Improving rigor and reproducibility in nonhuman primate research.

Nonhuman primates (NHPs) are a critical component of translational/preclinical biomedical research due to the strong similarities between NHP and human physiology and disease pathology. In some cases, NHPs represent the most appropriate, or even the only, animal model for complex metabolic, neurological, and infectious diseases. The increased demand for and limited availability of these valuable research subjects requires that rigor and reproducibility be a prime consideration to ensure the maximal utility of this scarce resource. Here, we discuss a number of approaches that collectively can contribute to enhanced rigor and reproducibility in NHP research.

Intraspecific and interspecific investigations of skeletal DNA methylation and femur morphology in primates.

OBJECTIVES: Epigenetic mechanisms influence the development and maintenance of complex phenotypes and may also contribute to the evolution of species-specific phenotypes. With respect to skeletal traits, little is known about the gene regulation underlying these hard tissues or how tissue-specific patterns are associated with bone morphology or vary among species. To begin exploring these topics, this study evaluates one epigenetic mechanism, DNA methylation, in skeletal tissues from five nonhuman primate species which display anatomical and locomotor differences representative of their phylogenetic groups. MATERIALS AND METHODS: First, we test whether intraspecific variation in skeletal DNA methylation is associated with intraspecific variation in femur morphology. Second, we identify interspecific differences in DNA methylation and assess whether these lineage-specific patterns may have contributed to species-specific morphologies. Specifically, we use the Illumina Infinium MethylationEPIC BeadChip to identify DNA methylation patterns in femur trabecular bone from baboons (n = 28), macaques (n = 10), vervets (n = 10), chimpanzees (n = 4), and marmosets (n = 6). RESULTS: Significant differentially methylated positions (DMPs) were associated with a subset of morphological variants, but these likely have small biological effects and may be confounded by other variables associated with morphological variation. Conversely, several species-specific DMPs were identified, and these are found in genes enriched for functions associated with complex skeletal traits. DISCUSSION: Overall, these findings reveal that while intraspecific epigenetic variation is not readily associated with skeletal morphology differences, some interspecific epigenetic differences in skeletal tissues exist and may contribute to evolutionarily distinct phenotypes. This work forms a foundation for future explorations of gene regulation and skeletal trait evolution in primates.

Shades of complexity: New perspectives on the evolution and genetic architecture of human skin.

Like many highly variable human traits, more than a dozen genes are known to contribute to the full range of skin color. However, the historical bias in favor of genetic studies in European and European-derived populations has blinded us to the magnitude of pigmentation's complexity. As deliberate efforts are being made to better characterize diverse global populations and new sequencing technologies, better measurement tools, functional assessments, predictive modeling, and ancient DNA analyses become more widely accessible, we are beginning to appreciate how limited our understanding of the genetic bases of human skin color have been. Novel variants in genes not previously linked to pigmentation have been identified and evidence is mounting that there are hundreds more variants yet to be found. Even for genes that have been exhaustively characterized in European populations like MC1R, OCA2, and SLC24A5, research in previously understudied groups is leading to a new appreciation of the degree to which genetic diversity, epistatic interactions, pleiotropy, admixture, global and local adaptation, and cultural practices operate in population-specific ways to shape the genetic architecture of skin color. Furthermore, we are coming to terms with how factors like tanning response and barrier function may also have influenced selection on skin throughout human history. By examining how our knowledge of pigmentation genetics has shifted in the last decade, we can better appreciate how far we have come in understanding human diversity and the still long road ahead for understanding many complex human traits.

Modeling 3D facial shape from DNA.

Human facial diversity is substantial, complex, and largely scientifically unexplained. We used spatially dense quasi-landmarks to measure face shape in population samples with mixed West African and European ancestry from three locations (United States, Brazil, and Cape Verde). Using bootstrapped response-based imputation modeling (BRIM), we uncover the relationships between facial variation and the effects of sex, genomic ancestry, and a subset of craniofacial candidate genes. The facial effects of these variables are summarized as response-based imputed predictor (RIP) variables, which are validated using self-reported sex, genomic ancestry, and observer-based facial ratings (femininity and proportional ancestry) and judgments (sex and population group). By jointly modeling sex, genomic ancestry, and genotype, the independent effects of particular alleles on facial features can be uncovered. Results on a set of 20 genes showing significant effects on facial features provide support for this approach as a novel means to identify genes affecting normal-range facial features and for approximating the appearance of a face from genetic markers.

Exome sequences of multiplex, multigenerational families reveal schizophrenia risk loci with potential implications for neurocognitive performance.

Schizophrenia is a serious mental illness, involving disruptions in thought and behavior, with a worldwide prevalence of about one percent. Although highly heritable, much of the genetic liability of schizophrenia is yet to be explained. We searched for susceptibility loci in multiplex, multigenerational families affected by schizophrenia, targeting protein-altering variation with in silico predicted functional effects. Exome sequencing was performed on 136 samples from eight European-American families, including 23 individuals diagnosed with schizophrenia or schizoaffective disorder. In total, 11,878 non-synonymous variants from 6,396 genes were tested for their association with schizophrenia spectrum disorders. Pathway enrichment analyses were conducted on gene-based test results, protein-protein interaction (PPI) networks, and epistatic effects. Using a significance threshold of FDR < 0.1, association was detected for rs10941112 (p = 2.1 × 10-5 ; q-value = 0.073) in AMACR, a gene involved in fatty acid metabolism and previously implicated in schizophrenia, with significant cis effects on gene expression (p = 5.5 × 10-4 ), including brain tissue data from the Genotype-Tissue Expression project (minimum p = 6.0 × 10-5 ). A second SNP, rs10378 located in TMEM176A, also shows risk effects in the exome data (p = 2.8 × 10-5 ; q-value = 0.073). PPIs among our top gene-based association results (p < 0.05; n = 359 genes) reveal significant enrichment of genes involved in NCAM-mediated neurite outgrowth (p = 3.0 × 10-5 ), while exome-wide SNP-SNP interaction effects for rs10941112 and rs10378 indicate a potential role for kinase-mediated signaling involved in memory and learning. In conclusion, these association results implicate AMACR and TMEM176A in schizophrenia risk, whose effects may be modulated by genes involved in synaptic plasticity and neurocognitive performance.

The Evolution of Tanning Needs Its Day in the Sun.

Variation in human pigmentation has long been an area of interest in biological anthropology, with the advent of genetic technologies allowing deeper plumbing of its evolutionary history. Genome-wide scans of selection show that pigmentation genes have undergone some of the strongest selection in many geographically distant populations. A variety of hypotheses for the photoprotective effects of melanin have been developed, but these hypotheses, as well as genetic studies, focus nearly exclusively on constitutive (basal) pigmentation levels. Failing to consider the contribution of the ultraviolet radiation (UVR) environment neglects the true interface between humans and our environment. Data drawn largely from dermatology demonstrate that constitutive pigmentation and tanning response are weakly coupled in populations from East Asia and the Americas. This suggests a possible role for persistent, UVR-induced pigmentation as a convergent adaptation akin to the protective effect of constitutive pigmentation. The adaptive potential of tanned skin, particularly in the Americas, where constitutive pigmentation is lower than expected, may fill in an important gap in our understanding of the evolution of skin color.

Natural selection for the Duffy-null allele in the recently admixed people of Madagascar.

While gene flow between distantly related populations is increasingly recognized as a potentially important source of adaptive genetic variation for humans, fully characterized examples are rare. In addition, the role that natural selection for resistance to vivax malaria may have played in the extreme distribution of the protective Duffy-null allele, which is nearly completely fixed in mainland sub-Saharan Africa and absent elsewhere, is controversial. We address both these issues by investigating the evolution of the Duffy-null allele in the Malagasy, a recently admixed population with major ancestry components from both East Asia and mainland sub-Saharan Africa. We used genome-wide genetic data and extensive computer simulations to show that the high frequency of the Duffy-null allele in Madagascar can only be explained in the absence of positive natural selection under extreme demographic scenarios involving high genetic drift. However, the observed genomic single nucleotide polymorphism diversity in the Malagasy is incompatible with such extreme demographic scenarios, indicating that positive selection for the Duffy-null allele best explains the high frequency of the allele in Madagascar. We estimate the selection coefficient to be 0.066. Because vivax malaria is endemic to Madagascar, this result supports the hypothesis that malaria resistance drove fixation of the Duffy-null allele in mainland sub-Saharan Africa.

ALDH2 is associated to alcohol dependence and is the major genetic determinant of "daily maximum drinks" in a GWAS study of an isolated rural Chinese sample.

Alcohol dependence (AD) is a moderately heritable phenotype with a small number of known risk genes mapped via linkage or candidate gene studies. We considered 313 males from among 595 members of documented, extended pedigrees in which AD segregates collected in Northern Hunan Province, China. A joint analysis of both males and females could not be performed as the difference in alcohol consumption variance was too large. Genome-wide association analyses were performed for approximately 300,000 single nucleotide polymorphisms (SNPs). Significant associations found in the ALDH2 region for AD (minimum P = 4.73 × 10(-8)) and two AD-related phenotypes: flushing response (minimum P = 4.75 × 10(-26)) and maximum drinks in a 24-hr period (minimum P = 1.54 × 10(-16)). Association of previous candidate SNP, rs10774610 in CCDC63, was confirmed but resulted from linkage disequilibrium with ALDH2. ALDH2 is strongly associated with flushing response, AD, and maximum drinks in males, with nonsynonymous SNP rs671 explaining 29.2%, 7.9%, and 22.9% of phenotypic variation, respectively, in this sample. When rs671 was considered as a candidate SNP in females, it explained 23.6% of the variation in flushing response, but alcohol consumption rates were too low among females-despite familial enrichment for AD-for an adequate test of association for either AD or maximum drinks. These results support a mediating effect of aldehyde dehydrogenase deficiency on alcohol consumption in males and a secondary, culturally mediated limitation on alcohol consumption by females that should be appropriately modeled in future studies of alcohol consumption in populations where this may be a factor.

Novel QTL at chromosome 6p22 for alcohol consumption: Implications for the genetic liability of alcohol use disorders.

Linkage studies of alcoholism have implicated several chromosome regions, leading to the successful identification of susceptibility genes, including ADH4 and GABRA2 on chromosome 4. Quantitative endophenotypes that are potentially closer to gene action than clinical endpoints offer a means of obtaining more refined linkage signals of genes that predispose alcohol use disorders (AUD). In this study we examine a self-reported measure of the maximum number of drinks consumed in a 24-hr period (abbreviated Max Drinks), a significantly heritable phenotype (h(2) = 0.32 ± 0.05; P = 4.61 × 10(-14)) with a strong genetic correlation with AUD (ρg = 0.99 ± 0.13) for the San Antonio Family Study (n = 1,203). Genome-wide SNPs were analyzed using variance components linkage methods in the program SOLAR, revealing a novel, genome-wide significant QTL (LOD = 4.17; P = 5.85 × 10(-6)) for Max Drinks at chromosome 6p22.3, a region with a number of compelling candidate genes implicated in neuronal function and psychiatric illness. Joint analysis of Max Drinks and AUD status shows that the QTL has a significant non-zero effect on diagnosis (P = 4.04 × 10(-3)), accounting for 8.6% of the total variation. Significant SNP associations for Max Drinks were also identified at the linkage region, including one, rs7761213 (P = 2.14 × 10(-4)), obtained for an independent sample of Chinese families. Thus, our study identifies a potential risk locus for AUD at 6p22.3, with significant pleiotropic effects on the heaviness of alcohol consumption that may not be population specific.

Technical note: comparing von Luschan skin color tiles and modern spectrophotometry for measuring human skin pigmentation.

Prior to the introduction of reflectance spectrophotometry into anthropological field research during the 1950s, human skin color was most commonly classified by visual skin color matching using the von Luschan tiles, a set of 36 standardized, opaque glass tiles arranged in a chromatic scale. Our goal was to establish a conversion formula between the tile-based color matching method and modern reflectance spectrophotometry to make historical and contemporary data comparable. Skin pigmentation measurements were taken on the forehead, inner upper arms, and backs of the hands using both the tiles and a spectrophotometer on 246 participants showing a broad range of skin pigmentation. From these data, a second-order polynomial conversion formula was derived by jackknife analysis to estimate melanin index (M-index) based on tile values. This conversion formula provides a means for comparing modern data to von Luschan tile measurements recorded in historical reports. This is particularly important for populations now extinct, extirpated, or admixed for which tile-based measures of skin pigmentation are the only data available.

Resilience, aging, and response to radiation exposure (RARRE) in nonhuman primates: a resource review.

The Wake Forest nonhuman primate (NHP) Radiation Late Effects Cohort (RLEC) is a unique and irreplaceable population of aging NHP radiation survivors which serves the nation's need to understand the late effects of radiation exposure. Over the past 16 years, Wake Forest has evaluated > 250 previously irradiated rhesus macaques (Macaca mulatta) that were exposed to single total body irradiation (IR) doses of 1.14-8.5 Gy or to partial body exposures of up to 10 Gy (5% bone marrow sparing) or 10.75 Gy (whole thorax). Though primarily used to examine IR effects on disease-specific processes or to develop radiation countermeasures, this resource provides insights on resilience across physiologic systems and its relationship with biological aging. Exposure to IR has well documented deleterious effects on health, but the late effects of IR are highly variable. Some animals exhibit multimorbidity and accumulated health deficits, whereas others remain relatively resilient years after exposure to total body IR. This provides an opportunity to evaluate biological aging at the nexus of resilient/vulnerable responses to a stressor. Consideration of inter-individual differences in response to this stressor can inform individualized strategies to manage late effects of radiation exposure, and provide insight into mechanisms underlying systemic resilience and aging. The utility of this cohort for age-related research questions was summarized at the 2022 Trans-NIH Geroscience Interest Group's Workshop on Animal Models for Geroscience. We present a brief review of radiation injury and its relationship to aging and resilience in NHPs with a focus on the RLEC.

Social genomics, cognition, and well-being during the COVID-19 pandemic.

INTRODUCTION: Adverse psychosocial exposure is associated with increased proinflammatory gene expression and reduced type-1 interferon gene expression, a profile known as the conserved transcriptional response to adversity (CTRA). Little is known about CTRA activity in the context of cognitive impairment, although chronic inflammatory activation has been posited as one mechanism contributing to late-life cognitive decline. METHODS: We studied 171 community-dwelling older adults from the Wake Forest Alzheimer's Disease Research Center who answered questions via a telephone questionnaire battery about their perceived stress, loneliness, well-being, and impact of COVID-19 on their life, and who provided a self-collected dried blood spot sample. Of those, 148 had adequate samples for mRNA analysis, and 143 were included in the final analysis, which including participants adjudicated as having normal cognition (NC, n = 91) or mild cognitive impairment (MCI, n = 52) were included in the analysis. Mixed effect linear models were used to quantify associations between psychosocial variables and CTRA gene expression. RESULTS: In both NC and MCI groups, eudaimonic well-being (typically associated with a sense of purpose) was inversely associated with CTRA gene expression whereas hedonic well-being (typically associated with pleasure seeking) was positively associated. In participants with NC, coping through social support was associated with lower CTRA gene expression, whereas coping by distraction and reframing was associated with higher CTRA gene expression. CTRA gene expression was not related to coping strategies for participants with MCI, or to either loneliness or perceived stress in either group. DISCUSSION: Eudaimonic and hedonic well-being remain important correlates of molecular markers of stress, even in people with MCI. However, prodromal cognitive decline appears to moderate the significance of coping strategies as a correlate of CTRA gene expression. These results suggest that MCI can selectively alter biobehavioral interactions in ways that could potentially affect the rate of future cognitive decline and may serve as targets for future intervention efforts.

OPRM1 and EGFR contribute to skin pigmentation differences between Indigenous Americans and Europeans.

Contemporary variation in skin pigmentation is the result of hundreds of thousands years of human evolution in new and changing environments. Previous studies have identified several genes involved in skin pigmentation differences among African, Asian, and European populations. However, none have examined skin pigmentation variation among Indigenous American populations, creating a critical gap in our understanding of skin pigmentation variation. This study investigates signatures of selection at 76 pigmentation candidate genes that may contribute to skin pigmentation differences between Indigenous Americans and Europeans. Analysis was performed on two samples of Indigenous Americans genotyped on genome-wide SNP arrays. Using four tests for natural selection--locus-specific branch length (LSBL), ratio of heterozygosities (lnRH), Tajima's D difference, and extended haplotype homozygosity (EHH)--we identified 14 selection-nominated candidate genes (SNCGs). SNPs in each of the SNCGs were tested for association with skin pigmentation in 515 admixed Indigenous American and European individuals from regions of the Americas with high ground-level ultraviolet radiation. In addition to SLC24A5 and SLC45A2, genes previously associated with European/non-European differences in skin pigmentation, OPRM1 and EGFR were associated with variation in skin pigmentation in New World populations for the first time.

A New Method of Bone Stromal Cell Characterization by Flow Cytometry.

The bone microenvironment cellular composition plays an essential role in bone health and is disrupted in bone pathologies, such as osteoporosis, osteoarthritis, and cancer. Flow cytometry protocols for hematopoietic stem cell lineages are well defined and well established. Additionally, a consensus for mesenchymal stem cell flow markers has been developed. However, flow cytometry markers for bone-residing cells-osteoblasts, osteoclasts, and osteocytes-have not been proposed. Here, we describe a novel partial digestion method to separate these cells from the bone matrix and present new markers for enumerating these cells by flow cytometry. We optimized bone digestion and analyzed markers across murine, nonhuman primate, and human bone. The isolation and staining protocols can be used with either cell sorting or flow cytometry. Our method allows for the enumeration and collection of hematopoietic and mesenchymal lineage cells in the bone microenvironment combined with bone-residing stromal cells. Thus, we have established a multi-fluorochrome bone marrow cell-typing methodology. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Partial digestion for murine long bone stromal cell isolation Alternate Protocol 1: Partial digestion for primate vertebrae stromal cell isolation Alternate Protocol 2: Murine vertebrae crushing for bone stromal cell isolation Basic Protocol 2: Staining of bone stromal cells Support Protocol 1: Fluorescence minus one control, isotype control, and antibody titration Basic Protocol 3: Cell sorting of bone stromal cells Alternate Protocol 3: Flow cytometry analysis of bone stromal cells Support Protocol 2: Preparing compensation beads.

Circulating miRNAs associated with bone mineral density in healthy adult baboons.

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and circulate in the blood, making them attractive biomarkers of disease state for tissues like bone that are challenging to interrogate directly. Here, we report on five miRNAs-miR-197-3p, miR-320a, miR-320b, miR-331-5p, and miR-423-5p-associated with bone mineral density (BMD) in 147 healthy adult baboons. These baboons ranged in age from 15 to 25 years (45-75 human equivalent years) and 65% were female with a broad range of BMD values including a minority of osteopenic animals. miRNAs were generated via RNA sequencing from buffy coats collected at necropsy and areal BMD (aBMD) measured postmortem via dual-energy X-ray absorptiometry (DXA) of the lumbar vertebrae. Differential expression analysis controlled for the underlying pedigree structure of these animals to account for genetic variation which may drive miRNA abundance and aBMD values. While many of these miRNAs have been associated with the risk of osteoporosis in humans, this finding is of interest because the cohort represents a model of normal aging and bone metabolism rather than a disease cohort. The replication of miRNA associations with osteoporosis or other bone metabolic disorders in animals with healthy aBMD suggests an overlap in normal variation and disease states. We suggest that these miRNAs are involved in the regulation of cellular proliferation, apoptosis, and protein composition in the extracellular matrix throughout life; and age-related dysregulation of these systems may lead to disease. These miRNAs may be early indicators of progression to disease in advance of clinically detectible osteoporosis.

Iris texture traits show associations with iris color and genomic ancestry.

OBJECTIVES: This study seeks to identify associations among genomic biogeographic ancestry (BGA), quantitative iris color, and iris texture traits contributing to population-level variation in these phenotypes. METHODS: DNA and iris photographs were collected from 300 individuals across three variably admixed populations (Portugal, Brazil, and Cape Verde). Two raters scored the photos for pigmentation spots, Fuchs' crypts, contraction furrows, and Wolflinn nodes. Iris color was quantified from RGB values. Maximum likelihood estimates of individual BGA were calculated from 176 ancestry informative markers. RESULTS: Pigmentation spots, Fuchs' crypts, contraction furrows, and iris color show significant positive correlation with increasing European BGA. Only contraction furrows are correlated with iris color. CONCLUSIONS: The relationship between BGA and iris texture illustrates a genetic contribution to this population-level variation.

An evolutionary perspective of DNA methylation patterns in skeletal tissues using a baboon model of osteoarthritis.

Epigenetic factors, such as DNA methylation, play an influential role in the development of the degenerative joint disease osteoarthritis (OA). These molecular mechanisms have been heavily studied in humans, and although OA affects several other animals in addition to humans, few efforts have taken an evolutionary perspective. This study explores the evolution of OA epigenetics by assessing the relationship between DNA methylation variation and knee OA development in baboons (Papio spp.) and by comparing these findings to human OA epigenetic associations. Genome-wide DNA methylation patterns were identified in bone and cartilage of the right distal femora from 56 pedigreed, adult baboons (28 with and 28 without knee OA) using the Illumina Infinium MethylationEPIC BeadChip. Several significantly differentially methylated positions (DMPs) and regions were found between tissue types. Substantial OA-related differential methylation was also identified in cartilage, but not in bone, suggesting that cartilage epigenetics may be more influential in OA than bone epigenetics. Additionally, some genes containing OA-related DMPs overlap with and display methylation patterns similar to those previously identified in human OA, revealing a mixture of evolutionarily conserved and divergent OA-related methylation patterns in primates. Overall, these findings reinforce the current etiological perspectives of OA and enhance our evolutionary understanding of epigenetic mechanisms associated with OA. This study further establishes baboons as a valuable nonhuman primate model of OA, and continued investigations in baboons will help to disentangle the molecular mechanisms contributing to OA and their evolutionary histories.

Heterozygosity of the major histocompatibility complex predicts later self-reported pubertal maturation in men.

Individual variation in the age of pubertal onset is linked to physical and mental health, yet the factors underlying this variation are poorly understood. Life history theory predicts that individuals at higher risk of mortality due to extrinsic causes such as infectious disease should sexually mature and reproduce earlier, whereas those at lower risk can delay puberty and continue to invest resources in somatic growth. We examined relationships between a genetic predictor of infectious disease resistance, heterozygosity of the major histocompatibility complex (MHC), referred to as the human leukocyte antigen (HLA) gene in humans, and self-reported pubertal timing. In a combined sample of men from Canada (n = 137) and the United States (n = 43), MHC heterozygosity predicted later self-reported pubertal development. These findings suggest a genetic trade-off between immunocompetence and sexual maturation in human males.

Insights into the genetic architecture of the human face.

The human face is complex and multipartite, and characterization of its genetic architecture remains challenging. Using a multivariate genome-wide association study meta-analysis of 8,246 European individuals, we identified 203 genome-wide-significant signals (120 also study-wide significant) associated with normal-range facial variation. Follow-up analyses indicate that the regions surrounding these signals are enriched for enhancer activity in cranial neural crest cells and craniofacial tissues, several regions harbor multiple signals with associations to different facial phenotypes, and there is evidence for potential coordinated actions of variants. In summary, our analyses provide insights into the understanding of how complex morphological traits are shaped by both individual and coordinated genetic actions.

Use of Metabolomic Profiling to Understand Variability in Adiposity Changes Following an Intentional Weight Loss Intervention in Older Adults.

Inter-individual response to dietary interventions remains a major challenge to successful weight loss among older adults. This study applied metabolomics technology to identify small molecule signatures associated with a loss of fat mass and overall weight in a cohort of older adults on a nutritionally complete, high-protein diet. A total of 102 unique metabolites were measured using liquid chromatography-mass spectrometry (LC-MS) for 38 adults aged 65-80 years randomized to dietary intervention and 36 controls. Metabolite values were analyzed in both baseline plasma samples and samples collected following the six-month dietary intervention to consider both metabolites that could predict the response to diet and those that changed in response to diet or weight loss.Eight metabolites changed over the intervention at a nominally significant level: D-pantothenic acid, L-methionine, nicotinate, aniline, melatonin, deoxycarnitine, 6-deoxy-L-galactose, and 10-hydroxydecanoate. Within the intervention group, there was broad variation in the achieved weight-loss and dual-energy x-ray absorptiometry (DXA)-defined changes in total fat and visceral adipose tissue (VAT) mass. Change in the VAT mass was significantly associated with the baseline abundance of α-aminoadipate (p = 0.0007) and an additional mass spectrometry peak that may represent D-fructose, myo-inositol, mannose, α-D-glucose, allose, D-galactose, D-tagatose, or L-sorbose (p = 0.0001). This hypothesis-generating study reflects the potential of metabolomic biomarkers for the development of personalized dietary interventions.