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1.
Nat Protoc ; 2024 Jul 18.
Article in English | MEDLINE | ID: mdl-39026121

ABSTRACT

Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan-microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health.

2.
Sci Rep ; 14(1): 11261, 2024 05 17.
Article in English | MEDLINE | ID: mdl-38760405

ABSTRACT

Here, we focused on the role of Nucleobindin 2 (NUCB2), a multifunctional protein, in gastric carcinoma (GC) progression. NUCB2 expression was investigated in 150 GC cases (20 non-invasive (pT1) and 130 invasive (pT2/pT3/pT4) tumors) by immunohistochemistry (IHC), and in situ hybridization for detection of the mRNA in 21 cases. Using GC cell lines, we determined whether NUCB2 expression was associated with specific cellular phenotypes. In GC clinical samples, NUCB2 was transcriptionally upregulated when compared to normal tissues. High NUCB2 expression was associated with clinicopathological factors including deep tumor invasion, lymphovascular invasion, lymph node metastasis, and advanced clinical stages, and was a significant independent predictor of unfavorable progression-free survival in 150 non-invasive and invasive GC patients. Similar findings were also evident in 72 invasive GC cases in which patients received post-operative chemotherapy, but not in 58 invasive tumors from patients who did not receive the chemotherapy. In cell lines, NUCB2 knockout inhibited proliferation, susceptibility to apoptosis, and migration capability by inducting cellular senescence; this was consistent with higher proliferation and apoptotic indices in the NUCB2 IHC-high compared to NUCB2 IHC-low GC cases. NUCB2-dependent inhibition of senescence in GC engenders aggressive tumor behavior by modulating proliferation, apoptosis, and migration.


Subject(s)
Cellular Senescence , Nucleobindins , Stomach Neoplasms , Humans , Stomach Neoplasms/pathology , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Nucleobindins/metabolism , Female , Male , Cell Line, Tumor , Middle Aged , Aged , Cell Proliferation , Gene Expression Regulation, Neoplastic , Apoptosis , Cell Movement , Prognosis
3.
PLoS One ; 19(4): e0296840, 2024.
Article in English | MEDLINE | ID: mdl-38625852

ABSTRACT

Kale is a nutrient-dense leafy vegetable associated with wide-ranging health benefits. It is tolerant of drought and temperature fluctuations, and could thus serve an increasingly important role in providing a safe and nutritious food supply during the climate crisis, while kale's ease of cultivation and ability to be grown in a wide range of soils make it a good fit for urban agriculture. In this pilot study we explored potential differences between kale grown at urban versus rural farms. We planted kale seedlings (Darkibor variety) at three urban and four rural farms in and around Baltimore City, Maryland, instructed farmers to cultivate them using their usual growing practices, harvested the kale from fields and points of distribution, and analyzed it for concentrations of carotenoids, vitamins C and K1, ten nutritional elements, and eight non-essential metals. Although sample sizes for some analyses were in some cases too small to produce statistically significant results, we identified potentially meaningful differences in concentrations of several components between urban and rural kale samples. Compared to urban samples, mean concentrations of carotenoids and vitamins were 22-38% higher in rural field samples. By contrast, mean concentrations for eight nutritional elements were higher in urban field samples by as much as 413% for iron. Compared to rural field samples, mean concentrations of nine non-essential metals were higher in urban samples, although lead and cadmium concentrations for all samples were below public health guidelines. Some urban-rural differences were more pronounced than those identified in prior research. For six elements, variance within urban and rural farms was greater than variance between urban and rural farms, suggesting urbanicity may not be the primary driver of some observed differences. For some nutrients, mean concentrations were higher than upper ranges reported in prior estimates, suggesting kale may have the potential to be more nutrient-dense than previously estimated. The nutritive and metals composition of this important crop, and the factors that influence it, merit continued investigation given its growing popularity.


Subject(s)
Brassica , Pilot Projects , Farms , Nutrients , Vitamins , Carotenoids
4.
Am J Pathol ; 194(3): 459-470, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38096983

ABSTRACT

Notch signaling contributes to tissue development and homeostasis, but little is known about its role in morular differentiation of endometrial carcinoma (Em Ca) cells. The current study focused on crosstalk between Notch and ß-catenin signaling in Em Ca with morules. Promoters of hairy and enhancer of split 1 (Hes1) and mastermind-like 2 (MAML2) were activated by Notch intracellular domain 1 but not ß-catenin, and a positive feedback loop between Hes1 and MAML2 was observed. Immunoreactivities for nuclear ß-catenin, Hes1, and MAML2, as well as the interaction between ß-catenin and Hes1 or MAML2, were significantly higher in morular lesions compared with surrounding carcinoma in Em Ca. Inhibition of glycogen synthase kinase-3ß (GSK-3ß) increased expression of total nuclear and cytoplasmic GSK-3ß and its phosphorylated forms, as well as Notch intracellular domain 1, Hes1, and active ß-catenin. GSK-3ß inhibition also decreased proliferation and migration, consistent with the response of cells stably overexpressing Hes1. Finally, the nuclear/cytoplasmic GSK-3ß score was significantly higher in morules compared with surrounding carcinoma in Em Ca, and it was positively correlated with nuclear ß-catenin, Hes1, and MAML2 scores. This complex interplay between Notch effectors and ß-catenin signaling through GSK-3ß inhibition contributes to the establishment and maintenance of ß-catenin-mediated morular differentiation, which is, in turn, associated with reduced proliferation and inhibition of migration in Em Ca.


Subject(s)
Carcinoma , Endometrial Neoplasms , Female , Humans , beta Catenin/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Endometrial Neoplasms/pathology , Signal Transduction/physiology
5.
Chemosphere ; 346: 140480, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37879369

ABSTRACT

Maternal exposure to particulate matter derived from diesel exhaust has been shown to cause metabolic dysregulation, neurological problems, and increased susceptibility to diabetes in the offspring. Diesel exhaust is a major source of air pollution and the use of biodiesel (BD) and its blends have been progressively increasing throughout the world; however, studies on the health impact of BD vs. petrodiesel combustion-generated exhaust have been controversial in part, due to differences in the chemical and physical nature of the associated particulate matter (PM). To explore the long-term impact of prenatal exposure, pregnant mice were exposed to PM generated by combustion of petrodiesel (B0) and a 20% soy BD blend (B20) by intratracheal instillation during embryonic days 9-17 and allowed to deliver. Offspring were then followed for 52 weeks. We found that mother's exposure to B0 and B20 PM manifested in striking sex-specific phenotypes with respect to metabolic adaptation, maintenance of glucose homeostasis, and medial hypothalamic glial cell makeup in the offspring. The data suggest PM exposure limited to a narrower critical developmental window may be compensated for by the mother and/or the fetus by altered metabolic programming in a marked sex-specific and fuel-derived PM-specific manner, leading to sex-specific risk for diseases related to environmental exposure later in life.


Subject(s)
Air Pollutants , Air Pollution , Male , Female , Mice , Animals , Particulate Matter/toxicity , Particulate Matter/analysis , Vehicle Emissions/toxicity , Vehicle Emissions/analysis , Biofuels/toxicity , Biofuels/analysis , Environmental Exposure , Gasoline/analysis , Air Pollutants/toxicity
6.
Adv Nutr ; 14(6): 1255-1269, 2023 11.
Article in English | MEDLINE | ID: mdl-37722488

ABSTRACT

Our objective was to convene interdisciplinary experts from government, academia, and industry to develop a Research Roadmap to identify research priorities about processed food intake and risk for obesity and cardiometabolic diseases (CMD) among United States populations. We convened attendees at various career stages with diverse viewpoints in the field. We held a "Food Processing Primer" to build foundational knowledge of how and why foods are processed, followed by presentations about how processed foods may affect energy intake, obesity, and CMD risk. Breakout groups discussed potential mechanistic and confounding explanations for associations between processed foods and obesity and CMD risk. Facilitators created research questions (RQs) based on key themes from discussions. Different breakout groups convened to discuss what is known and unknown for each RQ and to develop sub-RQs to address gaps. Workshop attendees focused on ultra-processed foods (UPFs; Nova Group 4) because the preponderance of evidence is based on this classification system. Yet, heterogeneity and subjectivity in UPF classification was a challenge for RQ development. The 6 RQs were: 1) What objective methods or measures could further categorize UPFs, considering food processing, formulation, and the interaction of the two? 2) How can exposure assessment of UPF intake be improved? 3) Does UPF intake influence risk for obesity or CMDs, independent of diet quality? 4) What, if any, attributes of UPFs influence ingestive behavior and contribute to excess energy intake? 5) What, if any, attributes of UPFs contribute to clinically meaningful metabolic responses? 6) What, if any, external environmental factors lead people to consume high amounts of UPFs? Uncertainty and complexity around UPF intake warrant further complementary and interdisciplinary causal, mechanistic, and methodological research related to obesity and CMD risk to understand the utility of applying classification by degree of processing to foods in the United States.


Subject(s)
Fast Foods , Food, Processed , Humans , Fast Foods/adverse effects , Diet , Energy Intake , Obesity/etiology , Food Handling
7.
BMC Cancer ; 23(1): 765, 2023 Aug 17.
Article in English | MEDLINE | ID: mdl-37592266

ABSTRACT

BACKGROUND: Although anaplastic lymphoma kinase (ALK) is overexpressed in several primary solid tumor types, its role in endometrial carcinoma (Em Ca) remains unclear. METHODS: We evaluated expression of ALK and its related molecules in clinical samples consisting of 168 Em Ca tissues. We also used Em Ca cell lines to evaluate the functional role of ALK. RESULTS: Cytoplasmic ALK immunoreactivity in the absence of chromosomal rearrangement was positively correlated with ALK mRNA expression, and was significantly higher in Grade (G) 3 Em Ca than in G1 or G2 tumors. ALK immunoreactivity was also significantly associated with expression of cancer stem cell (CSC)-related molecules (cytoplasmic CD133, ALDH1, Sox2) and neuroendocrine markers (CD56 and synaptophysin). Although the proliferative index was significantly higher in ALK-positive Em Ca when compared to ALK- negative malignancies, there was no association between ALK expression and other clinicopathological factors in this disease. In Em Ca cell lines, full-length ALK overexpression increased proliferation, decreased susceptibility to apoptosis, enhanced cancer stem cell features, and accelerated cell mobility, whereas these phenotypes were abrogated in ALK-knockdown cells. Finally, patients with tumors harboring either wild-type ALK or high ALK mRNA expression had a poorer prognosis than those with either mutant ALK or low ALK mRNA expression. CONCLUSION: Full-length ALK overexpression occurs in a subset of Em Ca, particularly in G3 tumors, and contributes to the establishment and maintenance of aggressive phenotypic characteristics through modulation of several biological processes.


Subject(s)
Anaplastic Lymphoma Kinase , Endometrial Neoplasms , Female , Humans , Anaplastic Lymphoma Kinase/genetics , Cytoplasm , Endometrial Neoplasms/genetics , Phenotype , RNA, Messenger
8.
Mol Oncol ; 17(10): 2168-2182, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37539980

ABSTRACT

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a scaffold protein that is required for epithelial polarity. Knockout (KO) of membranous EBP50 (Me-EBP50) in ovarian clear cell carcinoma (OCCC) cells induced an epithelial-mesenchymal transition (EMT)-like phenotype, along with decreased proliferation, accelerated migration capability, and induction of cancer stem cell (CSC)-like properties. Shotgun proteomics analysis of proteins that co-immunoprecipitated with EBP50 revealed that Me-EBP50 strongly interacts with myosin 9 (MYH9). Specific inhibition of MYH9 with blebbistatin phenocopied Me-EBP50 KO, and blebbistatin treatment potentiated the effects of Me-EBP50 KO. In OCCC cells from clinical samples, Me-EBP50 and MYH9 were co-localized at the apical plasma membrane. Patients with a combination of Me-EBP50-high and MYH9-high scores had the best prognosis for overall and progression-free survival. Our data suggest that Me-EBP50 has tumor-suppressive effects through the establishment and maintenance of epithelial polarization. By contrast, loss of Me-EBP50 expression induces EMT-like phenotypes, probably due to MYH9 dysfunction; this results in increased cell mobility and enhanced CSC-like properties, which in turn promote OCCC progression.

9.
Am J Pathol ; 193(8): 1116-1128, 2023 08.
Article in English | MEDLINE | ID: mdl-37169340

ABSTRACT

Epithelial-mesenchymal transition is a hallmark of uterine carcinosarcoma (UCS). Here, shotgun proteomics analysis used to identify biomarkers associated with blebbistatin-mediated epithelial-mesenchymal transition in UCS indicated up-regulation of nucleobindin-2 (NUCB2) in endometrial carcinoma (Em Ca) cells. Expression of N-cadherin, Snail, Slug, and ZEB1 was reduced in NUCB2 knockout Em Ca cells, whereas ZEB1, Twist1, and vimentin were up-regulated in NUCB2-overexpressing Em Ca cells. NUCB2 knockout reduced cell proliferation and migration, whereas NUCB2 overexpression had the opposite effect. Treatment of Em Ca cells with transforming growth factor (TGF)-ß1 dramatically altered morphology toward a fibroblastic appearance; concomitantly, expression of NUCB2 and ZEB1 increased. The NUCB2 promoter was also activated by transfection of Smad2. In UCS tissues, NUCB2 expression was significantly higher in sarcomatous compared with carcinomatous components, which was consistent with increased TGF-ß1 mRNA expression in stromal and sarcomatous components compared with carcinomatous components. In addition, NUCB2 score correlated positively with ZEB1 and vimentin scores, whereas ZEB1 score correlated positively with Slug and vimentin scores and inversely with the E-cadherin score. Collectively, these data indicate that TGF-ß-dependent up-regulation of NUCB2 and ZEB1 contributes to the phenotypic characteristics of sarcomatous components in UCS.


Subject(s)
Carcinosarcoma , Uterine Neoplasms , Humans , Female , Zinc Finger E-box-Binding Homeobox 1/genetics , Zinc Finger E-box-Binding Homeobox 1/metabolism , Nucleobindins/genetics , Nucleobindins/metabolism , Transforming Growth Factor beta1/metabolism , Vimentin/metabolism , Genes, Homeobox , Cadherins/genetics , Cadherins/metabolism , Uterine Neoplasms/genetics , Uterine Neoplasms/pathology , Phenotype , Carcinosarcoma/genetics , Carcinosarcoma/pathology , Zinc Fingers , Epithelial-Mesenchymal Transition/genetics , Cell Line, Tumor
10.
Microbiol Spectr ; 11(3): e0402022, 2023 06 15.
Article in English | MEDLINE | ID: mdl-37074179

ABSTRACT

Milk oligosaccharides (MOs) can be prebiotic and antiadhesive, while fatty acids (MFAs) can be antimicrobial. Both have been associated with milk microbes or mammary gland inflammation in humans. Relationships between these milk components and milk microbes or inflammation have not been determined for cows and could help elucidate a novel approach for the dairy industry to promote desired milk microbial composition for improvement of milk quality and reduction of milk waste. We aimed to determine relationships among milk microbiota, MFAs, MOs, lactose, and somatic cell counts (SCC) from Holstein cows, using our previously published data. Raw milk samples were collected at three time points, ranging from early to late lactation. Data were analyzed using linear mixed-effects modeling and repeated-measures correlation. Unsaturated MFA and short-chain MFA had mostly negative relationships with potentially pathogenic genera, including Corynebacterium, Pseudomonas, and an unknown Enterobacteriaceae genus but numerous positive relationships with symbionts Bifidobacterium and Bacteroides. Conversely, many MOs were positively correlated with potentially pathogenic genera (e.g., Corynebacterium, Enterococcus, and Pseudomonas), and numerous MOs were negatively correlated with the symbiont Bifidobacterium. The neutral, nonfucosylated MO composed of eight hexoses had a positive relationship with SCC, while lactose had a negative relationship with SCC. One interpretation of these trends might be that in milk, MFAs disrupt primarily pathogenic bacterial cells, causing a relative increase in abundance of beneficial microbial taxa, while MOs respond to and act on pathogenic taxa primarily through antiadhesive methods. Further research is needed to confirm the potential mechanisms driving these correlations. IMPORTANCE Bovine milk can harbor microbes that cause mastitis, milk spoilage, and foodborne illness. Fatty acids found in milk can be antimicrobial and milk oligosaccharides can have antiadhesive, prebiotic, and immune-modulatory effects. Relationships among milk microbes, fatty acids, oligosaccharides, and inflammation have been reported for humans. To our knowledge, associations among the milk microbial composition, fatty acids, oligosaccharides, and lactose have not been reported for healthy lactating cows. Identifying these potential relationships in bovine milk will inform future efforts to characterize direct and indirect interactions of the milk components with the milk microbiota. Since many milk components are associated with herd management practices, determining if these milk components impact milk microbes may provide valuable information for dairy cow management and breeding practices aimed at minimizing harmful and spoilage-causing microbes in raw milk.


Subject(s)
Microbiota , Milk , Animals , Female , Humans , Cattle , Milk/microbiology , Lactation , Fatty Acids , Lactose , Inflammation , Corynebacterium
11.
Diabetes Care ; 46(2): e39-e50, 2023 02 01.
Article in English | MEDLINE | ID: mdl-36701590

ABSTRACT

Individuals with an elevated fasting glucose level, elevated glucose level after glucose challenge, or elevated hemoglobin A1c level below the diagnostic threshold for diabetes (collectively termed prediabetes) are at increased risk for type 2 diabetes. More than one-third of U.S. adults have prediabetes but fewer than one in five are aware of the diagnosis. Rigorous scientific research has demonstrated the efficacy of both intensive lifestyle interventions and metformin in delaying or preventing progression from prediabetes to type 2 diabetes. The National Clinical Care Commission (NCCC) was a federal advisory committee charged with evaluating and making recommendations to improve federal programs related to the prevention of diabetes and its complications. In this article, we describe the recommendations of an NCCC subcommittee that focused primarily on prevention of type 2 diabetes in people with prediabetes. These recommendations aim to improve current federal diabetes prevention activities by 1) increasing awareness of and diagnosis of prediabetes on a population basis; 2) increasing the availability of, referral to, and insurance coverage for the National Diabetes Prevention Program and the Medicare Diabetes Prevention Program; 3) facilitating Food and Drug Administration review and approval of metformin for diabetes prevention; and 4) supporting research to enhance the effectiveness of diabetes prevention. Cognizant of the burden of type 1 diabetes, the recommendations also highlight the importance of research to advance our understanding of the etiology of and opportunities for prevention of type 1 diabetes.


Subject(s)
Diabetes Mellitus, Type 1 , Diabetes Mellitus, Type 2 , Metformin , Prediabetic State , Aged , Adult , Humans , United States , Prediabetic State/diagnosis , Diabetes Mellitus, Type 2/prevention & control , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 1/drug therapy , Medicare , Metformin/therapeutic use , Glucose/therapeutic use
12.
Diabetes Care ; 46(2): 255-261, 2023 02 01.
Article in English | MEDLINE | ID: mdl-36701592

ABSTRACT

The National Clinical Care Commission (NCCC) was established by Congress to make recommendations to leverage federal policies and programs to more effectively prevent and treat diabetes and its complications. The NCCC developed a guiding framework that incorporated elements of the Socioecological and Chronic Care Models. It surveyed federal agencies and conducted follow-up meetings with representatives from 10 health-related and 11 non-health-related federal agencies. It held 12 public meetings, solicited public comments, met with numerous interested parties and key informants, and performed comprehensive literature reviews. The final report, transmitted to Congress in January 2022, contained 39 specific recommendations, including 3 foundational recommendations that addressed the necessity of an all-of-government approach to diabetes, health equity, and access to health care. At the general population level, the NCCC recommended that the federal government adopt a health-in-all-policies approach so that the activities of non-health-related federal agencies that address agriculture, food, housing, transportation, commerce, and the environment be coordinated with those of health-related federal agencies to affirmatively address the social and environmental conditions that contribute to diabetes and its complications. For individuals at risk for type 2 diabetes, including those with prediabetes, the NCCC recommended that federal policies and programs be strengthened to increase awareness of prediabetes and the availability of, referral to, and insurance coverage for intensive lifestyle interventions for diabetes prevention and that data be assembled to seek approval of metformin for diabetes prevention. For people with diabetes and its complications, the NCCC recommended that barriers to proven effective treatments for diabetes and its complications be removed, the size and competence of the workforce to treat diabetes and its complications be increased, and new payment models be implemented to support access to lifesaving medications and proven effective treatments for diabetes and its complications. The NCCC also outlined an ambitious research agenda. The NCCC strongly encourages the public to support these recommendations and Congress to take swift action.


Subject(s)
Diabetes Mellitus, Type 2 , Prediabetic State , Humans , Policy , Housing
13.
Diabetes Care ; 46(2): e24-e38, 2023 02 01.
Article in English | MEDLINE | ID: mdl-36701595

ABSTRACT

The etiology of type 2 diabetes is rooted in a myriad of factors and exposures at individual, community, and societal levels, many of which also affect the control of type 1 and type 2 diabetes. Not only do such factors impact risk and treatment at the time of diagnosis but they also can accumulate biologically from preconception, in utero, and across the life course. These factors include inadequate nutritional quality, poor access to physical activity resources, chronic stress (e.g., adverse childhood experiences, racism, and poverty), and exposures to environmental toxins. The National Clinical Care Commission (NCCC) concluded that the diabetes epidemic cannot be treated solely as a biomedical problem but must also be treated as a societal problem that requires an all-of-government approach. The NCCC determined that it is critical to design, leverage, and coordinate federal policies and programs to foster social and environmental conditions that facilitate the prevention and treatment of diabetes. This article reviews the rationale, scientific evidence base, and content of the NCCC's population-wide recommendations that address food systems; consumption of water over sugar-sweetened beverages; food and beverage labeling; marketing and advertising; workplace, ambient, and built environments; and research. Recommendations relate to specific federal policies, programs, agencies, and departments, including the U.S. Department of Agriculture, the Food and Drug Administration, the Federal Trade Commission, the Department of Housing and Urban Development, the Environmental Protection Agency, and others. These population-level recommendations are transformative. By recommending health-in-all-policies and an equity-based approach to governance, the NCCC Report to Congress has the potential to contribute to meaningful change across the diabetes continuum and beyond. Adopting these recommendations could significantly reduce diabetes incidence, complications, costs, and inequities. Substantial political resolve will be needed to translate recommendations into policy. Engagement by diverse members of the diabetes stakeholder community will be critical to such efforts.


Subject(s)
Diabetes Mellitus, Type 2 , Sugar-Sweetened Beverages , Humans , Diabetes Mellitus, Type 2/epidemiology , Diabetes Mellitus, Type 2/prevention & control , Policy , Beverages , Housing
14.
Anal Chem ; 95(2): 1008-1015, 2023 01 17.
Article in English | MEDLINE | ID: mdl-36542787

ABSTRACT

Carbohydrates are the most abundant biomolecules in nature, and specifically, polysaccharides are present in almost all plants and fungi. Due to their compositional diversity, polysaccharide analysis remains challenging. Compared to other biomolecules, high-throughput analysis for carbohydrates has yet to be developed. To address this gap in analytical science, we have developed a multiplexed, high-throughput, and quantitative approach for polysaccharide analysis in foods. Specifically, polysaccharides were depolymerized using a nonenzymatic chemical digestion process followed by oligosaccharide fingerprinting using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS). Both label-free relative quantitation and absolute quantitation were done based on the abundances of oligosaccharides produced. Method validation included evaluating recovery for a range of polysaccharide standards and a breakfast cereal standard reference material. Nine polysaccharides (starch, cellulose, ß-glucan, mannan, galactan, arabinan, xylan, xyloglucan, chitin) were successfully quantitated with sufficient accuracy (5-25% bias) and high reproducibility (2-15% CV). Additionally, the method was used to identify and quantitate polysaccharides from a diverse sample set of food samples. Absolute concentrations of nine polysaccharides from apples and onions were obtained using an external calibration curve, where varietal differences were observed in some of the samples. The methodology developed in this study will provide complementary polysaccharide-level information to deepen our understanding of the interactions of dietary polysaccharides, gut microbial community, and human health.


Subject(s)
Glycomics , Tandem Mass Spectrometry , Humans , Tandem Mass Spectrometry/methods , Reproducibility of Results , Polysaccharides/chemistry , Chromatography, Liquid/methods , Oligosaccharides/chemistry , Chromatography, High Pressure Liquid/methods
15.
Food Chem ; 409: 135267, 2023 May 30.
Article in English | MEDLINE | ID: mdl-36586264

ABSTRACT

Oligosaccharides are known for several bioactivities on health, however, in sensitive individuals, can cause intestinal discomfort. This study aimed to investigate the oligosaccharide profiles in selected plant-based food products. A quantification method based on high-performance anion-exchange chromatography-pulsed amperometric detection was developed, validated, and used to measure major oligosaccharides. Additional low-abundant oligosaccharides and glycosides were characterized by liquid chromatography-tandem mass spectrometry and glycosidases. The summed concentration of raffinose, stachyose, and verbascose ranged from 0.12-0.19 mg/g in almond milk, 3.6-6.4 mg/g in soy milk, and 74-77 and 4.8-57 mg/g in defatted and full-fat soy four. Over 80 different oligosaccharides were characterized. Novel compounds, 2,3-butanediol glycosides, were identified in almond milk. Low-abundant oligosaccharides represented 25 %, 6 %, and 10 % of total OS in almond milk, soy milk, and soy flour, respectively. The data here are useful to estimate oligosaccharide consumption from dietary intake and facilitate further studies on their bioactivity.


Subject(s)
Prunus dulcis , Soy Milk , Humans , Flour , Oligosaccharides/chemistry , Glycosides
16.
Cancers (Basel) ; 16(1)2023 Dec 29.
Article in English | MEDLINE | ID: mdl-38201610

ABSTRACT

Ezin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a scaffold protein that interacts with several partner molecules including ß-catenin. Here, we examined the crosstalk between EBP50 and nuclear catenin during colorectal carcinoma (CRC) progression. In clinical samples, there were no correlations between the subcellular location of EBP50 and any clinicopathological factors. However, EBP50 expression was significantly lower specifically in the outer areas of tumor lesions, in regions where tumor budding (BD) was observed. Low EBP50 expression was also significantly associated with several unfavorable prognostic factors, suggesting that EBP50 depletion rather than its overexpression or subcellular distribution plays an important role in CRC progression. In CRC cell lines, knockout of EBP50 induced epithelial-mesenchymal transition (EMT)-like features, decreased proliferation, accelerated migration capability, and stabilized nuclear ß-catenin due to disruption of the interaction between EBP50 and ß-catenin at the plasma membrane. In addition, Slug expression was significantly higher in outer lesions, particularly in BD areas, and was positively correlated with nuclear ß-catenin status, consistent with ß-catenin-driven transactivation of the Slug promoter. Together, our data suggest that EBP50 depletion releases ß-catenin from the plasma membrane in outer tumor lesions, allowing ß-catenin to accumulate and translocate to the nucleus, where it transactivates the Slug gene to promote EMT. This in turn triggers tumor budding and contributes to the progression of CRC to a more aggressive phase.

17.
J Agric Food Chem ; 70(45): 14559-14570, 2022 Nov 16.
Article in English | MEDLINE | ID: mdl-36382383

ABSTRACT

Dietary fiber has long been known to be an essential component of a healthy diet, and recent investigations into the gut microbiome-health paradigm have identified fiber as a prime determinant in this interaction. Further, fiber is now known to impact the gut microbiome in a structure-specific manner, conferring differential bioactivities to these specific structures. However, current analytical methods for food carbohydrate analysis do not capture this important structural information. To address this need, we utilized rapid-throughput LC-MS methods to develop a novel analytical pipeline to determine the structural composition of soluble and insoluble fiber fractions from two AOAC methods (991.43 and 2017.16) at the total monosaccharide, glycosidic linkage, and free saccharide level. Two foods were chosen for this proof-of-concept study: oats and potato starch. For oats, both AOAC methods gave similar results. Insoluble fiber was found to be comprised of linkages corresponding to ß-glucan, arabinoxylan, xyloglucan, and mannan, while soluble fiber was found to be mostly ß-glucan, with small amounts of arabinogalactan. For raw potato starch, each AOAC method gave markedly different results in the soluble fiber fractions. These observed differences are attributable to the resistant starch content of potato starch and the different starch digestion conditions used in each method. Together, these tools are a means to obtain the complex structures present within dietary fiber while retaining "classical" determinations such as soluble and insoluble fiber. These efforts will provide an analytical framework to connect gravimetric fiber determinations with their constituent structures to better inform gut microbiome and clinical nutrition studies.


Subject(s)
Glycomics , beta-Glucans , Dietary Fiber/analysis , Carbohydrates/analysis , Starch/chemistry , Edible Grain/chemistry
18.
Cell Commun Signal ; 20(1): 181, 2022 11 21.
Article in English | MEDLINE | ID: mdl-36411429

ABSTRACT

BACKGROUND: Although a lack of functional PTEN contributes to tumorigenesis in a wide spectrum of human malignancies, little is known about the functional role of its overexpression in the tumors. The current study focused on PTEN overexpression in endometrial carcinoma (Em Ca). METHODS: The functional impact of PTEN overexpression was assessed by Em Ca cell lines. Immunohistochemical analyses were also conducted using 38 Em Ca with morular lesions. RESULTS: Em Ca cell lines stably overexpressing PTEN (H6-PTEN) exhibited epithelial-mesenchymal transition (EMT)-like features, probably through ß-catenin/Slug-meditated suppression of E-cadherin. PTEN overexpression also inhibited cell proliferation, accelerated cellular senescence, increased apoptotic features, and enhanced migration capability. Moreover, H6-PTEN cells exhibited cancer stem cell (CSC)-like properties, along with high expression of aldehyde dehydrogenase 1 and CD44s, a large ALDH 1high population, enriched spheroid formation, and ß-catenin-mediated upregulation of cyclin D2, which is required for persistent CSC growth. In clinical samples, immunoreactivities for PTEN, as well as CSC-related molecules, were significantly higher in morular lesions as compared to the surrounding carcinomas. PTEN score was positively correlated with expression of nuclear ß-catenin, cytoplasmic CD133, and CD44v6, and negatively with cell proliferation. Finally, estrogen receptor-α (ERα)-dependent expression of Ezrin-radixin-moesin-binding phophoprotein-50 (EBP50), a multifunctional scaffolding protein, acts as a negative regulator of morular formation by Em Ca cells through interacting with PTEN and ß-catenin. CONCLUSION: In the abscess of ERα/EBP50 expression, PTEN overexpression and nuclear ß-catenin stabilization promote the establishment and maintenance of morular phenotype associated with EMT/CSC-like features in Em Ca cells. Video Abstract.


Subject(s)
Endometrial Neoplasms , PTEN Phosphohydrolase , Animals , Female , Humans , beta Catenin , Endometrial Neoplasms/metabolism , Endometrial Neoplasms/pathology , Epithelial-Mesenchymal Transition , Estrogen Receptor alpha , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology
19.
Int J Mol Sci ; 23(20)2022 Oct 19.
Article in English | MEDLINE | ID: mdl-36293398

ABSTRACT

Adipose stem cells (ASCs) are reported to play a role in normal physiology as well as in inflammation and disease. The objective of this work was to elucidate inter-individual differences in growth, gene expression and response to inflammatory stimuli in ASCs from different donors. Human ASC1 (male donor) and ASC2 (female donor) were purchased from Lonza (Walkersville, MD). Cell proliferation was determined by the sulforhodamine B assay. After time-dependent treatment of ASCs with or without bacterial lipopolysaccharide (LPS), marker gene mRNAs for proliferation, steroid hormones, and xenobiotic and immune pathways were determined using RT-PCR, and secreted cytokine levels in media were measured using the Bio-Plex cytokine assay kit. ASCs from both donors expressed androgen receptors but not estrogen receptors. ASC2 had a 2-fold higher proliferation rate and a 6-fold higher level of proliferation marker Ki67 mRNA than ASC1. ASC2 exhibited significantly greater fold induction of TNF-α and CCL2 by LPS compared to ASC1. TNF-α and GM-CSF protein levels were also significantly higher in the LPS-induced ASC2 media, but IL-6 secretion was higher in the LPS-induced ASC1 media. Our findings suggest that inter-individual variability and/or possible sex differences exist in ASCs, which may serve as a key determinant to inflammatory responses of ASCs.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor , Lipopolysaccharides , Female , Male , Humans , Lipopolysaccharides/pharmacology , Lipopolysaccharides/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Ki-67 Antigen/genetics , Ki-67 Antigen/metabolism , Interleukin-6/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Receptors, Androgen/metabolism , Xenobiotics/metabolism , Adipose Tissue/metabolism , Cell Proliferation , RNA, Messenger/metabolism , Cytokines/genetics , Cytokines/metabolism , Hormones/metabolism , Gene Expression
20.
Adv Nutr ; 13(4): 1324-1393, 2022 08 01.
Article in English | MEDLINE | ID: mdl-35802522

ABSTRACT

The ASN Board of Directors appointed the Nutrition Research Task Force to develop a report on scientific methods used in nutrition science to advance discovery, interpretation, and application of knowledge in the field. The genesis of this report was growing concern about the tone of discourse among nutrition professionals and the implications of acrimony on the productive study and translation of nutrition science. Too often, honest differences of opinion are cast as conflicts instead of areas of needed collaboration. Recognition of the value (and limitations) of contributions from well-executed nutrition science derived from the various approaches used in the discipline, as well as appreciation of how their layering will yield the strongest evidence base, will provide a basis for greater productivity and impact. Greater collaborative efforts within the field of nutrition science will require an understanding that each method or approach has a place and function that should be valued and used together to create the nutrition evidence base. Precision nutrition was identified as an important emerging nutrition topic by the preponderance of task force members, and this theme was adopted for the report because it lent itself to integration of many approaches in nutrition science. Although the primary audience for this report is nutrition researchers and other nutrition professionals, a secondary aim is to develop a document useful for the various audiences that translate nutrition research, including journalists, clinicians, and policymakers. The intent is to promote accurate, transparent, verifiable evidence-based communication about nutrition science. This will facilitate reasoned interpretation and application of emerging findings and, thereby, improve understanding and trust in nutrition science and appropriate characterization, development, and adoption of recommendations.


Subject(s)
Nutritional Sciences , Research Design , Advisory Committees , Humans
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