Intense recent interest in understanding how the human gut microbiome influences health has kindled a concomitant interest in linking dietary choices to microbiome variation. Diet is known to be a driver of microbiome variation, and yet the precise mechanisms by which certain dietary components modulate the microbiome, and by which the microbiome produces byproducts and secondary metabolites from dietary components, are not well-understood. Interestingly, despite the influence of diet on the gut microbiome, the majority of microbiome studies published to date contain little or no analysis of dietary intake. Although an increasing number of microbiome studies are now collecting some form of dietary data or even performing diet interventions, there are no clear standards in the microbiome field for how to collect diet data or how to design a diet-microbiome study. In this article, we review the current practices in diet-microbiome analysis and study design and make several recommendations for best practices to provoke broader discussion in the field. We recommend that microbiome studies include multiple consecutive microbiome samples per study timepoint or phase and multiple days of dietary history prior to each microbiome sample whenever feasible. We find evidence that direct effects of diet on the microbiome are likely to be observable within days, while the length of an intervention required for observing microbiome-mediated effects on the host phenotype or host biomarkers, depending on the outcome, may be much longer, on the order of weeks or months. Finally, recent studies demonstrating that dietmicrobiome interactions are personalized suggest that diet-microbiome studies should either include longitudinal sampling within individuals to identify personalized responses, or should include an adequate number of participants spanning a range of microbiome types to identify generalized responses.

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

Dietary fiber, a nutrient derived mainly from whole grains, vegetables, fruits, and legumes, is known to confer a number of health benefits, yet most Americans consume less than half of the daily recommended amount. Convenience and affordability are key factors determining the ability of individuals to incorporate fiber-rich foods into their diet, and many Americans struggle to access, afford, and prepare foods rich in fiber. The objective of this clinical study was to test the changes in microbial community composition, human metabolomics, and general health markers of a convenient, easy to use prebiotic supplement in generally healthy young participants consuming a diet low in fiber. Twenty healthy adults participated in this randomized, placebo-controlled, double-blind, crossover study which was registered at clinicaltrials.gov as NCT03785860. During the study participants consumed 12 g of a prebiotic fiber supplement and 12 g of placebo daily as a powder mixed with water as part of their habitual diet in randomized order for 4 weeks, with a 4-week washout between treatment arms. Fecal microbial DNA was extracted and sequenced by shallow shotgun sequencing on an Illumina NovaSeq. Plasma metabolites were detected using liquid chromatography–mass spectrometry with untargeted analysis. The phylum Actinobacteria, genus Bifidobacterium, and several Bifidobacterium species (B. bifidum, B. adolescentis, B. breve, B. catenulatum, and B. longum) significantly increased after prebiotic supplementation when compared to the placebo. The abundance of genes associated with the utilization of the prebiotic fiber ingredients (sacA, xfp, xpk) and the production of acetate (poxB, ackA) significantly changed with prebiotic supplementation. Additionally, the abundance of genes associated with the prebiotic utilization (xfp, xpk), acetate production (ackA), and choline to betaine oxidation (gbsB) were significantly correlated with changes in the abundance of the genus Bifidobacterium in the prebiotic group. Plasma concentrations of the bacterially produced metabolite indolepropionate significantly increased. The results of this study demonstrate that an easy to consume, low dose (12 g) of a prebiotic powder taken daily increases the abundance of beneficial bifidobacteria and the production of health-promoting bacteria-derived metabolites in healthy individuals with a habitual low-fiber diet.Clinical Trial Registrationwww.clinicaltrials.gov/, identifier: NCT03785860

BackgroundHigh‐density lipoproteins (HDL) in circulation perform a crucial role in maintaining cholesterol homeostasis by removing cholesterol from peripheral tissues for excretion. High HDL has been linked with a lower risk of developing Alzheimer’s Disease (AD). However, the concentration of HDL only partially accounts for the variation in the ability of HDL to perform their primary function to efflux cholesterol. Apolipoprotein E (APOE) is the strongest genetic risk factor for AD, and its role in regulating cholesterol is implicated in AD. However, the complex relationship between APOE genotype and the function of peripheral HDL in AD patients remains largely unknown.MethodWe isolated HDL from plasma of 194 age‐ and sex‐matched, ApoE genotyped participants (non‐demented controls, n = 83; mild cognitive impairment, n = 40; AD dementia, n = 71) using two‐step flotation ultracentrifugation, followed by size exclusion chromatography. HDL cholesterol efflux capacity (CEC) and lecithin‐cholesterol acyltransferase (LCAT) activity were measured. We further explored the relationships between HDL functional capacity and cognitive, functional, and imaging scores.ResultRegardless of diagnosis, ApoE3/E4 carriers had significantly lower CEC index (p = 0.003) and LCAT activity (p < 0.001) vs. ApoE3/E3 carriers. When participants were stratified by APOE genotype and diagnosis, ApoE3/E3 MCI patients had significantly lower CEC index than controls (p = 0.042). In contrast, the CEC index of ApoE3/E4 AD patients was paradoxically increased compared to controls (p = 0.016). ApoE3/E3 MCI and AD patients had lower LCAT activity than controls (p = 0.012 and p = 0.004, respectively), but there were no differences in LCAT activity in ApoE3/E4 participants by diagnosis group. LCAT activity was positively correlated with verbal memory score (p = 0.033) and negatively correlated with clinical dementia rating (p = 0.025).ConclusionTo our knowledge, our study revealed for the first time an APOE genotype‐dependent alteration in peripheral HDL functional capacity among AD and MCI patients compared with non‐demented controls and an association between HDL LCAT activity and cognitive and functional scores.

BackgroundHigh density lipoproteins (HDL) are protective against a wide array of diseases and have recently also been found to be associated with reduced risk of Alzheimer’s Disease (AD). However, the amount of circulating HDL‐cholesterol does not correlate closely with function, explaining only about 40% of the variance in HDL’s ability to perform its primary role of cholesterol efflux. HDL particle composition, size and structure are important determinants of functional capacity, however little is known about HDL particle physical characteristics in AD patients, in particular whether there are apolipoprotein E (APOE) genotype‐dependent effects.MethodPlasma samples from a total of 172 APOE genotyped patients, n = 37 ApoE3/E3 and n = 34 ApoE3/E4 with diagnosed AD, n = 22 ApoE3/E3 and n = 18 ApoE3/E4 with diagnosed MCI, and n = 44 ApoE/E3 and n = 39 ApoE3/E4 age‐ and sex‐matched non‐AD controls were obtained from the UC Davis Alzheimer’s Disease Research Center biobank. HDL particles were isolated from plasma by sequential density‐adjusted ultracentrifugation followed by size‐exclusion chromatography. The size of HDL from each patient was characterized using negative‐stained transmission electron microscopy (NS‐TEM) followed by computational particle size analysis.ResultOn average, 34 TEM micrographs were obtained, and over 33,000 individual particles were selected and analyzed from each HDL sample using pre‐determined image quality and image selection criteria. The average particle size were compared between AD patients and controls stratified by APOE genotype. The mean particle diameter of HDL from MCI and AD patients was smaller compared to the control group (8.28 +/‐ 0.587, 8.30 +/‐ 0.662 vs. 8.84 +/‐ 0.587, respectively). The differences remained statistically significant when stratified by genotype. Both MCI and AD group had smaller HDL particle size compared to the control group in the ApoE3/E3 genotype (8.37 +/‐ 0.452, 8.48 +/‐ 0.613 vs. 8.90 +/‐ 0.602, respectively), and in the ApoE3/E4 genotype (8.17 +/‐ 0.707, 8.11 +/‐ 0.667 vs. 8.80 +/‐ 0.582, respectively).ConclusionThese results reveal that patients with Alzheimer’s dementia and mild cognitive impairment have disrupted HDL particle distribution, with smaller average HDL particle size, highlighting the need for further studies of lipoprotein metabolism in AD.