Blood-brain barrier (BBB) permeability is reportedly increased in patients exhibiting postoperative delirium. Changes in either cerebrospinal fluid (CSF) or blood metabolites have also been reported in cases of delirium. This investigation examined whether BBB permeability influences metabolite concentrations on either side of the barrier, and whether this changes in cases of delirium. This study is a nested case-control cohort (n = 54) drawn from the entire cohort of n = 315 participants. Preoperative CSF and plasma from postoperative delirium and control participants matched for age and gender underwent metabolomic profiling. Concentrations of metabolites in plasma and in CSF were obtained, and where measurable, a CSF/plasma metabolite ratio (Qmetab) was calculated. CSF/plasma albumin ratio (Qalb), a recognized indicator of BBB permeability, was also calculated. The above parameters were compared between delirium and control groups. Finally, each Qmetab was correlated against Qalb for control or delirium cases. In cases where delirium would later occur there were significant elevations in the Qmetabs of amino acids (Gln, His, Met, Phe, Tyr and Val), and one phosphatidylcholine (PC). Many other PC Qmetabs significantly correlated with Qalb and this mainly occurred in individuals subsequently experiencing delirium. In persons prone to postoperative delirium, the equilibrium of certain species of PC across the BBB correlates with BBB permeability. It is unclear whether this occurs because of changes in PC metabolism or transit, but understanding this could improve our understanding of the mechanisms underlying delirium.

Dietary biomarkers may help objectively assessing dietary pattern adherence. This study performed K-means clustering analysis on quantitative food diary data from a dietary intervention study. Standardised dietary data (134 food diaries) from 57 participants were K-means clustered stepwise until fully optimised and cross-validated. The primary endpoint was to develop distinct dietary clusters and to evaluate the performanceof 90 plasma metabolites. The secondary endpoint was to analyse the biomarker-food groups relationships from those distinct dietary patterns. The final two cluster models comprised of 6 specific food types. Cluster 1 included participants with higher intake of fruit and vegetables, legumes, fish and whole grain cereals, and lower intake of meat and sweet foods than Cluster 2. Ten plasma metabolites significantly differed between the clusters (p < 0.05; q < 0.05) with reasonable biomarker performance (receiver operating characteristic (ROC): 0.64-0.72). Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), α-linolenic acid, citric acid and vitamin C were significantly higher in Cluster 1, whereas adrenic acid, osbond acid, cholesterol, dihomo-γ-linolenic acid (DGLA) and triglycerides were higher in Cluster 2. Five additional metabolites also showed significant differences (p < 0.02; q < 0.11) and were included: palmitic acid, tyrosine, β-carotene, α-carotene and betaine. The DHA-to-Osbond acid ratio was an optimal indicator distinguishing healthy from unhealthy dietary patterns (ROC: 0.78). Combining clustering and metabolite profiling methods effectively identifies biomarkers of particular dietary patterns and highlights several robust food-metabolite correlations.

Postoperative delirium (POD) is a common complication in older surgical patients, linked to long-term cognitive decline and progression to dementia, yet its mechanisms remain unclear. We investigated arginine-related metabolites (ARMs) in cerebrospinal fluid (CSF) from 248 patients undergoing elective-surgery: 25 developed POD. Targeted mass spectrometry, gene expression profiling, and machine learning were applied to identify metabolic predictors. POD patients showed significant correlations with citrulline, ornithine, and glutamine, while models highlighted glutamine, glutamic acid, putrescine, N1-acetylspermidine, and spermidine as key biomarkers, achieving >77% predictive accuracy. Cluster and pathway analyses revealed POD-specific shifts in GABA synthesis and polyamine metabolism, contrasting with urea cycle dominance in non-POD cases. Associations persisted after adjusting for age and CSF Aβ42. Preoperative profiles in polyamine metabolism, ammonia detoxification, and neurotransmitter regulation suggest underlying neuroinflammatory and oxidative stress vulnerabilities that reduce resilience. Targeting polyamine biosynthesis may offer novel preventative and therapeutic strategies to mitigate POD and dementia risk.

An arabinan polysaccharide of low molecular weight (SC-S50, 3.2 KDa) and a structurally-related high molecular weight arabinan (SC-P50, 66 KDa) were isolated from Sida cordifolia root. NMR spectroscopy revealed the two polysaccharides were structurally similar consisting of three motifs, the main one being an α-L-arabinofuranose disaccharide (A-(1 → 5)-D) in the linear backbone; the A unit is further substituted at O-2 and O-3 positions by α-L-arabinofuranose. The two minority motifs also consist of α-1 → 5 linked L-arabinofuranose disaccharide, but in contrast, the A unit is replaced by the E or F unit, which is substituted by α-L-arabinofuranose only at the O-2 or O-3 position. SC-S50 did not activate macrophages cells in vitro, but in contrast, SC-P50 potently stimulated nitric oxide production in RAW264.7 macrophages (EC50: 7.92 μg/mL). SC-P50 significantly increased pinocytosis (phagocytosis), cell proliferation and cytokine secretion (IL-2, IL-6, TNF-α, IL-8), with minimal cytotoxicity. SC-P50 was then screened in 12 human primary cell-systems simulating various human tissue and disease characteristics. In 10 of these cell systems SC-P50 increased the production of IL-8. Mechanism-of-action studies unpicked the receptors, intracellular signalling and metabolic responses underlying SC-P50's immunomodulatory actions. SC-P50 is a novel immunomodulatory plant-derived polysaccharide which should be evaluated for its ability to prevent/treat infection.

BackgroundLewy body diseases, including dementia with Lewy bodies (DLB), are characterized by alpha-synuclein accumulation, leading to dementia. Previous studies suggest distinct epigenetic and metabolomic profiles in DLB.ObjectiveThis study aims to identify diagnostic biomarkers by analyzing the methylome and metabolome in the Brodmann area 7 of postmortem brain tissues from DLB patients and control subjects using multiomics approaches.MethodsMethylation analysis was performed using the Illumina EPIC array, and metabolomics profiling was conducted via 1H nuclear magnetic resonance (NMR) and direct injection/liquid chromatography coupled with mass spectrometry. Differential methylation and metabolite analysis were conducted, followed by pathway enrichment to explore biological relevance.ResultsWe identified 3478 significantly differentially methylated cytosines, mostly hypermethylated, enriched in CpG islands near transcription start sites. Pathway enrichment analysis showed significant pathways, primarily linked to olfactory and synaptic functions. Metabolomics profiling identified 15 significantly altered metabolites, with Phosphatidylethanolamine (PE) Biosynthesis being the most affected pathway. Key correlations between differentially methylated cytosines and metabolites, particularly in the PE Biosynthesis pathway involving PTDSS1 and PCYT2 genes, were observed.ConclusionsNotably, sex-specific differences were found, with females exhibiting more epigenetic and metabolomic changes than males. Increased hypermethylation, linked to transcriptional silencing, and disruptions in PE biosynthesis suggest a role in synaptic dysfunction and olfactory deficits. In addition, alpha-aminoadipic acid was strongly associated with vascular functions, hinting at a possible overlap between vascular health and DLB. This study provides new insights into DLB mechanisms and potential therapeutic targets. (c) 2024 International Parkinson and Movement Disorder Society.

The nucleobase queuine (q) and its nucleoside queuosine (Q) are micronutrients derived from bacteria that are acquired from the gut microbiome and/or diet in humans. Following cellular uptake, Q is incorporated at the wobble base (position 34) of tRNAs that decode histidine, tyrosine, aspartate, and asparagine codons, which is important for efficient translation. Early studies suggested that cytosolic uptake of queuine is mediated by a selective transporter that is regulated by mitogenic signals, but the identity of this transporter has remained elusive. Here, through a cross-species bioinformatic search and genetic validation, we have identified the solute carrier family member SLC35F2 as a unique transporter for both queuine and queuosine in Schizosaccharomyces pombe and Trypanosoma brucei. Furthermore, gene disruption in human HeLa cells revealed that SLC35F2 is the sole transporter for queuosine (Km 174 nM) and a high-affinity transporter for the queuine nucleobase (Km 67 nM), with the additional presence of second low-affinity queuine transporter (Km 259 nM). Ectopic expression of labeled SLC35F2 reveals localization to the cell membrane and Golgi apparatus via immunofluorescence. Competition uptake studies show that SLC35F2 is not a general transporter for other canonical ribonucleobases or ribonucleosides but selectively imports q and Q. The identification of SLC35F2, an oncogene, as the transporter of both q and Q advances our understanding of how intracellular levels of queuine and queuosine are regulated and how their deficiency contributes to a variety of pathophysiological conditions, including neurological disorders and cancer.

BackgroundBiofortified Zinc Flour to Eliminate Deficiency in Pakistan (BiZiFED) is a nutritional research program that evaluates the impact of consuming zinc biofortified wheat flour on zinc status and associated health outcomes of vulnerable communities in northwest Pakistan. Measuring zinc status from blood samples is fraught with problems. This feasibility study evaluated whether metabolite changes in tear biofluids could be used to understand zinc status.MethodsZinc deficiency is particularly prevalent amongst the female population in Pakistan. Therefore, a crossover trial was developed in which 25 women of reproductive age received standard, wheat flour, and another 25 received zinc-biofortified wheat flour for 8 weeks. At the end of this period, the nutritional intervention was switched between the groups for another 8 weeks. Tear biofluid was collected using Schirmer strips at baseline and after 8 and 16 weeks. Metabolomic analysis was conducted using the MxP® Quant 500 kit on the tear biofluid from a subset of the study participants.ResultsTwo metabolites had a significantly negative correlation with plasma zinc concentration: tiglylcarnitine and valine. Compared to baseline metabolite concentrations, acetylcarnitine, glutamine, two lysophosphatidylcholines (lysoPC a C16:0 and lysoPC a C18:1), and four sphingomyelins (SM (OH) C16:1, SM C16:0, SM C16:1, and SM C24:0) were all significantly decreased post-zinc intervention, whilst a ceramide (Cer(d18:1/18:0) was significantly increased.ConclusionThese results highlight the potential of using tear biofluids as an alternative source for metabolomic biomarkers, both for the assessment of the zinc status of individuals enrolled in nutritional studies and for indicating physiological changes that arise from nutritional supplementation.

Queuosine (Q) is a hypermodified 7-deaza-guanosine nucleoside exclusively synthesized by bacteria. This micronutrient and its respective nucleobase form queuine (q) are salvaged by humans either from gut microflora or digested food. Depletion of Q-tRNA in human or mouse cells causes protein misfolding that triggers endoplasmic reticular stress and the activation of the unfolded protein responses. In vivo, this reduces the neuronal architecture of the mouse brain affecting learning and memory. Herein, a sensitive method for quantifying free q and Q in human blood was developed, optimised and validated. After evaluating q/Q extraction efficiency in several different solid-phase sorbents, Bond Elut PBA (phenylboronic acid) cartridges were found to have the highest extraction recovery for q (82

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus infect the respiratory tract through surface proteins, causing similar symptoms. Since the onset of the COVID-19 pandemic, both viruses have posed significant ongoing global health threats. Like the influenza virus, SARS-CoV-2 evolves into variants that can reduce vaccine efficacy. Thus, herbal medicines are being explored as supplementary options to enhance protection against these infections. This study aimed to investigate the therapeutic potential of chlorogenic acid (3-CQA) and related extracts from green coffee beans and Echinacea purpurea against SARS-CoV-2 variants and H1N1 infection. The methods employed included an ELISA-based trimeric spike protein binding assay, viral infection assays, plaque assays, and molecular docking studies. Results showed that 3-CQA blocked spike protein/angiotensin converting enzyme 2 (ACE2) binding for most SARS-CoV-2 variants of concern, except the Delta variant. Extracts from Green coffee bean and E. purpurea effectively blocked all variants tested. Additionally, antibodies blocked spike protein binding up to Omicron BA.2. Molecular docking suggested that 3-CQA binding to Omicron BA.1, BA.2, and BA.4, though not to the Delta spike protein, may lead to steric hindrance, preventing receptor-binding domain interactions with ACE2. Finally, both 3-CQA and E. purpurea extract showed preventive effects against H1N1 viral infection, though with lower potency compared to SARS-CoV-2. In conclusion, 3-CQA has potential as a phytoconstituent marker for herbs with bioactive properties against SARS-CoV-2 and H1N1 viral infections.

BackgroundIn multifactorial diseases, alterations in the concentration of metabolites can identify novel pathological mechanisms at the intersection between genetic and environmental influences. This study aimed to profile the plasma metabolome of patients with dementia with Lewy bodies (DLB) and Alzheimer’s disease (AD), two neurodegenerative disorders for which our understanding of the pathophysiology is incomplete. In the clinical setting, DLB is often mistaken for AD, highlighting a need for accurate diagnostic biomarkers. We therefore also aimed to determine the overlapping and differentiating metabolite patterns associated with each and establish whether identification of these patterns could be leveraged as biomarkers to support clinical diagnosis.MethodsA panel of 630 metabolites (Biocrates MxP Quant 500) and a further 232 metabolism indicators (biologically informative sums and ratios calculated from measured metabolites, each indicative for a specific pathway or synthesis; MetaboINDICATOR) were analyzed in plasma from patients with probable DLB (n = 15; age 77.6 ± 8.2 years), probable AD (n = 15; 76.1 ± 6.4 years), and age-matched cognitively healthy controls (HC; n = 15; 75.2 ± 6.9 years). Metabolites were quantified using a reversed-phase ultra-performance liquid chromatography column and triple-quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, or by using flow injection analysis in MRM mode. Data underwent multivariate (PCA analysis), univariate and receiving operator characteristic (ROC) analysis. Metabolite data were also correlated (Spearman r) with the collected clinical neuroimaging and protein biomarker data.ResultsThe PCA plot separated DLB, AD and HC groups (R2 = 0.518, Q2 = 0.348). Significant alterations in 17 detected metabolite parameters were identified (q ≤ 0.05), including neurotransmitters, amino acids and glycerophospholipids. Glutamine (Glu; q = 0.045) concentrations and indicators of sphingomyelin hydroxylation (q = 0.039) distinguished AD and DLB, and these significantly correlated with semi-quantitative measurement of cardiac sympathetic denervation. The most promising biomarker differentiating AD from DLB was Glu:lysophosphatidylcholine (lysoPC a 24:0) ratio (AUC = 0.92; 95%CI 0.809–0.996; sensitivity = 0.90; specificity = 0.90).DiscussionSeveral plasma metabolomic aberrations are shared by both DLB and AD, but a rise in plasma glutamine was specific to DLB. When measured against plasma lysoPC a C24:0, glutamine could differentiate DLB from AD, and the reproducibility of this biomarker should be investigated in larger cohorts.

The impact of environmental factors on epigenetic changes is well established, and cellular function is determined not only by the genome but also by interacting partners such as metabolites. Given the significant impact of metabolism on disease progression, exploring the interaction between the metabolome and epigenome may offer new insights into Huntington's disease (HD) diagnosis and treatment. Using fourteen post-mortem HD cases and fourteen control subjects, we performed metabolomic profiling of human postmortem brain tissue (striatum and frontal lobe), and we performed DNA methylome profiling using the same frontal lobe tissue. Along with finding several perturbed metabolites and differentially methylated loci, Aminoacyl-tRNA biosynthesis (adj p-value = 0.0098) was the most significantly perturbed metabolic pathway with which two CpGs of the SEPSECS gene were correlated. This study improves our understanding of molecular biomarker connections and, importantly, increases our knowledge of metabolic alterations driving HD progression.

>Biological models of Alzheimer's disease(AD):Non-human models have contributed tremendously to the understanding of AD and its underlying pathological processes.These models have aided the investigation of the genetic and environmental risk factors.They also have enabled

Eukaryotic life benefits from-and ofttimes critically relies upon-the de novo biosynthesis and supply of vitamins and micronutrients from bacteria. The micronutrient queuosine (Q), derived from diet and/or the gut microbiome, is used as a source of the nucleobase queuine, which once incorporated into the anticodon of tRNA contributes to translational efficiency and accuracy. Here, we report high-resolution, substrate-bound crystal structures of the Sphaerobacter thermophilus queuine salvage protein Qng1 (formerly DUF2419) and of its human ortholog QNG1 (C9orf64), which together with biochemical and genetic evidence demonstrate its function as the hydrolase releasing queuine from queuosine-5'-monophosphate as the biological substrate. We also show that QNG1 is highly expressed in the liver, with implications for Q salvage and recycling. The essential role of this family of hydrolases in supplying queuine in eukaryotes places it at the nexus of numerous (patho)physiological processes associated with queuine deficiency, including altered metabolism, proliferation, differentiation and cancer progression.

• Introduction: Consumption of a Mediterranean diet (MD) has established health benefits, and the identification of novel biomarkers could enable objective monitoring of dietary pattern adherence. • Objectives: The present investigation performed untargeted metabolomics on blood plasma from a controlled study of MD adherence, to identify novel blood-based metabolite biomarkers associated with the MD pattern, and to build a logistic regression model that could be used to characterise MD adherence. • Methods: A hundred and thirty-five plasma samples from n = 58 patients collected at different time points were available. Using a 14-point scale MD Score (MDS) subjects were divided into ‘high’ or ‘low’ MDS adherence groups and liquid chromatography-mass spectrometry (LC-MS/MS) was applied for analysis. • Results: The strongest association with MDS was pectenotoxin 2 seco acid (r = 0.53; ROC = 0.78), a non-toxic marine xenobiotic metabolite. Several lipids were useful biomarkers including eicosapentaenoic acid, the structurally related lysophospholipid (20:5(5Z,8Z,11Z,14Z,17Z)/0:0), a phosphatidylcholine (P-18:1(9Z)/16:0) and also xi-8-hydroxyhexadecanedioic acid. Two metabolites negatively correlated with MDS, these were the monoacylglycerides (0:0/16:1(9Z)/0:0) and (0:0/20:3(5Z,8Z,11Z)/0:0). By stepwise elimination we selected a panel of 3 highly discriminatory metabolites and developed a linear regression model which identified ‘high MDS’ individuals with high sensitivity and specificity (AUC (95% CI) = 0.83 (0.76–0.97)). • Conclusion: Our study highlights the utility of metabolomics as an approach for developing novel panels of dietary biomarkers. Quantitative profiling of these metabolites is required to validate their utility for evaluating dietary adherence.

Dementia with Lewy bodies (DLB) is a common form of dementia with known genetic and environmental interactions. However, the underlying epigenetic mechanisms which reflect these gene-environment interactions are poorly studied. Herein, we measure genome-wide DNA methylation profiles of post-mortem brain tissue (Broadmann area 7) from 15 pathologically confirmed DLB brains and compare them with 16 cognitively normal controls using Illumina MethylationEPIC arrays. We identify 17 significantly differentially methylated CpGs (DMCs) and 17 differentially methylated regions (DMRs) between the groups. The DMCs are mainly located at the CpG islands, promoter and first exon regions. Genes associated with the DMCs are linked to “Parkinson’s disease” and “metabolic pathway”, as well as the diseases of “severe intellectual disability” and “mood disorders”. Overall, our study highlights previously unreported DMCs offering insights into DLB pathogenesis with the possibility that some of these could be used as biomarkers of DLB in the future.

Inhibition of DPP-4 and stimulation of GLP-1 secretion are therapeutic strategies for controlling glycaemia in type 2 diabetes. The present study assessed the DPP-4 inhibitory activity and GLP-1 secretory action of pigmented and non-pigmented rice (Oryza sativa L.), along with an extruded food product. Cereal-based extruded food products, with or without passion fruit powder, were prepared from red rice using a twin extruder. Optimal extrusion conditions were determined using a CCD of response surface methodology resulting in optimal conditions to be 97.5 °C, a screw speed of 250 rpm, feed moisture of 25.2% and addition of 11.25% passion fruit powder. Samples were sequentially extracted in n-hexane, ethanol (50%) and water. Ethanol/water (50:50) extracts of rice bran significantly inhibited DPP-4 activity by 70.48 ± 1.06%, comparing favourably with RR (42.55 ± 0.84%), PRR (35.91 ± 1.27%) and PA (29.14 ± 1.23%). DPP-4 inhibitory activity was retained in both extruded products albeit at reduced levels. GLP-1 secretion was stimulated mostly by extruded products extracted with n-hexane or ethanol which upregulated basal secretion by 6.1-fold and 4.2-fold, respectively. ICP-MS results showed that extruded food items have a lower arsenic content. In conclusion, there are potential opportunities for the nutraceuticals and functional food products using pigmented red rice.

Abstract Background The overuse of antibiotics has led to increased antimicrobial resistance, but plant-derived biological response modifiers represent a potential alternative to these drugs. This investigation examined the immunomodulatory and antibacterial activities of Sida cordifolia (used in ethnomedicinal systems to treat infectious disease). Methods Successive extractions were performed from the roots of these plants in hexane, chloroform, methanol and water. Immunomodulatory activity was determined in a series of experiments measuring the responses of splenocytes, macrophages and an in vivo model of innate immunity (Galleria mellonella). Antibacterial activity was assessed by determining minimum inhibitory/bactericidal concentrations (MIC/MBCs) for various Gram-positive and Gram-negative bacterial strains. Results Immunomodulatory activity was confined to the aqueous extract, and further fractionation and biochemical analysis yielded a highly potent polysaccharide-enriched fraction (SCAF5). SCAF5 is a complex mixture of different polysaccharides with multiple immunomodulatory effects including immune cell proliferation, antibody secretion, phagocytosis, nitric oxide production, and increased expression of pro-inflammatory cytokines. Furthermore, Galleria mellonella pre-treated with SCAF5 produced more haemocytes and were more resistant (P < 0.001) to infection with methicillin-resistant Staphylococcus aureus (MRSA) with a 98% reduction in bacterial load in pre-treated larvae compared to the negative control. The antibacterial activity of Sida cordifolia was confined to the methanolic fraction. Extensive fractionation identified two compounds, rosmarinic acid and its 4-O-β-d-glucoside derivative, which had potent activity against Gram-positive antibiotic-resistant bacteria, including MRSA. Conclusions Sida cordifolia counters bacterial infections through a dual mechanism, and immunomodulatory polysaccharides from this plant should be isolated and characterised to realise their potential as anti-infective agents. Such properties could be developed as an antibiotic alternative (1) in the clinic and (2) alternative growth promoter for the agri-food industry.

Sprouting is a traditional processing method which has been used for centuries to improve the nutritional value of cereals and legumes. There has been growing interest in sprouted products in recent years due to a high demand for more natural and healthy foods. Phytate is the primary storage form of phosphorus in plants. It is long recognised to affect human health as it forms insoluble complexes with minerals such as iron and zinc in cereals and legumes, thereby preventing their absorption in the body. Sprouting activates the enzyme phytase, which degrades phytate, thereby improving mineral bioaccessibility and bioavailability. The extent of phytate reduction varies depending on the sprouting conditions, cereal/legume species, cultivar and native phytase activity. Sprouting has been associated with increased iron, zinc and calcium bioaccessibility in many studies, but this appears to differ in cereals and legumes, which possibly is due to the presence of other 'antinutrients'. Protein digestibility also appears to be positively correlated with phytate reduction albeit less than for minerals. It is not possible to accurately predict the influence of sprouting on nutrient bioavailability because so few studies have been conducted. Further research is required to determine whether the commercial production of sprouted cereals and legumes can increase the nutritional value and health benefits of commercial end products.

Characterizing the gut microbiota of free-range and alternative poultry production systems provides information, which can be used to improve poultry welfare, performance, and environmental sustainability. Gut microbiota influence not only the health and metabolism of the host but also the presence of zoonotic agents contaminating food of animal origin. In this study, the composition and diversity of the cecal microbiota community of free-range grown chickens were characterized by 16S rDNA high-throughput Illumina sequencing. Significant differences were observed in the composition of chicken cecal microbiota at the time points of 28 days of age (Indoor group) and 56 days of age (Outdoor group), i.e., before and after the outdoor access period of chicken groups. The Outdoor group showed a richer and more complex microbial community, characterized by the onset of new phyla such as Deferribacterota and Synergistota, while the Indoor group showed an increase in Campylobacterota. At the species level, it is noteworthy that the occurrence of Mucispirillum schaedleri in Outdoor group is known to potentially stimulate mucus layer formation in the distal intestinal tract, thus being associated with a healthy gut. We also report a significant decrease in the Outdoor group of Helicobacter pullorum, highlighting that the lower abundance at the age of slaughter reduced the possibility to contaminate chickens' carcasses and, consequently, its zoonotic potential. As revealed by a mutual exclusion study in network analysis, H. pullorum was present only if Bacteroides barnesiae, an uncultured organism of the genus Synergistes, and Bacteroides gallinaceum were absent. Finally, microbiome predictive analysis revealed an increase of vitamins and micronutrient biosyntheses such as queuosine (Q) and its precursor pre Q0, in the Outdoor group, suggesting that the outdoor evolved microbiota of chickens do contribute to the vitamin pool of the gut and the biosynthesis of micronutrients involved in vital cell processes.