42 OMICS INNOVATIONS AND APPLICATIONS FOR PUBLIC HEALTH NUTRITION

Omics Innovatons and Applicatons for Public Health Nutiton An integrated view

Sun Eun Lee Introduction Center for Human Nutrition, Department of Let’s assume that you have been taken to a place you have nev- International Health, Johns Hopkins Bloomberg School er been before, and are not sure how you got there. The frst of Public Health, Baltimore, MD, USA thing you might do is look at a Google map, which may reveal several roads leading to the place, each with outlying inter- sections and roads fom other towns. Diferent overlays may reveal varying terrain, weather or road conditions throughout Key messages the region leading to your destination. Now, imagine a health problem afecting a population at your destination, and the > Omics technologies comprise sets of molecular mapping plexus of pathways (routes), biochemical networks (towns), tools that can help us understand and navigate to or fnction (trafc fow) and other infuential conditions (ter- from states of health, including nutriture. rain, road upkeep, weather, etc.) that may lead to the health problem, overlaid on the map. Omics technologies comprise > Single studies explore sets of genes, epigenetic sets of molecular mapping tools for each overlay that can marks, transcripts, , or metabolites, as well as help us understand and navigate to or fom states of health, microbial communities, in an unbiased manner. including nutriture. This breakthrough approach has become possible due to advances in the development and application > Trans-omics studies ofer opportunities to connect, of high-throughput technologies, which allow us to analyze integrate, and map a group of molecules across multiple large-scale to form new molecular maps to omics layers to identify pathways, interactions and health and disease. feedback loops that may more fully reveal the and, likely, suites of diagnostic markers, therapeutic targets “Omics technologies can help and pathways to and from disease states. us understand and navigate to or > Omics approaches are transitioning from the theoretical fom states of health” level to the level of practical application to beneft vulnerable populations. Conventional hypothesis-driven studies typically focus on a > Human studies need to be greatly expanded few specifc molecules of interest based on prior knowledge: a in number and breadth and rigorously designed to nutritional defciency or excess may set into motion a genomic overcome methodological, analytical and biological aberration or epigenetic change that afects RNA expression, complexities in omics data. synthesis, metabolite production, or certain bacterial growth (Figure 1A). Single omics studies explore sets (individ- ual overlays) of genes, epigenetic marks, transcripts, proteins, or metabolites, or microbial communities, in an unbiased man- ner. They are data-driven and provide opportunities to discov- SIGHT AND | VOL. 31(2) | 2017 OMICS INNOVATIONS AND APPLICATIONS FOR PUBLIC HEALTH NUTRITION 43

figure 1: Simplifed fameworks in molecular nutrition studies

Nutritional defciency or excess

Epigenome |

Proteome State of health

Metabolome

Microbiome

A B C Conventional disease Single (“planar”)-omics Trans-omics pathway search biomarker exploration pathways to health

Each layer represents the genome, epigenome, transcriptome, , , or , and circles or rounded rectangles indicate genes, epigenetic marks, transcripts, proteins, metabolites, or , depending on the layer. Solid and dashed arrows represent associations within and across layers, respectively. Please note that the epigenome and transcriptome, which are separate omics-layers, are combined for the sake of simplicity.

Modifed fgure fom Yugi et al.1 Reproduced with permission obtained fom the publisher.

er unknown factors and biological networks at each molecular Genome level associated with a phenotype (Figure 1B). There has been a growing interest in understanding efects of A limited but increasing number of trans-omics studies genetic variation on child health, enabled by genotyping sin- ofer opportunities to connect, integrate and map a group of gle nucleotide polymorphisms that may predispose biological molecules across multiple omics layers to identif pathways, pathways to misfre. For example, a genome-wide association interactions and feedback loops that may more flly reveal the meta-analysis study reported that the secretor genotype of the biology and, likely, suites of diagnostic markers, therapeutic FUT2 gene, which encodes an enzyme for histo-blood group targets and pathways to and fom disease states (Figure 1C).1 antigen production (a host defense mechanism), portends a Omics technologies are now poised to fll gaps in biological higher risk of diarrheal disease in children.2 The fnding illus- knowledge and provide roadmaps to prevention strategies in trates the potential importance of genetic variants in the FUT2 public health nutrition. gene that may identif children susceptible to enteropathogen- In the “Frontiers in Nutrition” edition of Sight and Life ic organisms. (1/2015), the principles of each omics technology and emerging In contrast to inherited genetic predispositions, studies of opportunity were discussed. The aim of this article is to highlight DNA damage biomarkers have revealed genome integrity can some of the recent progress and innovations in omics, mainly be modifed by nutritional status and diet early in life. Micro- focusing on infant and child nutrition and health (Table 1). In nucleated cells, which indicate missing genetic information and addition, this review will project the translation of omics-based chromosomal damage, have been observed more fequently in discovery into potential public health applications, drawing on malnourished than well-nourished Egyptian children.3 Large plasma as an example. birth size has also been associated with increased cytogenetic 44 OMICS INNOVATIONS AND APPLICATIONS FOR PUBLIC HEALTH NUTRITION

DNA damage biomarkers in the cord blood lymphocytes of oth- numerous immunologic responses and impaired mucosal repair. erwise well-nourished Australian infants.4 length attri- This study provides potential biomarker candidates of gut fnc- tion is considered a genomic marker of chromosomal instability tion and a noninvasive approach that can be used in the fture and fture disease risk. In Latino preschool children, exclusive to elucidate host- dynamics in the gastrointestinal breastfeeding and infant obesity were respectively negative- environment. ly and positively associated with telomere shortening.5 In the same study, soda consumption was associated with shortened Proteome telomere length. Thus, nutritional imbalance – leading to def- A plasma proteomics project using tandem ciency or excess – may be deleterious to genome health early was initiated, motivated by the need for a strategy to assess in life. Among fture challenges are the need to establish causal multiple nutritional defciencies in populations on a single an- relationships between nutritional exposures and DNA damage, alytic platform.10 Assuming that nutrient metabolism is coordi- efcacies of dietary interventions to attenuate or reverse DNA nately associated with a network of measurable proteins, and damage, short- and long-term health efects of early-life genome that such proteins possess sufcient variability to predict nutri- damage, and wider use of genome health assays in nutritional ent status, plasma nutriproteomes have been reported to date epidemiology and population . for vitamins A, D, E and K and selenium, comprising plasma transporters, immune and tissue repair proteins, and numerous “Nutritional imbalance may metabolic homeostatic factors in a population of school-aged Nepalese children.11–15 be deleterious to genome health The utility of plasma proteomics has been frther extend- early in life” ed to quantif clusters of proteins associated with attained stature and arm muscle mass, but not fat mass, refecting a lean, generally undernourished phenotype.16 Proteins asso- Epigenome and Transcriptome ciated with child height have included insulin-like growth Gene expression is regulated by chemical modifcations of his- factor (IGF)-1 and its three binding proteins, likely refecting tones and DNA without changing the underlying DNA sequence. the major role of IGF regulation in linear growth. Nearly 100 Maternal nutritional or other exposures during embryo-fetal plasma proteins originating in the liver and peripheral tissues development can lead to epigenetic changes in fetal DNA that involved in host defense, nutrient metabolism, and tissue re- may infuence disease risk later in life. The hypothesis of diet- pair have been linked to α₁-acid glycoprotein, a generalized epigenome interaction was tested in a rural Gambian population, infammatory biomarker, revealing a dynamic and informa- examining seasonal variations in maternal levels of nutrients in- tive proteome of subclinical infammation.17 Six stable and volved in methyl-donor pathways. The study revealed that DNA constitutive protein biomarkers of infammation have been methylation of infants difered by season of conception at spe- associated with intelligence test scores measured a year af- cifc metastable epialleles, which refect systemic (tissue-wide) ter blood collection, possibly refecting both chronicity and epigenetic development in early life.6 In the same cohort, ges- consequence of low-grade infammation.18 tational afatoxin exposure was associated with diferential DNA methylation in a set of genes, including growth-promoting “Plasma proteins have a capacity and immune-modulating genes, suggesting potential biological mechanisms of compromised host defense and growth impair- to refect short- and long-term 7 ment by afatoxin exposure in utero. Another epigenome-wide biological linkages to nutritional association study conducted in the United Kingdom showed that both maternal pre-pregnancy obesity and underweight were as- and health status” sociated with diferential DNA methylation in the fetus, shown to mediate intergenerational adiposity transmission.8 Among transcriptomics studies, which explore the complete The fndings collectively suggest that plasma proteins have a set of RNA molecules, Yu et al examined the pathogenesis of capacity to refect short- and long-term biological linkages to nu- environmental enteric dysfnction (EED), an asymptomatic tritional and health status. With biological validity of identifed condition of intestinal infammation, malabsorption and barri- biomarkers, widespread population use of proteomics methods er dysfnction, by profling the human intestinal transcriptome will depend on targeted, portable, low-volume, and inexpensive isolated fom fecal samples of Malawian children.9 Transcripts analytic methods (see box, A Proteomics Technological Transi- associated with EED suggest that EED can be characterized by tion: Biomarker discovery to public health application). SIGHT AND LIFE | VOL. 31(2) | 2017 OMICS INNOVATIONS AND APPLICATIONS FOR PUBLIC HEALTH NUTRITION 45

A B D

ceruloplasmin α-2-macroglobulin

hemopexin

vitamin D-binding protein A and B: Children in Sarlahi, Nepal were visited in their homes for assessment of nutritional and health status including pregnancy-zone protein anthropometric measurements C and blood sample collection. C: Blood samples were processed Apo A-1 lipoprotein by trained technicians at a local laboratory in Sarlahi. D: Our initial proteomics work was transthyretin based on two-dimensional diference in-gel electrophoresis analysis of serum samples fom pregnant Nepalese women.

Photo credits: Keith P West Jr. (A, B, and C) and Robert N Cole (D)

Metabolome is now being applied to elucidate the pathogen- “As nutritional metabolomics esis of childhood undernutrition and identif associated meta- bolic signatures using mass spectrometry or nuclear magnetic studies expand, it is likely that resonance spectroscopy. Bartz et al revealed remarkable chang- common metabolites will be es in concentrations of amino acids, fatty acids, acylcarnitines, hormones, growth factors and cytokines in response to dietary identifed as specifc biomarkers 19 regimens among severely malnourished Ugandan children. for diagnostics” In this study, fatty acids played a central role in metabolic re- sponse to acute malnutrition and case fatality was predicted by a low concentration of leptin, refecting the importance of fat Microbiome storage and metabolism for energy supply and survival. Microbiome studies are steadily revealing the importance of gut In Malawi, alterations in the serum metabolome were found microbiota for child growth and health, achieved by combining to be more prominent in children with kwashiorkor than ma- high-throughput gene sequencing technologies with innovative rasmus.20 Nutritional rehabilitation stimulated the recovery of experimental designs, including use of germ-fee animals. Sub- most amino acid profles but sphingomyelins and phosphati- ramanian et al have, for example, created relative microbiota dylcholines were incompletely restored, suggesting metabolic maturation indices fom “age-discriminatory” microbial signa- defcits remained afer clinical stabilization. Alterations in ami- tures fom fecal samples of healthy Bangladesh children.24 The no acids and choline have also been observed in stunted chil- indices have shown that severe acute malnutrition was associ- dren,21,22 possibly refecting common metabolic and nutritional ated with relative microbiota immaturity, which was only in- disturbances in wasting and stunting syndromes. Among amino completely and transiently restored by conventional nutritional acids, tryptophan metabolism has been consistently perturbed therapies. Blanton et al have shown that immature microbiota in studies of malnutrition and EED.20,22,23 transplanted fom undernourished Malawian children resulted As nutritional metabolomics studies expand, it is likely that in reduced weight and lean body mass gain and metabolic ab- common metabolites will be identifed as specifc biomarkers for normalities in mice, and this negative efect was ameliorated by diagnostics, and used for monitoring specifc pathway efects of an invasion of “age and growth-discriminatory” microbiota fom therapeutic and public health interventions. healthy donors.25 46 OMICS INNOVATIONS AND APPLICATIONS FOR PUBLIC HEALTH NUTRITION

figure 2: Conceptual workfow of the proteomics-driven development of nutrition and health assays for populations

DISCOVERY QUALIFICATION ASSAY DEVELOPMENT PUBLIC HEALTH (Replication & Validation) & EVALUATION APPLICATION

BLOOD SAMPLES TARGETED CANDIDATES

Proteins

Plasma A AB B

Peptides B C CD A D

A MASS SPECTROMETRY ANALYSIS B

Full MS Scan Fragment MS Scan CHILD NUTRITION & HEALTH STATUS

Peptide Micronutrients Growth Cognitive development Systemic infammation Vit A Folate Vit B₁₂ Gut health m / z m / z of A and B in blood Concentrations Cardiometabolic health Peptide Mass Peptide Sequence Vit D Vit E Fe Intervention Control

Zn Se I

C A Defciency Sufciency D B Threshold Sensitivity

Gold standard Statistical signi f cance New candidates

Fold changes 1-Specifcity POPULATION-SPECIFIC INTERVENTIONS

Untargeted proteomics Targeted proteomics Multiplexed (semi-quantitative) (quantitative) or ELISA protein assay TECHNOLOGIES

Sialylated oligosaccharides, functioning as prebiotics, Challenges and Opportunities were depleted in the breast milk of Malawian mothers of se- Omics technologies hold promise to reveal intermediate path- verely stunted infants relative to that of mothers of healthy ways, mechanisms, and interactions of growth, development, infants.26 Supplementing purified sialylated bovine milk fnctional health, and disease, and thus to advance knowledge, oligosaccharides to a typical local diet improved lean body change thresholds of action, and transform public health prac- mass gain, bone morphology and metabolism in tissues of tice. Each omics poses technical, logistical, implementation, animal models colonized with microbiota from a stunted in- and cost challenges that impede rapid progress in discoveries fant. Collectively, these studies provide preclinical evidence and applications. Human studies need to be greatly expanded that immature development of gut microbiota is a cause of in number and breadth and rigorously designed to overcome growth impairment and suggest that probiotic or prebiotic methodological, analytical, and biological complexities in omics strategies may be needed to promote microbiota maturity data. Interdisciplinary collaborations and training opportuni- and child growth. ties should be enhanced to promote interactions across felds SIGHT AND LIFE | VOL. 31(2) | 2017 OMICS INNOVATIONS AND APPLICATIONS FOR PUBLIC HEALTH NUTRITION 47

of biomedical science, nutrition, , biostatistics, computational biology and epidemiology. Efective communi- Statistical analysis is performed to select correlated proteins cation across diverse stakeholders including academia, fnders, with low chance of false discovery. frms and governments will ensure common un- derstanding in this fast-changing feld of science and facilitate Validation: The goal of this phase is to establish candidate dissemination and utilization of omics research fndings. proteins, replicated across populations and measured in assays that determine absolute concentrations in plasma

samples (e.g., enzyme-linked immunosorbent assay [ELISA] “ Embracing the complexity of omics or targeted proteomics). Randomized trials or longitudinal will ofer novel strategies to address studies are desirable to establish both correlations and responses to nutritional supplementation. key challenges in global health” A ssay development and evaluation: This stage sets out to produce a protein assay that simultaneously quantifes Conclusions multiple protein biomarkers. For example, the technology of Studies in the omics feld have demonstrated the complexity of antibody-based multiplexed protein measurement can be biological response to suboptimal nutritional status. These ap- used to develop an array that has antibodies attached to cap- proaches are transitioning fom theoretical to practical applica- ture various proteins in each single of a plate.27 Multiplex tion to beneft vulnerable populations. Appreciating healthy skep- ELISA assays with customized combinations of biomarkers ticism around the utility of omics, our vision remains clear that have been already tested for vitamin A, iron, iodine status embracing the complexity will ofer novel strategies to address plus infammation, and malaria markers.28,29 High biological key challenges in global health. Because health promotion and dis- sample and analyte throughput is desirable compared to ease prevention are a long public health journey, omics approach- conventional immunoassays. es have potential to serve as a roadmap that assists travelers in ex- ploring new territories or identifing the most expeditious routes. Application: The feasibility of using a multiplex protein assay as an advanced tool for nutrition research and public health practice is established. Depending on the purpose of A Proteomics Technological Transition: studies, the assay can be utilized to (1) characterize nutritional Biomarker discovery to public health application status of populations (e.g., micronutrients), (2) evaluate efects of nutrition-specifc or nutrition-sensitive interventions on Vision and goal: Micronutrient defciencies are a global nutrition or health status (e.g., growth and gut health), and health problem but remain hidden for lack of assessment tools (3) provide prognostic insights into health and disease burden in low-resource settings. The vision of a plasma proteomics (e.g., cardiometabolic health). The assay will provide invalu- project, supported by the Bill & Melinda Gates Foundation, able information about nutritional and health status of target was that clusters of proteins predictive of micronutrient status, populations that can promote more context-based program and possibly infammation and other functions, could development and policy decisions. be discovered, targeted for quantifcation, and measured, eventually, within a single, inexpensive assay. Acknowledgements Process: The conceptual workfow (Figure 2) illustrates Bill & Melinda Gates Foundation [OPP5241 (Senior Ofcer: Yiwu ongoing eforts to promote discovery11–13,15 and transition He) and GH614 (Senior Ofcer: Ellen Piwoz)], the Sight and Life to quantifcation, a future inexpensive assay, and public Foundation and DSM Ltd and the George G Graham Professorship health application. Endowment Fund. I am most gratefl to Keith P West, Jr and Kerry Schulze for reviewing this article and providing their feedback. Discovery: The aim of discovery is to quantify the direction and strength of association between protein biomarkers Correspondence: Sun Eun Lee PhD, MS and conventional nutritional indicators (e.g., micronutrient, Assistant Scientist, Center for Human Nutrition, anthropometry) and infammation status. Proteins are digested Department of International Health, Johns Hopkins Bloomberg into peptides for detection and relative quantifcation. School of Public Health, 615 N. Wolfe St. W2505, Baltimore, MD 21202, USA Email: [email protected] 48 OMICS INNOVATIONS AND APPLICATIONS FOR PUBLIC HEALTH NUTRITION

table 1: Summary of selective recent omics studies on infant or child health

Study population Biospecimen (Omics) technologies Study design Exposure | outcome

Genome Children in 8 Blood samples High-density single Genome-wide association Reported or developed countries nucleotide polymorphism meta-analysis diagnosed diarrhea (0–18 mo and 12–30 mo) (SNP) arrays Egyptian children Peripheral red blood cells Flow cytometric Case-control Malnutrition (kwashiorkor (2–60 mo) micronuclei assessment vs. marasmus) and bacterial pneumonia Australian infants Peripheral blood Cytokinesis-block Prospective cohort Birth anthropometry (at birth, 3 and 6 mo) (cord blood) lymphocytes micronucleus (weight, length, and head cytome assay circumference) Urban Latino children in Dried blood spots Quantitative polymerase Prospective cohort Obesity, abdominal obesity, the US (fom birth to 5 yr) chain reaction breastfeeding, and dietary intake Epigenome Gambian mother-infant Peripheral blood leukocytes Quantitative bisulfte Prospective cohort Dry (“harvest”) or rainy (“hungry”) (2–8 mo) dyads and hair follicles pyrosequencing season of conception Gambian mother-infant White blood cells Bisulfte pyrosequencing Prospective cohort Afatoxin exposure during (2–8 mo) dyads and Infnium HumanMeth- early pregnancy ylation450 BeadChip British mother-ofspring Cord blood and childhood Infnium Prospective cohort Pre-pregnancy obesity (at birth, 7.5 yr, and and adolescent HumanMethylation450 or underweight 17.1 yr) pairs peripheral blood BeadChip Transcriptome Malawian children Feces High-density microarray Prospective cohort Environmental enteric (12–61 mo) (GeneChip Human dysfnction (EED) Transcriptome Array)

Proteomics Nepalese children Plasma Isobaric tag for Cross-sectional Vitamins A (retinol), (6–8 yr) relative and absolute D (25-hydroxyvitamin D), quantitation (iTRAQ) and E (α-tocopherol), copper, mass spectrometry and selenium Same as above Plasma iTRAQ mass spectrometry Cross-sectional Anthropometric measurements including (1) height-for-age z-score, (2) arm muscle area, and (3) arm fat area and triceps and subscapular skinfolds

Same as above Plasma iTRAQ mass spectrometry Cross-sectional Infammation (α1-acid glycoprotein)

Same as above Plasma iTRAQ mass spectrometry Prospective cohort General intelligence test scores (7–9 yr) SIGHT AND LIFE | VOL. 31(2) | 2017 OMICS INNOVATIONS AND APPLICATIONS FOR PUBLIC HEALTH NUTRITION 49

Biomarkers | main fndings Reference

The SNP rs8111874 at chromosome 19q13.33 reached a genome-wide signifcant association with Bustamante et al² diarrheal disease around 1 year of age. Children with the A allele of the causal variant rs601338 in the FUT2 gene (non-secretor phenotype) had lower risk of diarrhea. Micronucleated-reticulocytes and erythrocytes were more fequently observed in malnourished children Elsayh et al³ with pneumonia than well-nourished children with/without pneumonia. The fequencies of micronucleated cells were higher in children with kwashiorkor than children with marasmus. Birth weight and length, and maternal weight and BMI during pregnancy were positively associated with the Dass Singh et al⁴ fequency of one or more of DNA damage biomarkers including micronuclei, nucleoplasmic bridges and nuclear buds. Head circumference at birth was negatively associated with the fequency of apoptotic cells. Exclusive breastfeeding at 4–6 wk of age was associated with longer telomere length, while early exposure to Wojcicki et al⁵ foods other than breast milk before 4-6 wk of age and infant obesity at 6 mo of age were associated with shorter telomere length at 4 and 5 yr of age. High soda consumption at 3 yr of age was associated with shorter telomere length.

CpG methylation rates at six metastable epialleles in peripheral blood leukocytes and hair follicles were Dominguez-Salas et al⁶ consistently higher in infants conceived in the rainy season compared to those conceived in the dry season. Diferential DNA methylation was observed in 71 CpG sites including growth factor genes such as FGF12 and Hernandez-Vargas et al⁷ IGF1, and immune-related genes such as CCL28, TLR2, and TGBI. One afatoxin-associated methylation region (miR-4520b) was identifed. Twenty-eight and 1621 CpG sites were diferentially methylated in fetuses of pre-pregnancy obese and Sharp et al⁸ underweight mothers, respectively, compared with fetuses of normal weight mothers; diferentially methylated sites associated with maternal over- or undernutrition tended to be associated with ofspring adiposity.

Fify-one transcripts encoding a broad range of immune response, epithelial cell adhesion, and mucin production Yu et al⁹ were associated with and diferentially expressed by EED. Among these, 17 transcripts (AQP9, CLEC7A, FCGR2A, FCGR3B, IFTM1, IFITM2, IFTM3, LYN, LYZ, MNDA, MSN, NCF2, PLEK, PROK2, S100A8, SAMSN1, and SELL) were associated with change in height-for-age z-scores over the 3 months afer stool collection.

Retinol-binding protein 4, vitamin D binding protein, apolipoprotein C-III, ceruloplasmin, and Cole et al¹¹ selenoprotein P were correlated with plasma retinol, 25-hydroxyvitamin D, α-tocopherol, copper, and selenium, respectively (r=0.58~0.88). Adding newly identifed second-tier proteins explained additional variation in nutrient concentrations. (1) (+) IGF-1, IGFALS, IGFBP3, afamin, tetranectin, apolipoprotein L1, carnosinase1, and vasorin; Lee et al¹⁶ (-) S100A12 and IGFBP2; (2) 17 proteins including extracellular matrix proteins were associated with arm muscle area; (3) no proteins were associated with fat mass measurements.

(+) 41 proteins including positive acute phase proteins, complement factors, protease inhibitors, Lee et al¹⁷ and intracellular signaling molecules; (-) 58 proteins involved in transporting or binding to micronutrients, , growth factors, and sex hormones, and homeostasis of extracellular matrix. (+) APOA1, APOA2, APOC1, APOC3, APOM, APOD, IGFALS, IGFBP3, and transthyretin; Lee et al¹⁸ (-) AGP1, C2, C5, C9, CFI, ACT, LRG1, RCN1, TIMELESS, LBP, PKM, DNAAF1, and EVI5; Six proteins (AGP1, C9, CFI, CFHR5, ACT, and RCN1) involved in infammatory response remained negatively associated with general intelligence test scores in flly adjusted models. 50 OMICS INNOVATIONS AND APPLICATIONS FOR PUBLIC HEALTH NUTRITION

Study population Biospecimen (Omics) technologies Study design Exposure | outcome

Metabolome Severely malnourished Serum Tandem mass Clinical nutritional Inpatient and/or outpatient children admitted to hospital spectrometry (targeted), rehabilitation nutritional treatment (milk-based in Uganda (6 mo –5 yr) gas chromatography/mass formulas and/or ready-to-use foods) spectrometry (non- targeted), and conven- tional chemistry assay Severely malnourished Serum Liquid chromatography- Clinical nutritional (1) Kwashiorkor vs. marasmus children admitted to hospital tandem mass spectrometry stabilization; case-control (2) Nutritional stabilization and community controls in (targeted) (3) Severe acute malnutrition vs. Malawi (9–59 mo) community control

Malawian children Serum Liquid chromatography- Cross-sectional Stunting (12–59 mo) tandem mass spectrometry (targeted) Brazilian children Urine ¹H-nuclear magnetic Case-control (1) Undernutrition (stunting, (6–26 mo) resonance spectroscopy wasting, and underweight). (2) Changes in HAZ (2-5 mo) Malawian children Serum Liquid chromatography- Cross-sectional Environmental enteric dysfnction (12–59 mo) tandem mass spectrometry (targeted) Microbiome Bangladesh children with Feces 16S ribosomal RNA Randomized intervention Food interventions (ready-to-use severe or moderate acute gene sequencing trial and post-intervention therapeutic food or a locally produced malnutrition (6–20 mo) follow-up food combination) Healthy or undernourished Feces 16S ribosomal Animal experiment Fecal microbiota fom healthy Malawian children (fecal RNA gene sequencing, (5-week-old or undernourished children donors) (6 and 18 mo) Targeted mass germ-fee mice) spectrometry 6-month postpartum Human milk (fom mothers); Liquid chromatography Animal experiment Purifed bovine milk Malawian mothers fom Feces (fom a 6 mo-old time-of-fight mass (germ-fee mice oligosaccharides supplementation two birth cohorts stunted Malawian infant) spectrometry, and piglets) 16S ribosomal RNA gene sequencing, Microbial RNA-Seq Targeted MS/MS, and GC/MS

(+) and (-) indicate positive and negative associations with exposures/outcomes, respectively

References 02. Bustamante M, Standl M, Bassat Q, Vilor-Tejedor N, Medina-Gomez 01. Yugi K, Kubota H, Hatano A, Kuroda S. Trans-Omics: How To Recon- C, Bonilla C, et al. A genome-wide association meta-analysis of diar- struct Biochemical Networks Across Multiple 'Omic' Layers. Trends rhoeal disease in young children identifes FUT2 locus and provides Biotechnol 2016;34:276–90. plausible biological pathways. Hum Mol Genet 2016;25:4127–42. SIGHT AND LIFE | VOL. 31(2) | 2017 OMICS INNOVATIONS AND APPLICATIONS FOR PUBLIC HEALTH NUTRITION 51

Biomarkers | main fndings Reference

(+) Albumin, amino acids, propionyl carnitine, leptin, IGF-I, insulin, and phosphorous; Bartz et al¹⁹ (-) Fatty acids, ketones, even-chain acylcarnitines, growth hormone, ghrelin, cortisol, IL-6, glucagon-like peptide-1, and peptide YY; A low concentration of baseline leptin was most strongly associated with child mortality.

(1) Albumin, lysine, methionine, threonine, aspartate, tryptophan, kynurenine, acylcarnitines, and Di Giovanni et al²⁰ phosphatidylcholines were more depleted in children with kwashiorkor than in those with marasmus. (2) Albumin, amino acids, biogenic amines, phosphate, potassium, and some lysophosphatidylcholines increased, while most sphingomyelins and phosphatidylcholines were not improved. (3) Metabolic profles of severe acute malnutrition remained diferent fom those of community controls, even afer nutritional treatment. (-) All essential amino acids (tryptophan, isoleucine, leucine, valine, methionine, threonine, histidine, Semba et al²¹ phenylalanine, lysine), conditionally essential amino acids (arginine, glycine, glutamine), non-essential amino acids (asparagine, glutamate, serine), and biogenic amines and sphingomyelins; alterations in glycerophospholipids (1) Alterations in choline and tryptophan metabolisms, reduced energy expenditure, and increased Mayneris-Perxachs et al ²² proteolytic activity of the gut microbiome; (2) urinary N-methylnicotinamide and β-aminoisobutyric acid predicted short-term growth among undernourished children. (-) Tryptophan, citrulline, ornithine, phosphatidylcholines, and sphingomyelins; Semba et al²³ (+) Glutamate, taurine, and serotonin

Children with severe and moderate acute malnutrition had microbiota immaturity relative to healthy children Subramanian et al²⁴ of similar chronologic age; food interventions improved microbiota maturity, but the efects were not sustained afer cessation of treatment in severely malnourished children. Mice colonized with microbiota fom undernourished children gained less weight and lean body mass than mice Blanton et al²⁵ colonized with microbiota fom healthy children, and showed alterations in bone morphology and metabolic abnormalities; invasion of age- and growth-discriminatory strains fom mice with healthy donor microbiota or specifc species (Ruminococcus gnavus and Clostridium symbiosum) into the microbiota of mice with undernourished donors improved growth and metabolic phenotypes in mice. Sialylated human milk oligosaccharides were more abundant in the breast milk of mothers of healthy infants than Charbonneau et al²⁶ that of mothers of stunted infants; adding sialylated bovine milk oligosaccharides to a typical Malawian diet improved lean body mass and nutrient metabolisms in liver, muscle, and brain in mice and piglets colonized with bacterial strains fom a stunted Malawian infant.

03. Elsayh KI, Sayed DM, Zahran AM, Saad K, Badr G. Efects of 04. Dass Singh M, Thomas P, Hor M, Almond T, Owens J, Hague W, et al. pneumonia and malnutrition on the frequency of micronuclei Infant birth outcomes are associated with DNA damage biomarkers in peripheral blood of pediatric patients. Int J Clin Exp Med as measured by the cytokinesis block micronucleus cytome assay: 2013;6:942–50. the DADHI study. Mutagenesis 2017;32:355–70. 52 OMICS INNOVATIONS AND APPLICATIONS FOR PUBLIC HEALTH NUTRITION: AN INTEGRATED VIEW

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