Comparative Blood and Urine Metabolomics Analysis of Healthy Elderly and Young Male Singaporeans.

Comparative metabolomics analysis of biofluids could provide information about the metabolic alterations in aging. To investigate the signature of multiple metabolic profiles associated with aging in an Asian population, we performed a pilot study in healthy Singaporeans, including 33 elderly and 33 young males. Fasting whole bloods were analyzed by routine hematology; the serum and urine metabolome profiles were obtained using NMR-based nontargeted metabolomics analysis and targeted lipoprotein analysis. Among the 90 identified compounds in serum and urine samples, 32 were significantly different between the two groups. The most obvious age-related metabolic signatures include decreased serum levels of albumin lysyl and essential amino acids and derivatives but increased levels of N-acetyl glycoproteins and several lipids and elevated urine levels of trimethylamine N-oxide, scyllo-inositol, citrate, and ascorbic acid but decreased levels of several amino acids, acetate, etc. Among 112 lipoprotein subfractions, 65 were elevated, and 2 were lower in the elderly group. These significantly age-varying metabolites, especially in the amino acid and fatty acid metabolism pathways, suggest that the regulation of these pathways contributes to the aging process in Chinese Singaporeans. Further multiomics studies including the gut microbiome and intervention studies in a larger cohort are needed to elucidate the possible mechanisms in the aging process.

Gut microbiota in neonates with congenital gastrointestinal surgical conditions: a prospective study.

BACKGROUND: There is limited information on gut microbiota of neonates with congenital gastrointestinal surgical conditions (CGISCs) available. METHODS: This study compared stool microbiota and short-chain fatty acids (SCFAs) of 37 term infants with CGISCs with 36 term healthy infants (HIs). Two stool samples were collected from each infant: as soon as possible after birth (week 1) and 10-14 days of life (week 2). RESULTS: Bacterial richness and alpha diversity were comparable between CGISCs and HIs at week 1 and week 2 (all p > 0.05). Beta diversity analysis revealed that at week 1, CGISCs had similar community structures to HIs (p = 0.415). However, by week 2, community structures of CGISCs were significantly different from HIs (p = 0.003). At week 1, there were no significant differences in the relative abundances of genera Bifidobacterium and Bacteroides between CGISCs and HIs. At week 2, the relative abundance of Bifidobacterium was significantly lower in CGISCs (mean percentage 7.21 ± 13.49 vs. 28.96 ± 19.6; p = 0.002). Bacteroides were also less abundant in the CGISC group (mean percentage 0.12 ± 0.49 vs. 6.59 ± 8.62; p = 0.039). Relative abundance of genera Pseudomonas and Escherichia-Shigella were higher in CGISCs. At week 2, stool concentrations of all SCFAs were lower in CGISCs (all p < 0.001). CONCLUSIONS: During hospitalization, neonates with CGISCs develop gut dysbiosis and deficiency of SCFAs. IMPACT: During hospitalisation, neonates with congenital gastrointestinal surgical conditions develop gut dysbiosis with deficiency of Bifidobacteria and Bacteroides and increased abundance of Escherichia-Shigella and Pseudomonas. They also have low levels of short chain fatty acids in their stools compared to healthy infants. This is the first study evaluating the gut microbiota using 16S ribosomal RNA sequencing methods and stool short chain fatty acids in neonates with congenital gastrointestinal surgical conditions and comparing them to healthy infants. The findings of this study will pave the way for randomised trials of bifidobacterial supplementation in neonates with congenital gastrointestinal surgical conditions.

Identification of Arenin, a Novel Kunitz-Like Polypeptide from the Skin Secretions of Dryophytes arenicolor.

Amphibian skin secretions are enriched with complex cocktails of bioactive molecules such as proteins, peptides, biogenic amines, alkaloids guanidine derivatives, steroids and other minor components spanning a wide spectrum of pharmacological actions exploited for centuries in folk medicine. This study presents evidence on the protein profile of the skin secretions of the canyon tree frog, Dryophytes arenicolor. At the same time, it presents the reverse-phase liquid chromatography isolation, mass spectrometry characterization and identification at mRNA level of a novel 58 amino acids Kunitz-like polypeptide from the skin secretions of Dryophytes arenicolor, arenin. Cell viability assays performed on HDFa, CaCo2 and MCF7 cells cultured with different concentrations of arenin showed a discrete effect at low concentrations (2, 4, 8 and 16 µg/mL) suggesting a multi-target interaction in a hormetic-like dose-response. Further work is required to investigate the mechanisms underlying the variable effect on cell viability produced by different concentrations of arenin.

Honeys with anti-inflammatory capacity can alter the elderly gut microbiota in an ex vivo gut model.

The anti-inflammatory effect of different sourced honeys and the impact on elderly gut microbiota were studied in terms of chemical compositions, anti-inflammatory effect and gut microbiota modulating capacities. All four honeys suppressed the production of pro-inflammatory markers NO, IL-1ß and IL-6 induced by lipopolysaccharide and promoted the expression of anti-inflammatory cytokines IL-10 in RAW 264.7 cells. Moreover, in the ex vivo batch gut model using elderly fecal microbiota (referred to as microcosm), it was showed that the addition of honeys increased the abundance of beneficial lactobacilli, decreased the abundance of potentially harmful Gram negative enteric bacteria, and exerted a beneficial effect on the production of short chain fatty acids. The concentration of gallic acid in honeys was positively correlated with the expression level of IL-10 and the abundance of lactobacilli. These findings indicate honeys with anti-inflammatory capacity have great potential for regulating the elderly gut microbiota which would lead to health benefits.

Integrative multiomics analysis reveals host-microbe-metabolite interplays associated with the aging process in Singaporeans.

The age-associated alterations in microbiomes vary across populations due to the influence of genetics and lifestyles. To the best of our knowledge, the microbial changes associated with aging have not yet been investigated in Singapore adults. We conducted shotgun metagenomic sequencing of fecal and saliva samples, as well as fecal metabolomics to characterize the gut and oral microbial communities of 62 healthy adult male Singaporeans, including 32 young subjects (age, 23.1 ± 1.4 years) and 30 elderly subjects (age, 69.0 ± 3.5 years). We identified 8 gut and 13 oral species that were differentially abundant in elderly compared to young subjects. By combining the gut and oral microbiomes, 25 age-associated oral-gut species connections were identified. Moreover, oral bacteria Acidaminococcus intestine and Flavonifractor plautii were less prevalent/abundant in elderly gut samples than in young gut samples, whereas Collinsella aerofaciens and Roseburia hominis showed the opposite trends. These results indicate the varied gut-oral communications with aging. Subsequently, we expanded the association studies on microbiome, metabolome and host phenotypic parameters. In particular, Eubacterium eligens increased in elderly compared to young subjects, and was positively correlated with triglycerides, which implies that the potential role of E. eligens in lipid metabolism is altered during the aging process. Our results demonstrated aging-associated changes in the gut and oral microbiomes, as well as the connections between metabolites and host-microbe interactions, thereby deepening the understanding of alterations in the human microbiome during the aging process in a Singapore population.

Development of a Dairy-Free Fermented Oat-Based Beverage With Enhanced Probiotic and Bioactive Properties.

Lactobacillus fermentum PC1 with proven probiotic properties was used to ferment oats with added honey to develop a probiotic beverage with enhanced bioactive ingredients. The viable Lactobacilli were enumerated during the fermentation and storage at 4°C, as well as after exposure to simulated gastrointestinal tract conditions. Good survival was noted both during storage as well as when exposed to the in vitro digestive tract conditions. Comparative analysis of the antioxidant activity, total phenolic content, and phenolic composition indicated fermentation improved the total antioxidant capacity and phenolic acid concentration. An increase of more than 50% of gallic acid, catechin, vanillic acid, caffeic acid, p-coumaric acid, and ferulic acid was observed in the methanol extracts. Moreover, no significant decrease in the ß-glucan content was noted during fermentation and storage. In conclusion, this fermented product has a great potential as a functional food with enhanced probiotic survival and increased bioactive ingredients.

Lactobacillus fermentum PC1 has the Capacity to Attenuate Joint Inflammation in Collagen-Induced Arthritis in DBA/1 Mice.

Lactobacillus strains have shown efficacy in attenuating inflammation. This study evaluated the potential of Lactobacillus fermentum PC1 for the treatment of rheumatoid arthritis (RA) using a murine model of collagen-induced arthritis. On Day 1, healthy DBA/1 mice (six to eight weeks of age) were immunized, with 100 µg of Chicken Type 11 collagen emulsified in complete Freund's adjuvant (CFA) by intradermal injection, at the base of the tail. On Day 21, the mice were immunized intraperitoneally with 100 µg of Bovine Type11 collagen in phosphate buffered saline (PBS). On Day 28, the mice were immunized intraperitoneally with 50 µg of lipopolysaccharide (LPS). Viable L. fermentum PC1 (1 × 108 colony forming units) was given daily from Day two until the end of the experiment. From Day 21 onwards, the mice were monitored daily for clinical signs of arthritis. On Day 44, the experiment was terminated. Paws were obtained for histology and serum for cytokine assays. L. fermentum PC1-fed mice had significantly reduced paw inflammation as well as decreased synovial infiltration and less cartilage damage. Circulating serum cytokine profiles revealed decreased IL-12 and increased anti-inflammatory cytokines, namely IL-4 and IL-10. Thus, early administration of L. fermentum PC1 could prove to be a valuable therapeutic agent in the management of RA.

Lactobacilli can attenuate inflammation in mouse macrophages exposed to polyethylene particles in vitro.

OBJECTIVE: It is well established that polyethylene (PE) wear particles induce macrophage production of cytokines and mediators associated with the pathogenesis of inflammatory osteolysis. The objective of this study was to examine the potential of three Lactobacillus strains to attenuate the TNF-α cytokine response of macrophages exposed to Ceridust 3615 PE particles. An in vitro experimental model using the RAW 246.7 macrophage cell line and PE particles was utilized. RESULTS: Lactobacillus strains were found to modulate the cytokines in a strain and dose specific manner. Only the Lactobacillus acidophilus strain that was tested was able to attenuate PE particle-induced TNF-α production by RAW 246.7 macrophages. This effect was independent of IL-10 cytokine levels since all three strains of lactobacilli yielded comparable levels of IL-10. It was concluded that some, but not all, Lactobacillus strains may be useful in reducing the risk of inflammatory osteolysis and that further studies in appropriate in vivo models are warranted. Furthermore, this in vitro model can be used to evaluate the inflammatory potential of new materials being tested for use as joint implants.

Characteristics of the adhesion of PCC Lactobacillus fermentum VRI 003 to Peyer's patches.

The characteristics of the adhesion of PCC Lactobacillus fermentum VRI 003 to Peyer's patches was studied in vitro. The adhesion of L. fermentum 003 was strongly inhibited in the presence of d-mannose and methyl-alpha-d-mannoside although other carbohydrates tested, such as N-acetyl-glucosamine, d-galactose, d-glucose and l-fucose, did not affect the adhesion. Lactobacillus fermentum 003 was shown to strongly attach to mannose immobilized on a surface using BSA, suggesting that L. fermentum 003 specifically adhered to mannose-containing molecule(s). Pretreatment of L. fermentum 003 with proteinase K and trypsin decreased the adhesive capacity and bacterial surface extracts diminished adhesion of L. fermentum 003 indicating that cell surface proteins are involved in adhesion to Peyer's patches. It was concluded that a mannose-specific protein mediated adhesion of L. fermentum 003 to the Peyer's patches.

Factors that Influence the Immunological Adjuvant Effect of Lactobacillus fermentum PC1 on Specific Immune Responses in Mice to Orally Administered Antigens.

This study examined the influences of the dosage of the adjuvant, the nature of the antigen and the host genetics on the capacity of L. fermentum PC1 (PC1) to function as an oral adjuvant. BALB/c and DBA/1 mice were vaccinated with either ovalbumin (OVA) or Salmonella Typhimurium on days 0 and 14, Mice were also dosed with the PC1 (108 CFU or 10(11) CFU per dose per mouse) with the antigens (days 0 and 14) and alone (days -1 and 13). The higher PC1 dose elicited a greater specific serum IgG2a response than IgG1 for both antigens and mice strains, indicating a Th1-biased humoral immune response. The Th1 bias was also observed at the cellular level with greater specific IFN-γ levels than IL-4 and IL-10 with both antigen types and mouse strains. With the particulate antigen, the lower dose of PC1 elicited a Th1 bias at the cellular level, but a balanced Th1/Th2 response at the systemic humoral level. With the soluble antigen, a strong Th1-biased response occurred at the cellular level while the systemic humoral response was Th2-biased. In conclusion, PC1 at the higher dose was an excellent Th1 adjuvant, which was unaffected by the nature of the antigen or the host's genetic background.

Adjuvant properties and colonization potential of adhering and non-adhering Lactobacillus spp following oral administration to mice.

This study aimed to determine whether adhesive strains of Lactobacillus possessed an increased ability to colonize the gastrointestinal tract and to examine the adjuvant capacities of these strains for the 50000 molecular-mass fragment C of tetanus toxin (TTFC) following oral administration. The three strains used in this study showed different patterns of adhesion to tissue from all regions of the gastrointestinal tract, with two strains adhering in high numbers and one strain showing negligible association with all tissue types. The colonization patterns in the gastrointestinal tract of C57BL/6 mice following oro-gastric dosing was also monitored, and it was found that adhesive Lactobacillus strains could be detected for at least 24 h, in association with either fecal material and/or with gastrointestinal tissue or contents. In addition, mice were given an oro-gastric dose of the lactobacilli (5 x 10(8) colony forming units) with TTFC (10 and 50 micro g), and the serum-specific IgM and IgG antibody responses monitored in serum. The adhesive strains, which persisted within the gastrointestinal tract for at least 24 h, showed enhanced antigen-specific serum IgG and IgM antibody responses in comparison to a non-adhesive strain that failed to be detected in the gastrointestinal tract. Adhesion to the gastrointestinal tract is a factor affecting the capacity of lactobacilli to persist within the gastrointestinal tract and to act as an adjuvant for orally administered antigen.

Purification and characterization of a surface protein from Lactobacillus fermentum 104R that binds to porcine small intestinal mucus and gastric mucin.

An adhesion-promoting protein involved in the binding of Lactobacillus fermentum strain 104R to small intestinal mucus from piglets and to partially purified gastric mucin was isolated and characterized. Spent culture supernatant fluid and bacterial cell wall extracts were fractionated by ammonium sulfate precipitation and gel filtration. The active fraction was purified by affinity chromatography. The adhesion-promoting protein was detected in the fractions by adhesion inhibition and dot blot assays and visualized by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, and Western blotting with horseradish peroxidase-labeled mucus and mucin. The active fraction was characterized by estimating the relative molecular weight and by assessing the presence of carbohydrates in, and heat sensitivity of, the active region of the adhesion-promoting protein. The purified protein was digested with porcine trypsin, and the peptides were purified in a SMART system. The peptides were tested for adhesion to horseradish peroxidase-labeled mucin by using the dot blot adhesion assay. Peptides which bound mucin were sequenced. It was shown that the purified adhesion-promoting protein on the cell surface of L. fermentum 104R is extractable with 1 M LiCl and low concentrations of lysozyme but not with 0.2 M glycine. The protein could be released to the culture supernatant fluid after 24 h of growth and had affinity for both small intestinal mucus and gastric mucin. In the native state this protein was variable in size, and it had a molecular mass of 29 kDa when denatured. The denatured protein did not contain carbohydrate moieties and was not heat sensitive. Alignment of amino acids of the adhering peptides with sequences deposited in the EMBL data library showed poor homology with previously published sequences. The protein represents an important molecule for development of probiotics.

Gastrointestinal microbial community shifts observed following oral administration of a Lactobacillus fermentum strain to mice.

The indigenous gastrointestinal microbiota acts as an integral defense against the colonisation of orally introduced microbes. Whilst this can be important in host protection, some introduced species, including lactobacilli, can have a positive impact on existing microbial communities. The interaction of a candidate probiotic strain of Lactobacillus fermentum within the gastrointestinal tract was monitored in a mouse model and its effect on the indigenous microbiota observed. L. fermentum KLD was administered via oro-gastric doses to mice with both a specific pathogen-free (SPF) and an ampicillin-depleted gut microbiota, containing no detectable lactobacilli. Its persistence was monitored by detection in faecal homogenates using culturing methods and polymerase chain reaction with L. fermentum specific primers. Microbial population shifts were observed using denaturing gradient gel electrophoresis (DGGE). L. fermentum KLD was detected within the gastrointestinal tract of SPF mice for up to 36 h, and for greater than 11 days in the ampicillin-treated mice. The administration resulted in substantial changes within the host Lactobacillus levels, determined by DGGE of 16S rDNA from faecal samples. Denaturing gradient profiles, from faecal samples collected at a range of pre- and post-dose intervals of groups of 10 SPF mice, indicated that several other constituents of the gastrointestinal community also fluctuated following dosing. These included Bifidobacterium and Eubacterium, which increased following KLD administration. The indigenous microbiota affected the persistence of L. fermentum KLD and in SPF mice the administration of this strain induced significant shifts in the indigenous microbial community.