Circadian orchestration of host and gut microbiota in infection.

Circadian rhythms are present in almost every organism and regulate multiple aspects of biological and physiological processes (e.g. metabolism, immune responses, and microbial exposure). There exists a bidirectional circadian interaction between the host and its gut microbiota, and potential circadian orchestration of both host and gut microbiota in response to invading pathogens. In this review, we summarize what is known about these intestinal microbial oscillations and the relationships between host circadian clocks and various infectious agents (bacteria, fungi, parasites, and viruses), and discuss how host circadian clocks prime the immune system to fight pathogen infections as well as the direct effects of circadian clocks on viral activity (e.g. SARS-CoV-2 entry and replication). Finally, we consider strategies employed to realign normal circadian rhythmicity for host health, such as chronotherapy, dietary intervention, good sleep hygiene, and gut microbiota-targeted therapy. We propose that targeting circadian rhythmicity may provide therapeutic opportunities for the treatment of infectious diseases.

Aspartate Metabolism Facilitates IL-1ß Production in Inflammatory Macrophages.

Increasing evidence support that cellular amino acid metabolism shapes the fate of immune cells; however, whether aspartate metabolism dictates macrophage function is still enigmatic. Here, we found that the metabolites in aspartate metabolism are depleted in lipopolysaccharide (LPS) plus interferon gamma (IFN-γ)-stimulated macrophages. Aspartate promotes interleukin-1ß (IL-1ß) secretion in M1 macrophages. Mechanistically, aspartate boosts the activation of hypoxia-inducible factor-1α (HIF-1α) and inflammasome and increases the levels of metabolites in aspartate metabolism, such as asparagine. Interestingly, asparagine also accelerates the activation of cellular signaling pathways and promotes the production of inflammatory cytokines from macrophages. Moreover, aspartate supplementation augments the macrophage-mediated inflammatory responses in mice and piglets. These results uncover a previously uncharacterized role for aspartate metabolism in directing M1 macrophage polarization.

Effects of dietary gamma-aminobutyric acid supplementation on amino acid profile, intestinal immunity, and microbiota in ETEC-challenged piglets.

Enterotoxigenic Escherichia coli (ETEC) infection is the most common cause of diarrhea in piglets, and ETEC could increase intestinal gamma-aminobutyric acid (GABA)-producing bacteria to affect intestinal immunity. However, the effect of GABA on ETEC-infected piglets is still unclear. This study aims at investigating the impact of dietary GABA supplementation on the growth performance, diarrhea, intestinal morphology, serum amino acid profile, intestinal immunity, and microbiota  in the ETEC-infected piglet model. Eighteen piglets were randomly divided into two groups, in which the piglets were fed with a basal diet with 20 mg kg-1 GABA supplementation or not. The experiment lasted for three weeks, and the piglets were challenged with ETEC K88 on the fifteenth day. The results showed that dietary GABA reduced the feed conversion ratio, promoted the kidney organ index but did not affect the diarrheal score and small intestinal morphology in ETEC-challenged piglets. Ileal mucosal amino acids (such as carnosine and anserine) and serum amino acids (including threonine and GABA) were increased upon GABA supplementation. GABA enhanced ileal gene expression of TNF-α, IFN-γ, pIgR, and MUC2, while inhibited the ileal expression of IL-18 in ETEC-challenged piglets. GABA supplementation also highly regulated the intestinal microbiota by promoting community richness and diversity and reducing the abundance of the dominant microbial population of the ileal microbiota. Collectively, GABA improves growth performance, regulates the serum amino acid profile, intestinal immunity, and gut microbiota in ETEC-challenged piglets. This study is a fine attempt to reveal the function of GABA in ETEC-infected piglets. It would contribute to the understanding of the roles of exogenous nutrition on the host response to ETEC infection.

Melatonin overcomes MCR-mediated colistin resistance in Gram-negative pathogens.

Background: Emergence, prevalence and widely spread of plasmid-mediated colistin resistance in Enterobacteriaceae strongly impairs the clinical efficacy of colistin against life-threatening bacterial infections. Combinations of antibiotics and FDA-approved non-antibiotic agents represent a promising means to address the widespread emergence of antibiotic-resistant pathogens. Methods: Herein, we investigated the synergistic activity between melatonin and antibiotics against MCR (mobilized colistin resistance)-positive Gram-negative pathogens through checkerboard assay and time-killing curve. Molecular mechanisms underlying its mode of action were elucidated. Finally, we assessed the in vivo efficacy of melatonin in combination with colistin against drug-resistant Gram-negative bacteria. Results: Melatonin, which has been approved for treating sleep disturbances and circadian disorders, substantially potentiates the activity of three antibiotics, particularly colistin, against MCR-expressing pathogens without enhancing its toxicity. This is evidence that the combination of colistin with melatonin enhances bacterial outer membrane permeability, promotes oxidative damage and inhibits the effect of efflux pumps. In three animal models infected by mcr-1-carrying E. coli, melatonin dramatically rescues colistin efficacy. Conclusion: Our findings revealed that melatonin serves as a promising colistin adjuvant against MCR-positive Gram-negative pathogens.

The relationship between urinary kidney injury molecule-1 and blood bone metabolism markers in patients with chronic kidney disease
.

OBJECTIVE: The aim of this study was to evaluate the changes of urinary kidney injury molecule-1(uKIM-1) in chronic kidney disease (CKD) at different stages, and to determine the relationships between uKIM-1 and circulating bone metabolism markers. MATERIALS AND METHODS: This cross-sectional study included CKD patients (n = 121) and controls (n = 65). CKD stages were assigned to each individual according to their estimated glomerular filtration rate (eGFR), which was calculated with the modification of diet in renal disease (MDRD) equation. We evaluated the relationships of bone metabolism markers (including calcium, phosphorus, intact parathyroid hormone (iPTH), 25 hydroxy vitamin D (25(OH)D), alkaline phosphatase (ALP), fibroblast growth factor 23 (FGF23), and α-Klotho), uKIM-1, and eGFR. We also compared the levels of bone metabolism markers and uKIM-1 at different CKD stages. The uKIM-1 level was standardized with urine creatinine (uCr). RESULTS: Compared with healthy controls, CKD patients had higher levels of uKIM-1/uCr, serum creatinine, urea, phosphorus, iPTH, and plasma FGF23, whereas they had lower levels of serum calcium, α-Klotho, and plasma 25(OH)D. In CKD patients, eGFR was positively correlated with levels of serum calcium, α-Klotho, and plasma 25(OH)D, whereas it was negatively correlated with serum phosphorus, iPTH, plasma FGF23, and uKIM-1/uCr. Serum calcium and α-Klotho were significantly decreased in patients with stage 5 CKD compared to those with stage 1 CKD. Serum phosphorus, iPTH, and plasma FGF23 were significantly elevated in patients with stage 4 CKD when compared to those with stage 1 CKD. UKIM-1/uCr was significantly elevated in patients with stage 5 CKD when compared to those with stage 1 CKD. In CKD patients, uKIM-1/uCr levels were positively correlated with levels of serum phosphorus and plasma FGF23, whereas they were negatively correlated with serum calcium and plasma 25(OH)D. CONCLUSION: UKIM-1/uCr levels are increased with the deterioration of CKD stage and are correlated with the development of CKD-mineral and bone disorder (CKD-MBD).

Effects of dietary gamma-aminobutyric acid supplementation on the intestinal functions in weaning piglets.

This study aims to investigate the effects of dietary gamma-aminobutyric acid (GABA) supplementation on the growth performance, intestinal immunity, intestinal GABAergic system, amino acid profiles and gut microflora of the weaned piglets. Totally sixteen healthy piglets were randomly assigned into two groups to be fed with the basal diet (Con group) or the basal diet with GABA (20 mg kg-1) supplementation. Body weights and feed intakes were monitored weekly. Piglets were sacrificed after 3 weeks of GABA supplementation to collect the blood, ileum, ileal mucosa and luminal content. Immune-associated factors, GABAergic system, amino acid profiles, and microbiota in the ileum and serum amino acid profiles were explored. The results showed that GABA supplementation improved the growth performance and modulated the intestinal immunity with inhibiting the gene expressions of IL-22, proinflammatory cytokines (IL-1 and IL-18), and Muc1, but promoted the expressions of anti-inflammatory cytokines (IFN-γ, IL-4, and IL-10), TLR6 and MyD88. GABA regulated a few components of the intestinal GABAergic system, increased the levels of most amino acids in the ileal mucosa but reduced the serum amino acid profiles. GABA regulated the population and diversity of intestinal microbiota, such as the abundances of the dominant microbial populations, the community richness, and diversity of the ileal microbiota. In conclusion, GABA supplementation modulated the intestinal functions, including intestinal immunity, intestinal amino acid profiles and gut microbiota, and the results can be helpful for understanding the functions of GABA in the intestine.

Melatonin reprogramming of gut microbiota improves lipid dysmetabolism in high-fat diet-fed mice.

Melatonin has been shown to improve lipid metabolism and gut microbiota communities in animals and humans; however, it remains to know whether melatonin prevents obesity through gut microbiota. Here, we found that high-fat diet promoted the lipid accumulation and intestinal microbiota dysbiosis in mice, while oral melatonin supplementation alleviated the lipid accumulation and reversed gut microbiota dysbiosis, including the diversity of intestinal microbiota, relative abundances of Bacteroides and Alistipes, and functional profiling of microbial communities, such as energy metabolism, lipid metabolism, and carbohydrate metabolism. Interestingly, melatonin failed to alleviate the high-fat-induced lipid accumulation in antibiotic-treated mice; however, microbiota transplantation from melatonin-treated mice alleviated high-fat diet-induced lipid metabolic disorders. Notably, short-chain fatty acids were decreased in high-fat diet-fed mice, while melatonin treatment improved the production of acetic acid. Correlation analysis found a marked correlation between production of acetic acid and relative abundances of Bacteroides and Alistipes. Importantly, sodium acetate treatment also alleviated high-fat diet-induced lipid metabolic disorders. Taken together, our results suggest that melatonin improves lipid metabolism in high-fat diet-fed mice, and the potential mechanisms may be associated with reprogramming gut microbiota, especially, Bacteroides and Alistipes-mediated acetic acid production. Future studies are needed for patients with metabolic syndrome to fully understand melatonin's effects on body weight and lipid profiles and the potential mechanism of gut microbiota.

Glutamine supplementation improves intestinal cell proliferation and stem cell differentiation in weanling mice.

BACKGROUND: Intestinal stem cells can be differentiated into absorptive enterocytes and secretory cells, including Paneth cells, goblet cells, and enteroendocrine cells. Glutamine is a primary metabolic fuel of small intestinal enterocytes and is essential for the viability and growth of intestinal cells. OBJECTIVE: Whether glutamine supplementation affects the differentiation of intestinal stem cells is unknown. DESIGN: Three-week-old ICR (Institute of Cancer Research) male mice were divided randomly into two groups: 1) mice receiving a basal diet and normal drinking water and 2) mice receiving a basal diet and drinking water supplemented with glutamine. After 2 weeks, the mice were sacrificed to collect the ileum for analysis. RESULTS: The study found that glutamine supplementation in weanling mice decreases the crypt depth in the ileum, leading to higher ratio of villus to crypt in the ileum, but promotes cell proliferation of intestinal cells and mRNA expression of Lgr5 (leucine-rich repeat-containing g-protein coupled receptor5) in the ileum. Glutamine has no effect on the number of Paneth cells and goblet cells, and the expression of markers for absorptive enterocytes, Paneth cells, goblet cells, and enteroendocrine cells. CONCLUSION: These findings reveal the beneficial effects of dietary glutamine supplementation to improve intestinal morphology in weanling mammals.

Metabolomic Profiles Reveal Potential Factors that Correlate with Lactation Performance in Sow Milk.

Sow milk contains necessary nutrients for piglets; however, the relationship between the levels of metabolites in sow milk and lactation performance has not been thoroughly elucidated to date. In this study, we analysed the metabolites in sow milk from Yorkshire sows with high lactation (HL) or low lactation (LL) performance; these categories were assigned based on the weight gain of piglets during the entire lactation period (D1 to D21). The concentration of milk fat in the colostrum tended to be higher in the HL group (P = 0.05), the level of mannitol was significantly lower in the HL group (P < 0.05) and the level of glucuronic acid lactone was significantly higher in the HL group (P < 0.05) compared to those in LL group. In mature milk, the levels of lactose, creatine, glutamine, glutamate, 4-hydroxyproline, alanine, asparagine, and glycine were significantly higher (P < 0.05) in the HL group than those in LL group. The level of fatty acids showed no significant difference between the two groups in both the colostrum and mature milk. This study suggested that lactation performance may be associated with the levels of lactose and several amino acids in sow milk, and these results can be used to develop new feed additives to improve lactation performance in sows.

Hyperhomocysteinemia and cardiovascular disease in animal model.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and is associated with primary causes of mortality and morbidity throughout the world. Several studies have been carried out to evaluate the effects of a diet inducing cystathionine-ß-synthase, methyltetrafolate, folic acid, and vitamin B supplemented with methionine on the homocysteine metabolism and in lowering the plasma total homocysteine levels. A large number of molecular and biomedical studies in numerous animals, such as mice, rabbits, and pigs, have sought to elevate the plasma total homocysteine levels and to identify a disease model for human hyperhomocysteinemia. However, a specific animal model is not suitable for hyperhomocysteinemia in terms of all aspects of cardiovascular disease. In this review article, the experimental progress of animal models with plasma total homocysteine levels is examined to identify a feasible animal model of hyperhomocysteinemia for different aspects.

Melatonin alleviates weanling stress in mice: Involvement of intestinal microbiota.

Melatonin influences intestinal microbiota and the pathogenesis of various diseases. This study was conducted to explore whether melatonin alleviates weanling stress through intestinal microbiota in a weanling mouse model. Melatonin supplementation in weanling mice (provided in the drinking water at a dosage of 0.2 mg/mL for 2 weeks) significantly improved body weight gain (1.4 ± 0.03 g/day in melatonin group vs 1.2 ± 0.06 g/day in control group) and intestinal morphology (ie, villus length, crypt depth, and villus to crypt ratio), but had little effect on the proliferation or apoptosis of intestinal cells, the numbers of Paneth cells and goblet cells, as well as the expression of makers related to enterocytes (sucrase) and endocrine cells (chromogranin A and peptide YY) in the ileum. Melatonin supplementation had little effect on serum levels of amino acids or stress-related parameters (eg, SOD, TNF-α, and angiotensin I). 16S rRNA sequencing suggested that melatonin supplementation increased the richness indices of intestinal microbiota (observed species, Chao 1, and ACE) and shaped the composition of intestinal microbiota (eg, increase in the abundance of Lactobacillus [19 ± 3% in melatonin group vs 6 ± 2% in control group]), which was demonstrated using an ex vivo proliferation assay and colonic loop proliferation assay. Melatonin supplementation also significantly influenced the metabolism of intestinal microbiota, such as amino acid metabolism and drug metabolism. More importantly, in antibiotic-treated weanling mice and germ-free weanling mice, melatonin failed to affect body weight gain or intestinal morphology. Melatonin significantly reduced (by about 60%) the bacterial load in enterotoxigenic Escherichia coli (ETEC)-infected weanling mice, but had little effect on ETEC load in antibiotic-pretreated animals. In conclusion, melatonin affects body weight gain, intestinal morphology, and intestinal ETEC infection through intestinal microbiota in weanling mice. The findings highlight the importance of intestinal microbiota in mediating the various physiological functions of melatonin in the host.

Lysine Restriction Affects Feed Intake and Amino Acid Metabolism via Gut Microbiome in Piglets.

BACKGROUND/AIMS: Our previous reports suggested that dietary supplementation with lysine influenced intestinal absorption and metabolism of amino acids. In this study, we further investigated the effect of lysine restriction (30%) on feed intake and we also tested the hypothesis that gut microbiome contributed to the potential mechanism of lysine restriction-mediated feeding behavior. Here, we profiled gut microbial communities by sequencing 16S ribosomal ribonucleic acid (rRNA) genes from gut samples as well as growth performance, serum hormones, and intestinal lysine transport in a piglet model. RESULTS: Piglets preferred to the lysine restricted diet when giving three diets and the feed intake was markedly higher in the lysine-restricted group than that in the control group. Altered hormones (leptin, CCK, and ghrelin) might contribute to the feeding behavior caused by lysine restriction. Meanwhile, lysine transporting ability (SLC7A1 and SLC7A2 expression, intestinal electrophysiological changes, and amino acid pool in mesenteric vein) was decreased in response to lysine restriction. Through deep sequencing of bacterial rRNA markers, we observed that bacterial diversity was enhanced in the lysine-restricted group (Shannon H, PD, and Chao1). At the phylum level, lysine restriction enhanced gut Actinobacteria, Saccharibacteria, and Synergistetes abundances. At the family level, Moraxellaceae, Halomonadaceae, Shewanellaceae, Corynebacteriaceae, Bacillaceae, Comamonadaceae, Microbacteriaceae, Caulobacteraceae, and Synergistaceae abundances were increased in response to lysine restriction. Predictive functional profiling of microbial communities by PICRUSt also confirmed that dietary lysine restriction affected gut microbiome, which might further mediate amino acid metabolism, membrane transport, and endocrine system. CONCLUSION: Our results indicated that lysine restriction inhibited intestinal lysine transport and promoted feed intake, which might be associated with gut microbiome.

Melatonin alters amino acid metabolism and inflammatory responses in colitis mice.

Inflammatory bowel disease is a chronic inflammatory dysfunction of the gastrointestinal tract. This study explored the hypothesis that melatonin has beneficial functions in the mouse model of colitis induced by dextran sodium sulfate (DSS), with a specific focus on the expression of intestinal inflammatory cytokines and the serum levels of amino acids. The results revealed that mice with melatonin supplementation had a reduction in weight loss and disease index induced by DSS treatment. Melatonin stifled the expression of colonic IL-17 in mice with DSS-induced colitis. Melatonin also lowered the serum levels of Asp, Ser, Met, and Leu (p < 0.05), but increased those of Glu and Cys (p < 0.05). Thus, melatonin treatment is promising and may function as a potential adjuvant therapy to alleviate the clinical symptoms of patients with inflammatory bowel disease.

Methionine restriction on oxidative stress and immune response in dss-induced colitis mice.

A strong correlation exists between inflammatory bowel disease (IBD) and oxidative stress involving alterations of several key signaling pathways. It is known that methionine promotes reactive oxygen species (ROS) production; we therefore hypothesize that a methionine restriction diet would reduce ROS production, inflammatory responses, and the course of IBD. We generated a murine colitis model by dextran sodium sulfate (DSS) treatment and tested the effects of the methionine restriction diet. Forty-eight mice were randomly divided into four groups of equal size, which included a control (CON) group, an MR (methionine restriction diet) group, a DSS treated group and an MR-DSS treated group. Mice in the first two groups had unrestricted access to water for one week. Mice in the two DSS-treated groups had unrestricted access to 5% DSS solution supplied in the drinking water for the same period. Mice in the CON and DSS groups were given a basal diet, whereas mice in the MR-DSS and MR groups were fed a 0.14% MR diet. We found that DSS reduced daily weight gain, suppressed antioxidant enzyme expression, increased histopathology scores and activated NF-κB and nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap1) signaling. We also showed that the MR diet upregulated catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities, decreased myeloperoxidase (MPO), TNF-α and IL-1ß, and reversed activation of the NF-κB signaling pathway in MR-DSS mice. Taken together, our results imply that the MR diet may be considered as an adjuvant in IBD therapeutics.

Alpha-ketoglutarate (AKG) lowers body weight and affects intestinal innate immunity through influencing intestinal microbiota.

Alpha-ketoglutarate (AKG), a precursor of glutamate and a critical intermediate in the tricarboxylic acid cycle, shows beneficial effects on intestinal function. However, the influence of AKG on the intestinal innate immune system and intestinal microbiota is unknown. This study explores the effect of oral AKG administration in drinking water (10 g/L) on intestinal innate immunity and intestinal microbiota in a mouse model. Mouse water intake, feed intake and body weight were recorded throughout the entire experiment. The ileum was collected for detecting the expression of intestinal proinflammatory cytokines and innate immune factors by Real-time Polymerase Chain Reaction. Additionally, the ileal luminal contents and feces were collected for 16S rDNA sequencing to analyze the microbial composition. The intestinal microbiota in mice was disrupted with an antibiotic cocktail. The results revealed that AKG supplementation lowered body weight, promoted ileal expression of mammalian defensins of the alpha subfamily (such as cryptdins-1, cryptdins-4, and cryptdins-5) while influencing the intestinal microbial composition (i.e., lowering the Firmicutes to Bacteroidetes ratio). In the antibiotic-treated mouse model, AKG supplementation failed to affect mouse body weight and inhibited the expression of cryptdins-1 and cryptdins-5 in the ileum. We concluded that AKG might affect body weight and intestinal innate immunity through influencing intestinal microbiota.

Dietary Saccharomyces cerevisiae Cell Wall Extract Supplementation Alleviates Oxidative Stress and Modulates Serum Amino Acids Profiles in Weaned Piglets.

This research aims to evaluate the effects of dietary supplementation with Saccharomyces cerevisiae cell wall extract (SCCWE) on growth performance, oxidative stress, intestinal morphology, and serum amino acid concentration in weaned piglets. Utilizing a completely randomized design, 40 healthy piglets weaned at 21 d were grouped into 4 experimental treatments with 10 pigs per treatment group. Treatments consisted of a basal diet (T0), a basal diet with a 0.05% SCCWE (T1), a basal diet with a 0.10% SCCWE (T2), and a basal diet with a 0.15% SCCWE (T3). SCCWE supplementation increased the average daily gain and final body weight compared with T0 (P < 0.05). SCCWE in T2 and T3 improved the average daily feed intake and decreased the feed/gain ratio compared with T1 and T2 (P < 0.05). SCCWE decreased serum malondialdehyde (MDA) and increased activities of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) significantly compared to T0 (P < 0.05). SCCWE increased the concentration of Ile compared to T0 (P < 0.05). Moreover, the concentrations of Leu, Phe, and Arg were higher in T2 and T3 (P < 0.05). These findings indicate beneficial effects of SCCWE supplementation on growth performance, the concentration of some essential amino acids, and alleviation of oxidative stress in weaned piglets.

L-Glutamine and L-arginine protect against enterotoxigenic Escherichia coli infection via intestinal innate immunity in mice.

Dietary glutamine (Gln) or arginine (Arg) supplementation is beneficial for intestinal health; however, whether Gln or Arg may confer protection against Enterotoxigenic Escherichia coli (ETEC) infection is not known. To address this, we used an ETEC-infected murine model to investigate the protective effects of Gln and Arg. Experimentally, we pre-treated mice with designed diet of Gln or Arg supplementation prior to the oral ETEC infection and then assessed mouse mortality and intestinal bacterial burden. We also determined the markers of intestinal innate immunity in treated mice, including secretory IgA response (SIgA), mucins from goblet cells, as well as antimicrobial peptides from Paneth cells. ETEC colonized in mouse small intestine, including duodenum, jejunum, and ileum, and inhibited the mRNA expression of intestinal immune factors, such as polymeric immunoglobulin receptor (pIgR), cryptdin-related sequence 1C (CRS1C), and Reg3γ. We found that dietary Gln or Arg supplementation decreased bacterial colonization and promoted the activation of innate immunity (e.g., the mRNA expression of pIgR, CRS1C, and Reg3γ) in the intestine of ETEC-infected mice. Our results suggest that dietary arginine or glutamine supplementation may inhibit intestinal ETEC infection through intestinal innate immunity.

DNA Methylation and the Potential Role of Methyl-Containing Nutrients in Cardiovascular Diseases.

Patients suffering from cardiovascular diseases (CVDs) experience a low quality of life and increase pressure on healthcare systems both nationally and globally. DNA methylation, which refers to the pathway by which DNA methyltransferase facilitates the addition of a methyl group to DNA, is of critical importance in this respect primarily because the epigenetic modification is implicated in a range of serious conditions including atherosclerosis, CVDs, and cancer. Research findings indicate that the number of epigenetic alterations can be elicited (both in utero and in adults) through the administration of certain nutritional supplements, including folic acid and methionine; this is partly attributable to the effect employed by methyl-containing nutrients in DNA methylation. Thus, for the purpose of illuminating viable therapeutic measures and preventive strategies for CVDs, research should continue to explore the intricate associations that exist between epigenetic regulation and CVD pathogenesis. This review centers on an exposition of the mechanism by which DNA methylation takes place, the impact it has on a range of conditions, and the potential clinical value of nutrition, driven mainly by the observation that nutritional supplements such as folic acid can affect DNA methylation.