Diet modulates gut microbiome composition and function. However, determining causal links between diet-gut microbiome interactions and human health is complicated by inconsistencies in the evidence, arising partially from variability in research methods and reporting. Widespread adoption of standardized best practices would advance the field but require those practices to be identified, consolidated, and discussed. This umbrella review aimed to identify recommended best practices, define existing gaps, and collate considerations for conducting research on diet-gut microbiome interactions and their impact on human health outcomes. Reviews meeting inclusion criteria and published after 2013 were identified using a systematic search. Recommendations, considerations, and gaps relating to the best practices associated with study design, participant selection, dietary intervention/assessment, biological sample collection, and data analysis and reporting were extracted and consolidated. Eight narrative reviews were included. Several general points of agreement were identified, and a recurring theme was that best practices are dependent upon the research aims, outcomes, and feasibility. Multiple gaps were also identified. Some, such as suboptimal diet assessment methods and lack of validated dietary intake biomarkers, are particularly relevant to nutrition science. Others, including defining a "healthy" gut microbiome and the absence of standardized sample and data collection/analysis protocols, were relevant specifically to gut microbiome research. Gaps specific to diet-gut microbiome research include the underrepresentation of microbiome-modulating dietary components in food databases, lack of knowledge regarding interventions eliciting changes in the gut microbiome to confer health benefits, lack of in situ measurement methods, and the need to further develop and refine statistical approaches for integrating diet and gut microbiome data. Future research and cross-disciplinary exchange will address these gaps and evolve the best practices. In the interim, the best practices and considerations discussed herein, and the publications from which that information was extracted provide a roadmap for conducting diet-gut microbiome research. This trial was registered at PROSPERO as CRD42023437645.

Supplementary Figure 2 from Deficiency in the 15-kDa Selenoprotein Inhibits Tumorigenicity and Metastasis of Colon Cancer Cells

The field of nutritional sciences has gone through metamorphoses during the past 80 years since Nutrition Reviews was first published. During this time, we have seen the transition from conducting proximate analyses for determining the macronutrient content of food to identifying the essential vitamins and minerals, using foodomics and biomarkers to help us estimate what type of food and nutrients we are consuming, and determining the impact dietary patterns have on health. As such, we have borne witness to the evolution of nutritional sciences from the golden age of the discovery of the essential nutrients required to prevent the certainty of deficiency diseases to the current age of optimizing nutrition to reduce the risk of communicable and non-communicable diseases in both populations and individuals. In the United States, the first dietary guidelines were published by Caroline Hunt and Helen Atwater in 1917 and titled “How to Select Foods.” These guidelines identified 5 food groups to provide 8 dietary substances. The food groups were fruits and vegetables, meats and other protein-rich foods, cereals and other starchy foods, sweets, and fatty foods. The dietary substances needed for health were identified as mineral substances, protein, starch, sugar, fat, cellulose, certain newly discovered substances in very small amounts, and flavorings and condiments. These original guidelines are separate from the Dietary Guidelines for Americans, which were first launched in 1980, included 7 Dietary Guidelines Statements, and were based on a 1977 report by the Senate Select Committee on Nutrition called “Dietary Goals for Americans.” Since that time, dietary guidelines have been regularly updated and are now the foundation of national nutrition programs and international food assistance. Over the past 80 years, studies on the impact of diet and nutrition have become more complex for animal studies, for studies with human subjects (including clinical trials), and for community-based interventions. We now use measures of nutritional status as both dependent and independent variables, as they can be outcome measures for interventions and predictors of health outcomes. Summarizing the evidence from these studies and incorporating the results into policies and programs have also evolved. Literature reviews support the development of nutrition recommendations for policies and programs. At the same time, the methods for reviewing the scientific literature have expanded and now include narrative reviews and standardized methods such as systematic

Selenoproteins play important roles in many cellular functions and biochemical pathways in mammals. Our previous study showed that the deficiency of the 15 kDa selenoprotein (Selenof) significantly reduced the formation of aberrant crypt foci (ACF) in a mouse model of azoxymethane (AOM)-induced colon carcinogenesis. The objective of this study was to examine the effects of Selenof on inflammatory tumorigenesis, and whether dietary selenium modified these effects. For 20 weeks post-weaning, Selenof-knockout (KO) mice and littermate controls were fed diets that were either deficient, adequate or high in sodium selenite. Colon tumors were induced with AOM and dextran sulfate sodium. Surprisingly, KO mice had drastically fewer ACF but developed a similar number of tumors as their littermate controls. Expression of genes important in inflammatory colorectal cancer and those relevant to epithelial barrier function was assessed, in addition to structural differences via tissue histology. Our findings point to Selenof’s potential role in intestinal barrier integrity and structural changes in glandular and mucin-producing goblet cells in the mucosa and submucosa, which may determine the type of tumor developing.

BACKGROUND AND AIMS Public interest in pre- , pro-, and synbiotic products is increasing because of interactions between gut microbiota and human health. Our aim was to describe non-food (from dietary supplements or medication) pre, pro-, and synbiotic use by U.S. adults and children and reported reasons. METHOD Using data from the National Health and Nutrition Examination Survey (NHANES), we text-mined dietary supplement and prescription medication labels and ingredients to identify pre-, pro-, and synbiotic products used in the past 30 days. We describe trends in use from 1999-2018 (n=101,199) and prevalence in 2015-2016 and 2017-2018 (n=19,215) by age groups, gender, ethnicity/race, education, income, self-reported diet and health quality, and prescription gastrointestinal medication use stratified by children (<19 years) and adults (19+ years). RESULTS Non-food pre-, pro-, and synbiotic use increased up to three-fold in recent cycles. Prevalence of use for all ages for prebiotics was 2.4% (95% confidence interval: 2.0, 2.9%), probiotics was 4.5% (3.5, 5.6%), and synbiotics was 1.1% (0.8%, 1.5%). Use was highest among older adults [8.8% (5.4, 13.3) among 60-69 years for probiotics], non-Hispanic Whites, higher educational attainment and income, more favorable self-reported diet or health quality, and concurrent prescription gastrointestinal medication use. The top reasons for use were for digestive health and to promote/maintain general health. Less than 30% reported using these products based on a healthcare provider recommendation. CONCLUSION One in twenty U.S. adults or children use non-food, pre-, pro-, or synbiotic products and use has sharply increased in recent years. Most individuals voluntarily take these products for general digestive or overall health reasons.

This review provides an overview of the interrelationships among the diet, gut microbiota, and health status and then focuses specifically on published research assessing the relationship of low-/no-calorie sweeteners (LNCSs) to selected aspects of the gut microbiota. Microbiome research is expanding as new data on its role in health and disease vulnerability emerge. The gut microbiome affects health, digestion, and susceptibility to disease. In the last 10 years, investigations of LNCS effects on the gut microbiota have proliferated, although results are conflicting and are often confounded by differences in study design such as study diet, the form of the test article, dosage, and study population. Staying current on microbiome research and the role of dietary inputs, such as LNCSs, will allow healthcare and nutrition practitioners to provide evidence-based guidance to the individuals they serve.

Dietary microRNAs (miRNAs) are thought to regulate a wide range of biological processes, including the gut microbiota. However, it is difficult to separate specific effect(s) of miRNA from that of the food matrix. This study aims to elucidate the specific effect(s) of dietary corn miRNAs, ingested as a whole food, on the gut microbiota. We developed an autoclave procedure to remove 98% of miRNA from corn. A mouse feeding study was conducted comparing autoclaved corn to nonautoclaved corn and purified corn miRNA. Compared to nonspecific nucleotides and corn devoid of miRNAs, feeding purified corn miRNAs or corn to C57BL/6 mice via gavage or diet supplementation for two weeks lead to a decrease in total bacteria in the cecum. The effect appeared to be due to changes in Firmicutes . Additionally, corn matrix minus miRNA and processing also affected gut bacteria. In silico analysis identified corn miRNAs that aligned to Firmicutes genome sequences lending further support to the interaction between corn miRNAs and this bacterium. These data support interactions between plant food miRNA, as well as matrix, and the gut microbiota exist but complex. However, it provides additional support for mechanism by which bioactive dietary components interact with the gut microbiota.

INTRODUCTION:To date, there has been little effort to develop standards for metabolome-based gut microbiome measurements despite the significant efforts toward standard development for DNA-based microbiome measurements.OBJECTIVES:The National Institute of Standards and Technology (NIST), The BioCollective (TBC), and the North America Branch of the International Life Sciences Institute (ILSI North America) are collaborating to extend NIST's efforts to develop a Human Whole Stool Reference Material for the purpose of method harmonization and eventual quality control.METHODS:The reference material will be rationally designed for adequate quality assurance and quality control (QA/QC) for underlying measurements in the study of the impact of diet and nutrition on functional aspects of the host gut microbiome and relationships of those functions to health. To identify which metabolites deserve priority in their value assignment, NIST, TBC, and ILSI North America jointly conducted a workshop on September 12, 2019 at the NIST campus in Gaithersburg, Maryland. The objective of the workshop was to identify metabolites for which evidence indicates relevance to health and disease and to decide on the appropriate course of action to develop a fit-for-purpose reference material.RESULTS:This document represents the consensus opinions of workshop participants and co-authors of this manuscript, and provides additional supporting information. In addition to developing general criteria for metabolite selection and a preliminary list of proposed metabolites, this paper describes some of the strengths and limitations of this initiative given the current state of microbiome research.CONCLUSIONS:Given the rapidly evolving nature of gut microbiome science and the current state of knowledge, an RM (as opposed to a CRM) measured for multiple metabolites is appropriate at this stage. As the science evolves, the RM can evolve to match the needs of the research community. Ultimately, the stool RM may exist in sequential versions. Beneficial to this evolution will be a clear line of communication between NIST and the stakeholder community to ensure alignment with current scientific understanding and community needs.

Folate, an essential nutrient found naturally in foods in a reduced form, is present in dietary supplements and fortified foods in an oxidized synthetic form (folic acid). There is widespread agreement that maintaining adequate folate status is critical to prevent diseases due to folate inadequacy (e.g., anemia, birth defects, and cancer). However, there are concerns of potential adverse effects of excess folic acid intake and/or elevated folate status, with the original concern focused on exacerbation of clinical effects of vitamin B-12 deficiency and its role in neurocognitive health. More recently, animal and observational studies have suggested potential adverse effects on cancer risk, birth outcomes, and other diseases. Observations indicating adverse effects from excess folic acid intake, elevated folate status, and unmetabolized folic acid (UMFA) remain inconclusive; the data do not provide the evidence needed to affect public health recommendations. Moreover, strong biological and mechanistic premises connecting elevated folic acid intake, UMFA, and/or high folate status to adverse health outcomes are lacking. However, the body of evidence on potential adverse health outcomes indicates the need for comprehensive research to clarify these issues and bridge knowledge gaps. Three key research questions encompass the additional research needed to establish whether high folic acid or total folate intake contributes to disease risk. 1) Does UMFA affect biological pathways leading to adverse health effects? 2) Does elevated folate status resulting from any form of folate intake affect vitamin B-12 function and its roles in sustaining health? 3) Does elevated folate intake, regardless of form, affect biological pathways leading to adverse health effects other than those linked to vitamin B-12 function? This article summarizes the proceedings of an August 2019 NIH expert workshop focused on addressing these research areas.

Phenolic compounds have been recognized as promising compounds for the prevention of chronic diseases, including neurodegenerative ones. However, phenolics like flavan-3-ols (F3O) are poorly absorbed along the gastrointestinal tract and structurally rearranged by gut microbiota, yielding smaller and more polar metabolites like phenyl-γ-valerolactones, phenylvaleric acids and their conjugates. The present work investigated the ability of F3O-derived metabolites to cross the blood-brain barrier (BBB), by linking five experimental models with increasing realism. First, an in silico study examined the physical-chemical characteristics of F3O metabolites to predict those most likely to cross the BBB. Some of these metabolites were then tested at physiological concentrations to cross the luminal and abluminal membranes of brain microvascular endothelial cells, cultured in vitro. Finally, three different in vivo studies in rats injected with pure 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone, and rats and pigs fed grapes or a F3O-rich cocoa extract, respectively, confirmed the presence of 5-(hydroxyphenyl)-γ-valerolactone-sulfate (3′,4′ isomer) in the brain. This work highlighted, with different experimental models, the BBB permeability of one of the main F3O-derived metabolites. It may support the neuroprotective effects of phenolic-rich foods in the frame of the “gut-brain axis”.

On December 17, 2018, the North American branch of the International Life Sciences Institute (ILSI North America) convened a workshop "Can We Begin to Define a Healthy Gut Microbiome Through Quantifiable Characteristics?" with >40 invited academic, government, and industry experts in Washington, DC. The workshop objectives were to 1) develop a collective expert assessment of the state of the evidence on the human gut microbiome and associated human health benefits, 2) see if there was sufficient evidence to establish measurable gut microbiome characteristics that could serve as indicators of "health," 3) identify short- and long-term research needs to fully characterize healthy gut microbiome-host relationships, and 4) publish the findings. Conclusions were as follows: 1) mechanistic links of specific changes in gut microbiome structure with function or markers of human health are not yet established; 2) it is not established if dysbiosis is a cause, consequence, or both of changes in human gut epithelial function and disease; 3) microbiome communities are highly individualized, show a high degree of interindividual variation to perturbation, and tend to be stable over years; 4) the complexity of microbiome-host interactions requires a comprehensive, multidisciplinary research agenda to elucidate relationships between gut microbiome and host health; 5) biomarkers and/or surrogate indicators of host function and pathogenic processes based on the microbiome need to be determined and validated, along with normal ranges, using approaches similar to those used to establish biomarkers and/or surrogate indicators based on host metabolic phenotypes; 6) future studies measuring responses to an exposure or intervention need to combine validated microbiome-related biomarkers and/or surrogate indicators with multiomics characterization of the microbiome; and 7) because static genetic sampling misses important short- and long-term microbiome-related dynamic changes to host health, future studies must be powered to account for inter- and intraindividual variation and should use repeated measures within individuals.

The current study seeks to resolve the discrepancy in the literature regarding the cross-kingdom transfer of plant microRNAs (miRNAs) into mammals using an improved miRNA processing and detection method. Two studies utilizing C57BL/6 mice were performed. In the first study, mice were fed an AIN-93M diet and gavaged with water, random deoxynucleotide triphosphates (dNTP) or isolated corn miRNAs for two weeks (n = 10 per group). In the second study, mice were fed an AIN-93M diet, or the diet supplemented with 3% fresh or autoclaved corn powder for two weeks (n = 10 per group). Corn miRNA levels were analyzed in blood and tissue samples by real-time PCR (RT-PCR) following periodate oxidation and β elimination treatments to eliminate artifacts. After removing false positive detections, there were no differences in corn miRNA levels between control and treated groups in cecal, fecal, liver and blood samples. Using an in vitro digestion system, corn miRNAs in AIN-93M diet or in the extracts were found to be extensively degraded. Less than 1% was recovered in the gastrointestinal tract after oral and gastric phases. In conclusion, no evidence of increased levels of corn miRNAs in whole blood or tissues after supplementation of corn miRNAs in the diet was observed in a mouse model.

A 2-day workshop organized by the National Institutes of Health and U.S. Department of Agriculture included 16 presentations focused on the role of diet in alterations of the gastrointestinal microbiome, primarily that of the colon. Although thousands of research projects have been funded by U.S. federal agencies to study the intestinal microbiome of humans and a variety of animal models, only a minority addresses dietary effects, and a small subset is described in sufficient detail to allow reproduction of a study. Whereas there are standards being developed for many aspects of microbiome studies, such as sample collection, nucleic acid extraction, data handling, etc., none has been proposed for the dietary component; thus this workshop focused on the latter specific point. It is important to foster rigor in design and reproducibility of published studies to maintain high quality and enable designs that can be compared in systematic reviews. Speakers addressed the influence of the structure of the fermentable carbohydrate on the microbiota and the variables to consider in design of studies using animals, in vitro models, and human subjects. For all types of studies, strengths and weaknesses of various designs were highlighted, and for human studies, comparisons between controlled feeding and observational designs were discussed. Because of the lack of published, best-diet formulations for specific research questions, the main recommendation is to describe dietary ingredients and treatments in as much detail as possible to allow reproduction by other scientists.

Although many Americans use dietary supplements, databases of dietary supplements sold in the United States have not been widely available. The Dietary Supplement Label Database (DSLD), an easily accessible public-use database, was created in 2008 to provide information on dietary supplement composition for use by researchers and consumers. We describe in this article the history, key features, recent enhancements, and common applications of the DSLD. Accessing current information easily and quickly is crucial for documenting exposures to dietary supplements because they contain nutrients and other bioactive ingredients that may have beneficial or adverse effects on human health. This article details recent developments with the DSLD to achieve this goal, and provides examples of how the DSLD has been used. With periodic updates to track changes in product composition and capture new products entering the market, the DSLD currently contains > 71,000 dietary supplement labels. Following usability testing with consumer and researcher user groups completed in 2016, improvements to the DSLD interface were made. As of 2018, both a desktop and mobile device version are available. Since its inception in 2008, the DSLD has been used for research, exposure monitoring, and for other purposes by users in the public and private sectors. Further refinement of the user interface and search features is planned to facilitate ease of use for stakeholders. The DSLD can be used to track changes in product composition and capture new products entering the market. With > 71,000 dietary supplement labels, it is a unique resource that policymakers, researchers, clinicians, and consumers may find valuable for multiple applications.

MicroRNAs (miRNAs) ubiquitously exist in microorganisms, plants, and animals, and appear to modulate a wide range of critical biological processes. However, no definitive conclusion has been reached regarding the uptake of exogenous dietary small RNAs into mammalian circulation and organs and cross-kingdom regulation. One of the critical issues is our ability to assess and distinguish the origin of miRNAs. Although periodate oxidation has been used to differentiate mammalian and plant miRNAs, validation of treatment efficiency and the inclusion of proper controls for this method were lacking in previous studies. This study aimed to address: 1) the efficiency of periodate treatment in a plant or mammalian RNA matrix, and 2) the necessity of inclusion of internal controls. We designed and tested spike-in synthetic miRNAs in various plant and mammalian matrices and showed that they can be used as a control for the completion of periodate oxidation. We found that overloading the reaction system with high concentration of RNA resulted in incomplete oxidation of unmethylated miRNA. The abundant miRNAs from soy and corn were analyzed in the plasma, liver, and fecal samples of C57BL/6 mice fed a corn and soy-based chow diet using our improved methodology. The improvement resulted in the elimination of the false positive detection in the liver, and we did not detect plant miRNAs in the mouse plasma or liver samples. In summary, an improved methodology was developed for plant miRNA detection that appears to work well in different sample matrices.

MicroRNAs (miRNAs) ubiquitously exist in microorganisms, plants and animals, and appear to modulate a wide range of critical biological processes. However, no definitive conclusion has been reached regarding the uptake of exogenous dietary small RNAs into mammalian circulation and organs and cross‐kingdom regulation. One of the critical issues is our ability to assess and distinguish the origin of miRNAs. Although periodate oxidation has been used to differentiate mammalian and plant miRNAs, validation of treatment efficiency and inclusion of proper controls for this method were lacking in previous studies. This study aimed to address: 1) the efficiency of periodate treatment in plant or mammalian RNA matrices, and 2) the necessity of inclusion of internal controls. We designed and tested spike‐in synthetic miRNAs in various plant and mammalian matrices and showed that they can be used as control for the completion of periodate oxidation. We found that overloading the reaction system with high concentration of RNA resulted in incomplete oxidation of unmethylated miRNA. The abundant miRNAs from soy and corn were analyzed in the plasma, liver, and fecal samples of C57BL/6 mice fed with a corn and soy based chow diet using our improved methodology. We did not detect plant miRNAs in the mouse plasma or liver samples. In summary, an improved methodology was developed for plant miRNA detection that appears to work well in different sample matrices. Support or Funding Information U.S. Department of Agriculture appropriated fund #8040‐51530‐056‐00D Office of Dietary Supplements Interagency Reimbursable Agreement #8040‐51530‐056‐20‐I

Consumption of the probiotic bacteria LactobacillusrhamnosusLGG and flavanol-rich cocoa have purported immune modulating effects. This study compared the host response to infection with Ascaris suum in three-month-old pigs fed a standard growth diet supplemented with a vehicle control: LGG, cocoa powder (CP) or LGG + CP. Pigs were inoculated with infective A. suum eggs during Week 5 of dietary treatment and euthanized 17 days later. Lactobacillus abundance was increased in pigs fed LGG or LGG + CP. Specific anti-A. suum IgG2 antibodies were decreased (p < 0.05) in LGG + CP-fed pigs compared to pigs fed CP alone. Pigs fed LGG had significantly reduced expression (p < 0.05) of Eosinophil peroxidase (EPX), Interleukin 13 (IL-13), Eotaxin 3 (CCL26), Toll-like receptor 2 (TLR2), TLR4, and TLR9 and Interleukin-1Beta (IL1B) in the tracheal-bronchial lymph node (TBLN) independent of CP treatment. These results suggested that feeding LGG significantly reduced the localized prototypical Th2-related markers of infection with A. suum in the TBLN. Although feeding CP does not appear to affect the A. suum-induced Th2-associated cytokine response, feeding LGG + CP reduced anti-A. suum antibodies and delayed intestinal expulsion of parasitic larvae from the intestine.

The science surrounding vitamin D presents both challenges and opportunities. Although many uncertainties are associated with the understandings concerning vitamin D, including its physiological function, the effects of excessive intake, and its role in health, it is at the same time a major interest in the research and health communities. The approach to evaluating and interpreting the available evidence about vitamin D should be founded on the quality of the data and on the conclusions that take into account the totality of the evidence. In addition, these activities can be used to identify critical data gaps and to help structure future research. The Office of Dietary Supplements (ODS) at the National Institutes of Health has as part of its mission the goal of supporting research and dialogues for topics with uncertain data, including vitamin D. This review considers vitamin D in the context of systematically addressing the uncertainty and in identifying research needs through the filter of the work of ODS. The focus includes the role of systematic reviews, activities that encompass considerations of the totality of the evidence, and collaborative activities to clarify unknowns or to fix methodological problems, as well as a case study using the relationship between cancer and vitamin D.