Fibre-rich Foods to Treat Obesity and Prevent Colon Cancer trial study protocol: a randomised clinical trial of fibre-rich legumes targeting the gut microbiome, metabolome and gut transit time of overweight and obese patients with a history of noncancerous adenomatous polyps.

INTRODUCTION: Recently published studies support the beneficial effects of consuming fibre-rich legumes, such as cooked dry beans, to improve metabolic health and reduce cancer risk. In participants with overweight/obesity and a history of colorectal polyps, the Fibre-rich Foods to Treat Obesity and Prevent Colon Cancer randomised clinical trial will test whether a high-fibre diet featuring legumes will simultaneously facilitate weight reduction and suppress colonic mucosal biomarkers of colorectal cancer (CRC). METHODS/DESIGN: This study is designed to characterise changes in (1) body weight; (2) biomarkers of insulin resistance and systemic inflammation; (3) compositional and functional profiles of the faecal microbiome and metabolome; (4) mucosal biomarkers of CRC risk and (5) gut transit. Approximately 60 overweight or obese adults with a history of noncancerous adenomatous polyps within the previous 3 years will be recruited and randomised to one of two weight-loss diets. Following a 1-week run-in, participants in the intervention arm will receive preportioned high-fibre legume-rich entrées for two meals/day in months 1-3 and one meal/day in months 4-6. In the control arm, entrées will replace legumes with lean protein sources (eg, chicken). Both groups will receive in-person and written guidance to include nutritionally balanced sides with energy intake to lose 1-2 pounds per week. ETHICS AND DISSEMINATION: The National Institutes of Health fund this ongoing 5-year study through a National Cancer Institute grant (5R01CA245063) awarded to Emory University with a subaward to the University of Pittsburgh. The study protocol was approved by the Emory Institutional Review Board (IRB approval number: 00000563). TRIAL REGISTRATION NUMBER: NCT04780477.

Is Salt at Fault? Dietary Salt Consumption and Inflammatory Bowel Disease.

Epidemiological trends have led to a growing consensus that diet plays a central role in the etiopathogenesis of inflammatory bowel diseases (IBD). A Western diet high in ultra-processed foods has been associated with an increased prevalence of IBD worldwide. Much attention has focused on components of the Western diet, including the high fat content, lack of fiber, added sugars, and use of additives, such as carrageenan and other emulsifiers. Less attention has been paid to the impact of high salt intake, an integral component of ultra-processed foods, which has increased dramatically in the US diet over the past 50 years. We review a growing body of literature linking the rise in dietary salt intake with the epidemiology of IBD, increased consumption of salt as a component of ultra-processed foods, high salt intake and imbalances in immune homeostasis, the effects of a high-salt diet on other inflammatory disorders, salt's impact on animal colitis models, salt as an underrecognized component in diet modification-induced remission of IBD, and directions for future investigation.

Recent studies have shown that high dietary salt intake is proinflammatory and contributes to chronic inflammatory conditions. Combined with investigations demonstrating low-salt exclusive enteral nutrition induced Crohn's remission, salt intake is likely a contributory factor to inflammatory bowel diseases' pathogenesis and severity.

Interactions between the environmental and human microbiota in the preservation of health and genesis of disease: symposium report.

PURPOSE OF REVIEW: The purpose of this symposium was to bring thought leaders in the microbiome from the west to Africa to share their unique experiences with African investigators in order to build the foundations for scientifically rigorous explorations into the African human and environmental microbiome that may explain why disease patterns are different in Africa where the chief killers are infectious diseases, whereas noncommunicable diseases (NCDs) are the major threat to healthcare resources in the developed world. RECENT FINDINGS: The application of new high throughput technologies to the investigation of the microbiome and its metabolome has revealed mechanisms whereby a traditional African high fiber diet can suppress NCDs which include colon cancer, inflammatory bowel diseases, obesity, type 2 diabetes and atherosclosis. There is concern that with migration and westernization, NCDs are becoming more common in Africa and that food security is becoming impaired by unbalanced obesogenic foods rather than inadequate food intake. SUMMARY: There is an urgent need for the formation of combined African-Western research programs to identify what is good and bad in the African diet-microbiome axis to develop strategies to prevent the incidence of NCDs rising to western levels in Africa, at the same time offering novel prevention strategies against the #1 healthcare threat in the developed world.

Disease-Related Malnutrition and Enteral Nutrition.

There are many misconceptions surrounding the diagnosing and treatment of malnutrition and around feeding people with enteral nutrition (EN). Often the decisions made by clinicians are made from anecdote or guidelines that may be out of date or supported by low-quality evidence. In this article, we will discuss different aspects of diagnosing malnutrition and delve deeper into the science and evidence behind certain recommendations. Our goal is to better equip the reader with the most current data-supported recommendation, such as indications, contraindications, complications of EN, tube and ostomy complications, types and use of specialized enteral formulas, and home management.

Parenteral Nutrition.

Parenteral nutrition (PN) is a therapy to nourish patients who cannot tolerate feeding via the gut. Though a life-saving intervention, it does have risks associated. In this article, we aim to dispel myths associated with PN. Practitioners who manage critically ill patients or patients with intestinal failure should be equipped with evidence-based knowledge of PN including the indications, contraindications, feasibility, complications, and long-term management of PN.

Dietary fibre to reduce colon cancer risk in Alaska Native people: the Alaska FIRST randomised clinical trial protocol.

INTRODUCTION: Diet, shown to impact colorectal cancer (CRC) risk, is a modifiable environmental factor. Fibre foods fermented by gut microbiota produce metabolites that not only provide food for the colonic epithelium but also exert regulatory effects on colonic mucosal inflammation and proliferation. We describe methods used in a double-blinded, randomised, controlled trial with Alaska Native (AN) people to determine if dietary fibre supplementation can substantially reduce CRC risk among people with the highest reported CRC incidence worldwide. METHODS AND ANALYSES: Eligible patients undergoing routine screening colonoscopy consent to baseline assessments and specimen/data collection (blood, urine, stool, saliva, breath and colon mucosal biopsies) at the time of colonoscopy. Following an 8-week stabilisation period to re-establish normal gut microbiota post colonoscopy, study personnel randomise participants to either a high fibre supplement (resistant starch, n=30) or placebo (digestible starch, n=30) condition, repeating stool sample collection. During the 28-day supplement trial, each participant consumes their usual diet plus their supplement under direct observation. On day 29, participants undergo a flexible sigmoidoscopy to obtain mucosal biopsy samples to measure the effect of the supplement on inflammatory and proliferative biomarkers of cancer risk, with follow-up assessments and data/specimen collection similar to baseline. Secondary outcome measures include the impact of a high fibre supplement on the oral and colonic microbiome and biofluid metabolome. ETHICS AND DISSEMINATION: Approvals were obtained from the Alaska Area and University of Pittsburgh Institutional Review Boards and Alaska Native Tribal Health Consortium and Southcentral Foundation research review bodies. A data safety monitoring board, material transfer agreements and weekly study team meetings provide regular oversight throughout the study. Study findings will first be shared with AN tribal leaders, health administrators, providers and community members. Peer-reviewed journal articles and conference presentations will be forthcoming once approved by tribal review bodies. TRIAL REGISTRATION NUMBER: NCT03028831.

Clinical Characteristics of Inflammatory Bowel Disease Patients Requiring Long-Term Parenteral Support in the Present Era of Highly Effective Biologic Therapy.

BACKGROUND: Despite advances in the medical management of inflammatory bowel disease (IBD), a subset of patients may require extensive surgery, leading to short-bowel syndrome/intestinal failure requiring long-term home parenteral nutrition (PN) or customized intravenous fluid (IVF) support. Our aim was to further define the characteristics of IBD patients requiring home PN/IVF. METHODS: This is an observational study from a prospective IBD research registry. Patients receiving long-term home PN/IVF support during 2009-2015 were identified and compared with remaining IBD patients. Demographics, surgical history, smoking, narcotic use, IBD treatment, healthcare charges, and presence of biomarkers were reviewed. The IBD-PN group was stratified into 3 groups based on median healthcare charges. RESULTS: Of 2359 IBD patients, there were 25 (1%, 24 with Crohn's disease) who required home PN/IVF, and 250 randomly selected IBD patients matched for disease type formed the control population. Median duration of PN use was 27 months (interquartile range, 11-66). PN use was significantly associated with smoking, narcotic use, IBD-related operations, and lower quality-of-life scores. Among IBD-PN patients, 7 of 25 (28%, 3 after use of teduglutide) were able to successfully discontinue this modality. Median healthcare charges in the IBD-PN group were $51,456 annually. Median charges in the controls were $3427. Period prevalence mortality was 11.5% in IBD-PN and 3.8% in controls. CONCLUSIONS: IBD patients requiring long-term home PN/IVF support are a small minority in the present era of immunomodulator/biologic therapy. These refractory patients have a 15-fold increase in annual median healthcare charges compared with control IBD patients.

A simplified method for the quantitation of short-chain fatty acids in human stool.

A one-vial extraction method for the quantitation of short-chain fatty acids (SCFAs) in human stool was developed. Samples were extracted with an acidified aqueous internal standard solution, sodium sulfate, and diethyl ether, followed by analysis with GC-FID. Accuracy, in terms of relative recovery, was typically between 90 and 110% for most analytes; without internal standard, the accuracy was about 5-34%; the linear dynamic range (LDR) was 0.05-50 µmol per gram; the limit of detection (LOD) was less than or equal to 0.05 µmol per gram; and the (lower) limit of quantitation (LOQ) was 1 µmol per gram. The method is suitable for quantitating acetic acid, propanoic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, isohexanoic acid, hexanoic acid, and heptanoic acid. It is not suitable for the quantitation of formic acid. Application to human biological research was tested by the measurement of SCFA in heathy humans. This confirmed that the method performed adequately, and even better than expected, with values up to 150 µmol per gram.

Dietary fat, bile acid metabolism and colorectal cancer.

Colorectal cancer (CRC) risk is predominantly driven by environmental factors, in particular diet. A high intake of dietary fat has been implicated as a risk factor inducing the formation of pre-neoplastic lesions (e.g., adenomatous polyps) and/or exacerbating colonic tumorigenesis. Recent data attributed the tumor-promoting activity of high-fat diets to their effects on gut microbiota composition and metabolism, in particular with regard to bile acids. Bile acids are synthesized in the liver in response to dietary fat and facilitate lipid absorption in the small intestine. The majority of bile acids is re-absorbed during small intestinal transit and subjected to enterohepatic circulation. Bile acids entering the colon undergo complex biotransformation performed by gut bacteria, resulting in secondary bile acids that show tumor-promoting activity. Excessive dietary fat leads to high levels of secondary bile acids in feces and primes the gut microbiota to bile acid metabolism. This promotes an altered overall bile acid pool, which activates or restricts intestinal and hepatic cross-signaling of the bile acid receptor, farnesoid X receptor (FXR). Recent studies provided evidence that FXR is a main regulator of bile acid-mediated effects on intestinal tumorigenesis integrating dietary, microbial and genetic risk factors for CRC. Selective FXR agonist or antagonist activity by specific bile acids depends on additional factors (e.g., bile acid concentration, composition of bile acid pool, genetic instability of cells) and, thus, may differ in healthy and tumorigenic conditions in the intestine. In conclusion, fat-mediated alterations of the gut microbiota link bile acid metabolism to CRC risk and colonic tumorigenesis, exemplifying how gut microbial co-metabolism affects colon health.

Weak Microbial Metabolites: a Treasure Trove for Using Biomimicry to Discover and Optimize Drugs.

For decades, traditional drug discovery has used natural product and synthetic chemistry approaches to generate libraries of compounds, with some ending as promising drug candidates. A complementary approach has been to adopt the concept of biomimicry of natural products and metabolites so as to improve multiple drug-like features of the parent molecule. In this effort, promiscuous and weak interactions between ligands and receptors are often ignored in a drug discovery process. In this Emerging Concepts article, we highlight microbial metabolite mimicry, whereby parent metabolites have weak interactions with their receptors that then have led to discrete examples of more potent and effective drug-like molecules. We show specific examples of parent-metabolite mimics with potent effects in vitro and in vivo. Furthermore, we show examples of emerging microbial ligand-receptor interactions and provide a context in which these ligands could be improved as potential drugs. A balanced conceptual advance is provided in which we also acknowledge potential pitfalls-hyperstimulation of finely balanced receptor-ligand interactions could also be detrimental. However, with balance, we provide examples of where this emerging concept needs to be tested. SIGNIFICANCE STATEMENT: Microbial metabolite mimicry is a novel way to expand on the chemical repertoire of future drugs. The emerging concept is now explained using specific examples of the discovery of therapeutic leads from microbial metabolites.

Diet and the Human Gut Microbiome: An International Review.

This review summarizes the key results of recently published studies on the effects of dietary change and nutritional intervention on the human microbiome from around the world, focusing on the USA, Canada, Europe, Asia, and Africa. It first explores mechanisms that might explain the ability of fiber-rich foods to suppress the incidence and mortality from westernized diseases, notably cancers of the colon, breast, liver, cardiovascular, infectious, and respiratory diseases, diabetes, and obesity (O'Keefe in Lancet Gastroenterol Hepatol 4(12):984-996, 2019; Am J Clin Nutr 110:265-266, 2019). It summarizes studies from Africa which suggest that disturbance of the colonic microbiome may exacerbate chronic malnutrition and growth failure in impoverished communities and highlights the importance of breast feeding. The American section discusses the role of the microbiome in the swelling population of patients with obesity and type 2 diabetes and examines the effects of race, ethnicity, geography, and climate on microbial diversity and metabolism. The studies from Europe and Asia extoll the benefits of whole foods and plant-based diets. The Asian studies examine the worrying changes from low-fat, high-carbohydrate diets to high-fat, low-carbohydrate ones and the increasing appearance of westernized diseases as in Africa and documents the ability of high-fiber traditional Chinese diets to reverse type 2 diabetes and control weight loss. In conclusion, most of the studies reviewed demonstrate clear changes in microbe abundances and in the production of fermentation products, such as short-chain fatty acids and phytochemicals following dietary change, but the significance of the microbiota changes to human health, with the possible exception of the stimulation of butyrogenic taxa by fiber-rich foods, is generally implied and not measured. Further studies are needed to determine how these changes in microbiota composition and metabolism can improve our health and be used to prevent and treat disease.

A prospective cohort analysis of gut microbial co-metabolism in Alaska Native and rural African people at high and low risk of colorectal cancer.

BACKGROUND: Alaska Native (AN) people have the world's highest recorded incidence of sporadic colorectal cancer (CRC) (∼91:100,000), whereas rural African (RA) people have the lowest risk (<5:100,000). Previous data supported the hypothesis that diet affected CRC risk through its effects on the colonic microbiota that produce tumor-suppressive or -promoting metabolites. OBJECTIVES: We investigated whether differences in these metabolites may contribute to the high risk of CRC in AN people. METHODS: A cross-sectional observational study assessed dietary intake from 32 AN and 21 RA healthy middle-aged volunteers before screening colonoscopy. Analysis of fecal microbiota composition by 16S ribosomal RNA gene sequencing and fecal/urinary metabolites by 1H-NMR spectroscopy was complemented with targeted quantification of fecal SCFAs, bile acids, and functional microbial genes. RESULTS: Adenomatous polyps were detected in 16 of 32 AN participants, but not found in RA participants. The AN diet contained higher proportions of fat and animal protein and less fiber. AN fecal microbiota showed a compositional predominance of Blautia and Lachnoclostridium, higher microbial capacity for bile acid conversion, and low abundance of some species involved in saccharolytic fermentation (e.g., Prevotellaceae, Ruminococcaceae), but no significant lack of butyrogenic bacteria. Significantly lower concentrations of tumor-suppressive butyrate (22.5 ± 3.1 compared with 47.2 ± 7.3 SEM µmol/g) coincided with significantly higher concentrations of tumor-promoting deoxycholic acid (26.7 ± 4.2 compared with 11 ± 1.9 µmol/g) in AN fecal samples. AN participants had lower quantities of fecal/urinary metabolites than RA participants and metabolite profiles correlated with the abundance of distinct microbial genera in feces. The main microbial and metabolic CRC-associated markers were not significantly altered in AN participants with adenomatous polyps. CONCLUSIONS: The low-fiber, high-fat diet of AN people and exposure to carcinogens derived from diet or environment are associated with a tumor-promoting colonic milieu as reflected by the high rates of adenomatous polyps in AN participants.

Fiber, Fat, and Colorectal Cancer: New Insight into Modifiable Dietary Risk Factors.

PURPOSE OF REVIEW: To review recent data on the role and interactions of fiber and fat as dietary risk factors associated with colorectal cancer (CRC) risk in humans. RECENT FINDINGS: Fiber intake shows convincing and linear dose-response negative correlation with CRC risk. Dietary fiber stimulates butyrogenic activity of the gut microbiota, providing high amounts of butyrate that shows extensive anti-neoplastic effects. A high-fat diet promotes CRC risk through stimulated bile acid metabolism, facilitating bile acid conversion by the gut microbiota to tumor-promoting deoxycholic acid. Comprehensive interactions of these microbial metabolites are likely to underlie mechanisms driving diet-dependent CRC risk in different populations, but require further experimental investigation. Dietary fiber and fat shape the composition and metabolic function of the gut microbiota, resulting in altered amounts of butyrate and deoxycholic acid in the colon. Fiber supplementation and restriction of fat intake represent promising strategies to reduce CRC risk in healthy individuals.

The association between dietary fibre deficiency and high-income lifestyle-associated diseases: Burkitt's hypothesis revisited.

In 1969, Denis Burkitt published an article titled "Related disease-related cause?", which became the foundation for Burkitt's hypothesis. Working in Uganda, he noted that middle-aged people (40-60 years old) had a much lower incidence of diseases that were common in similarly aged people living in England, including colon cancer, diverticulitis, appendicitis, hernias, varicose veins, diabetes, atherosclerosis, and asthma, all of which are associated with lifestyles commonly led in high-income countries (HICs; also known as western diseases). Following Cleave's common cause hypothesis-which suggests that if a group of diseases occur together in the same population or individual, they are likely to have a common cause-Burkitt attributed these diseases to the small quantities of dietary fibre consumed in HICs due mainly to the over-processing of natural foods. Nowadays, dietary fibre intake in HICs is around 15 g/day (well below the amount of fibre Burkitt advocated of >50 g/day-which is associated with diets from rural, southern and eastern sub-Sahalean Africa). Since Burkitt's death in 1993, his hypothesis has been verified and extended by large-scale epidemiological studies, which have reported that fibre deficiency increases the risk of colon, liver, and breast cancer and increases all cancer mortality and death from cardiovascular, infectious, and respiratory diseases, diabetes, and all non-cardiovascular, non-cancer causes. Furthermore, mechanistic studies have now provided molecular explanations for these associations, typified by the role of short-chain fatty acids, products of fibre fermentation in the colon, in suppressing colonic mucosal inflammation and carcinogenesis. Evidence suggests that short-chain fatty acids can affect the epigenome through metabolic regulatory receptors in distant organs, and that this can reduce obesity, diabetes, atherosclerosis, allergy, and cancer. Diseases associated with high-income lifestyles are the most serious threat to health in developed countries, and public and governmental awareness needs to be improved to urge an increase in intake of fibre-rich foods. This Viewpoint will summarise the evidence that suggests that increasing dietary fibre intake to 50 g/day is likely to increase lifespan, improve the quality of life during the added years, and substantially reduce health-care costs.

International Cancer Microbiome Consortium consensus statement on the role of the human microbiome in carcinogenesis.

OBJECTIVE: In this consensus statement, an international panel of experts deliver their opinions on key questions regarding the contribution of the human microbiome to carcinogenesis. DESIGN: International experts in oncology and/or microbiome research were approached by personal communication to form a panel. A structured, iterative, methodology based around a 1-day roundtable discussion was employed to derive expert consensus on key questions in microbiome-oncology research. RESULTS: Some 18 experts convened for the roundtable discussion and five key questions were identified regarding: (1) the relevance of dysbiosis/an altered gut microbiome to carcinogenesis; (2) potential mechanisms of microbiota-induced carcinogenesis; (3) conceptual frameworks describing how the human microbiome may drive carcinogenesis; (4) causation versus association; and (5) future directions for research in the field.The panel considered that, despite mechanistic and supporting evidence from animal and human studies, there is currently no direct evidence that the human commensal microbiome is a key determinant in the aetiopathogenesis of cancer. The panel cited the lack of large longitudinal, cohort studies as a principal deciding factor and agreed that this should be a future research priority. However, while acknowledging gaps in the evidence, expert opinion was that the microbiome, alongside environmental factors and an epigenetically/genetically vulnerable host, represents one apex of a tripartite, multidirectional interactome that drives carcinogenesis. CONCLUSION: Data from longitudinal cohort studies are needed to confirm the role of the human microbiome as a key driver in the aetiopathogenesis of cancer.

Switching to a fibre-rich and low-fat diet increases colonic folate contents among African Americans.

How dietary patterns impact colonic bacterial biosynthesis of vitamins and utilization by humans is poorly understood. Our aim was to investigate whether a reciprocal dietary switch between rural South Africans (traditionally high fibre, low fat) and African Americans (Western diet of low fibre, high fat) affects colonic folate synthesis. Colonic evacuants were obtained from 20 rural South Africans and 20 African Americans consuming their usual diets at baseline. For 2 weeks thereafter, rural South Africans were provided with a Western diet (protein, 27%; fat, 52%; carbohydrate, 20%; and fibre, 8 g/day) and African Americans were provided with a high fibre, low-fat diet (protein, 16%; fat, 17%; carbohydrate, 63%; and fibre, 43 g/day). Colonic evacuants were again collected. No difference between groups at baseline in the folate content of 3-h evacuants was observed. The high-fibre, low-fat diet consumed by African Americans during the intervention produced a 41% increase in mean total folate content compared with baseline values (p = 0.0037). No change was observed in rural South Africans consuming a Western diet. Mean total folate content of colonic evacuants was higher among African Americans at the end of the dietary switch (3107 ± 1811 µg) compared with rural South Africans (2157 ± 1956 µg) (p = 0.0409). In conclusion, consistent with animal studies, switching from a Western diet to one higher in fibre and lower in fat can be expected to result in greater colonic folate content. Future research should confirm that these observations are not transitory and understand the contribution of transit-time to the findings.

Association of Dietary Habits with Severity of Acute Pancreatitis.

BACKGROUND: The effect of diet on risk of acute pancreatitis (AP) has been suggested by prior studies, but the association of dietary habits with severity of AP has not been previously evaluated. OBJECTIVE: The objective of the study was to assess differences in reported dietary habits in patients with severe AP compared with those with mild or moderate AP. METHODS: A prospectively maintained cohort of patients with AP was utilized. A brief questionnaire on dietary habits was implemented. Dietary habits were categorized based on the overall type of diet, fruit/vegetable servings, fat content, dairy consumption, dessert/sweets consumption, and fluid intake. Patients were grouped into mild/moderate and severe AP. Multivariate analysis was used to determine whether dietary habits have an independent association with AP severity. RESULTS: 407 patients with AP were studied. Mean patient age was 51 y, and 202 (50%) were men. 29% of patients were smokers and 46% actively consumed alcohol. 225 patients had mild AP, 103 moderate AP, and 79 developed severe AP. The 3 groups were comparable in race, body mass index, etiology of AP, and comorbidities. Dietary factors were overall comparable between the groups except for diet type: subjects with severe AP had a higher percentage of consuming a meat-rich diet (84%) than patients with mild AP (72%) and moderate AP (67%) (P = 0.04). Based on multivariable logistic regression, the OR of developing severe AP was 2.5 (95% CI: 1.24-5.32, P = 0.01) between patients who eat a meat-rich diet and those who consume a vegetable-based diet. CONCLUSIONS: A meat-rich diet is independently associated with the development of persistent organ failure (severe disease) in patients with AP. These findings require further evaluation and could be useful for patient counseling, risk stratification, and disease prevention. This study is registered at clinicaltrials.gov as NCT03075605.

Considerations for best practices in studies of fiber or other dietary components and the intestinal microbiome.

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.