict tumor progression. Clin Cancer Res; 22(11); 2812-24. ©2016 AACR.