Cyclotides derived from Viola dalatensis Gagnep: A novel approach for enrichment and evaluation of antimicrobial activity.

Cyclotides, plant-derived cysteine-rich peptides, exhibit a wide range of beneficial biological activities and possess exceptional structural stability. Cyclotides are commonly distributed throughout the Violaceae family. Viola dalatensis Gagnep, a Vietnamese species, has not been well studied, especially for cyclotides. This pioneering research explores cyclotides from V. dalatensis as antimicrobials. This study used a novel approach to enhance cyclotides after extraction. The approach combined 30% ammonium sulfate salt precipitation and RP-HPLC. A comprehensive analysis was performed to ascertain the overall protein content, flavonoids content, polyphenol content, and free radical scavenging capacity of compounds derived from V. dalatensis. Six known cyclotides were sequenced utilizing MS tandem. Semi-purified cyclotide mixtures (M1, M2, and M3) exhibited antibacterial efficacy against Bacillus subtilis (inhibitory diameters: 19.67-23.50 mm), Pseudomonas aeruginosa (22.17-23.50 mm), and Aspergillus flavus (14.67-21.33 mm). The enriched cyclotide precipitate from the stem extract demonstrated a minimum inhibitory concentration (MIC) of 0.08 mg/mL against P. aeruginosa, showcasing significant antibacterial effectiveness compared to the stem extract (MIC: 12.50 mg/mL). Considerable advancements have been achieved in the realm of cyclotides, specifically in their application as antimicrobial agents.

A pilot study of dietary fibres on pathogen growth in an ex vivo colonic model reveals their potential ability to limit vancomycin-resistant Enterococcus expansion.

Aim: Dietary fibre is important for shaping gut microbiota. The aim of this pilot study was to investigate the impact of dietary fibres on pathogen performance in the presence of gut microbiota. Methods: In an ex vivo gut model, pooled faecal samples were spiked with a cocktail of representative gastrointestinal pathogens and fermented with yeast ß-glucan for 24 hours, after which 16S rRNA amplicon sequencing and short-chain and branched-chain fatty acid (SCFA and BCFA) analyses were performed. In addition, oat ß-glucan, arabinoxylan, yeast ß-glucan, and galactooligosaccharides were each tested against individual representative pathogens and pathogen growth was assessed via qPCR. Glucose served as a control carbon source. Results: Based on 16S rRNA amplicon sequencing, yeast ß-glucan selected for higher proportions of Bacteroides (P = 0.0005, ~6 fold) and Clostridia (P = 0.005, ~3.6 fold) while species of Escherichia/Shigella (P = 0.021, ~2.8 fold) and Lactobacillus (P = 0.007, ~ 15.7-fold) were higher in glucose. Pathogen relative abundance did not differ between glucose and yeast ß-glucan. In the absence of pathogens, higher production of BCFAs (P = 0.002) and SCFAs (P = 0.002) fatty acids was observed for fibre group(s). For individual pathogens, yeast ß-glucan increased growth of Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes (P < 0.05), arabinoxylan increased S. typhimurium (P < 0.05). Tested fibres decreased vancomycin-resistant Enterococcus faecium (P < 0.05), with yeast ß-glucan causing a 1-log reduction (P < 0.01), while galactooligosaccharides decreased L. monocytogenes (P < 0.05). Conclusion: Tested fibres differentially influenced the growth of pathogens, but yeast ß-glucan could represent a dietary strategy to help limit vancomycin-resistant enterococci (VRE) expansion in the gut.

Yeast ß-Glucan Improves Insulin Sensitivity and Hepatic Lipid Metabolism in Mice Humanized with Obese Type 2 Diabetic Gut Microbiota.

SCOPE: Gut microbiota alterations are associated with obesity and type 2 diabetes. Yeast ß-glucans are potential modulators of the innate immune-metabolic response, by impacting glucose, lipid, and cholesterol homeostasis. The study examines whether yeast ß-glucan interacts differentially with either an obese healthy or obese diabetic gut microbiome, to impact metabolic health through hepatic effects under high-fat dietary challenge. METHODS AND RESULTS: Male C57BL/6J mice are pre-inoculated with gut microbiota from obese healthy (OBH) or obese type 2 diabetic (OBD) subjects, in conjunction with a high-fat diet (HFD) with/without yeast ß-glucan. OBD microbiome colonization adversely impacts metabolic health compared to OBH microbiome engraftment. OBD mice are more insulin resistant and display hepatic lipotoxicity compared to weight matched OBH mice. Yeast ß-glucan supplementation resolves this adverse metabolic phenotype, coincident with increasing the abundance of health-related bacterial taxa. Hepatic proteomics demonstrates that OBD microbiome transplantation increases HFD-induced hepatic mitochondrial dysfunction, disrupts oxidative phosphorylation, and reduces protein synthesis, which are partly reverted by yeast ß-glucan supplementation. CONCLUSIONS: Hepatic metabolism is adversely affected by OBD microbiome colonization with high-fat feeding, but partially resolved by yeast ß-glucan. More targeted dietary interventions that encompass the interactions between diet, gut microbiota, and host metabolism may have greater treatment efficacy.

Loss of MMR and TGFBR2 Increases the Susceptibility to Microbiota-Dependent Inflammation-Associated Colon Cancer.

BACKGROUND AND AIMS: Mutations in DNA mismatch repair (MMR) genes are causative in Lynch syndrome and a significant proportion of sporadic colorectal cancers (CRCs). MMR-deficient (dMMR) CRCs display increased mutation rates, with mutations frequently accumulating at short repetitive DNA sequences throughout the genome (microsatellite instability). The TGFBR2 gene is one of the most frequently mutated genes in dMMR CRCs. Therefore, we generated an animal model to study how the loss of both TGFBR2 signaling impacts dMMR-driven intestinal tumorigenesis in vivo and explore the impact of the gut microbiota. METHODS: We generated VCMsh2/Tgfbr2 mice in which Msh2loxP and Tgfbr2loxP alleles are inactivated by Villin-Cre recombinase in the intestinal epithelium. VCMsh2/Tgfbr2 mice were analyzed for their rate of intestinal cancer development and for the mutational spectra and gene expression profiles of tumors. In addition, we assessed the impact of chemically induced chronic inflammation and gut microbiota composition on colorectal tumorigenesis. RESULTS: VCMsh2/Tgfbr2 mice developed small intestinal adenocarcinomas and CRCs with histopathological features highly similar to CRCs in Lynch syndrome patients. The CRCs in VCMsh2/Tgfbr2 mice were associated with the presence of colitis and displayed genetic and histological features that resembled inflammation-associated CRCs in human patients. The development of CRCs in VCMsh2/Tgfbr2 mice was strongly modulated by the gut microbiota composition, which in turn was impacted by the TGFBR2 status of the tumors. CONCLUSIONS: Our results demonstrate a synergistic interaction between MMR and TGFBR2 inactivation in inflammation-associated colon tumorigenesis and highlight the crucial impact of the gut microbiota on modulating the incidence of inflammation-associated CRCs.

A randomized controlled trial of a pharmacist-led intervention to enhance knowledge of Vietnamese patients with type 2 diabetes mellitus.

OBJECTIVES: We aimed to assess whether a pharmacist-led intervention enhances knowledge, medication adherence and glycemic control in patients with type 2 diabetes mellitus (T2DM). METHODS: We conducted a single-blinded randomized controlled trial in Vietnam. Individuals with T2DM were recruited from a general hospital and randomly allocated to intervention and routine care. The intervention group received routine care plus counselling intervention by a pharmacist, including providing drug information and answering individual patients' queries relating to T2DM and medications, which had not been done in routine care. We assessed the outcomes: knowledge score as measured by the Diabetes Knowledge Questionnaire, self-reported adherence and fasting blood glucose (FBG) at the 1-month follow-up. KEY FINDINGS: A total of 165 patients (83 intervention, 82 control) completed the study; their mean age was 63.33 years, and 49.1% were males. The baseline characteristics of the patients were similar between the groups. At 1-month follow-up, the pharmacist's intervention resulted in an improvement in all three outcomes: knowledge score [B = 5.527; 95% confidence intervals (CI): 3.982 to 7.072; P < 0.001], adherence [odds ratio (OR) = 9.813; 95% CI: 2.456 to 39.205; P = 0.001] and attainment of target FBG (OR = 1.979; 95% CI: 1.029 to 3.806; P = 0.041). CONCLUSIONS: The pharmacist-led intervention enhanced disease knowledge, medication adherence and glycemic control in patients with T2DM. This study provides evidence of the benefits of pharmacist counselling in addition to routine care for T2DM outpatients in a Vietnam population.

Microbiome Transfer Partly Overrides Lack of IL-1RI Signaling to Alter Hepatic but not Adipose Tissue Phenotype and Lipid Handling following a High-Fat Diet Challenge.

SCOPE: IL-1RI-mediated inflammatory signaling alters metabolic tissue responses to dietary challenges (e.g., high-fat diet [HFD]). Recent work suggests that metabolic phenotype is transferrable between mice in a shared living environment (i.e., co-housing) due to gut microbiome exchange. The authors examine whether the metabolic phenotype of IL-1RI-/- mice fed HFD or low-fat diet (LFD) could be transferred to wild-type (WT) mice through gut microbiome exchange facilitated by co-housing. METHODS AND RESULTS: Male WT (C57BL/J6) and IL-1RI-/- mice are fed HFD (45% kcal) or LFD (10% kcal) for 24 weeks and housed i) by genotype (single-housed) or ii) with members of the other genotype in a shared microbial environment (co-housed). The IL-1RI-/-  gut microbiome is dominant to WT, meaning that co-housed WT mice adopted the IL-1RI-/- microbiota profile. This is concomitant with greater body weight, hepatic lipid accumulation, adipocyte hypertrophy, and hyperinsulinemia in co-housed WT mice, compared to single-housed counterparts. These effects are most evident following HFD. Primary features of microbiome differences are Lachnospiraceae and Ruminococcaceae (known producers of SCFA). CONCLUSION: Transfer of SCFA-producing microbiota from IL-1RI-/- mice highlights a new connection between diet, inflammatory signaling, and the gut microbiome, an association that is dependent on the nature of the dietary fat challenge.

Whole Blueberry and Isolated Polyphenol-Rich Fractions Modulate Specific Gut Microbes in an In Vitro Colon Model and in a Pilot Study in Human Consumers.

Blueberry (BB) consumption is linked to improved health. The bioconversion of the polyphenolic content of BB by fermentative bacteria in the large intestine may be a necessary step for the health benefits attributed to BB consumption. The identification of specific gut microbiota taxa that respond to BB consumption and that mediate the bioconversion of consumed polyphenolic compounds into bioactive forms is required to improve our understanding of how polyphenols impact human health. We tested the ability of polyphenol-rich fractions purified from whole BB-namely, anthocyanins/flavonol glycosides (ANTH/FLAV), proanthocyanidins (PACs), the sugar/acid fraction (S/A), and total polyphenols (TPP)-to modulate the fecal microbiota composition of healthy adults in an in vitro colon system. In a parallel pilot study, we tested the effect of consuming 38 g of freeze-dried BB powder per day for 6 weeks on the fecal microbiota of 17 women in two age groups (i.e., young and older). The BB ingredients had a distinct effect on the fecal microbiota composition in the artificial colon model. The ANTH/FLAV and PAC fractions were more effective in promoting microbiome alpha diversity compared to S/A and TPP, and these effects were attributed to differentially responsive taxa. Dietary enrichment with BB resulted in a moderate increase in the diversity of the microbiota of the older subjects but not in younger subjects, and certain health-relevant taxa were significantly associated with BB consumption. Alterations in the abundance of some gut bacteria correlated not only with BB consumption but also with increased antioxidant activity in blood. Collectively, these pilot data support the notion that BB consumption is associated with gut microbiota changes and health benefits.

Exploratory analysis of covariation of microbiota-derived vitamin K and cognition in older adults.

BACKGROUND: Vitamin K has multiple important physiological roles, including blood coagulation and beneficial effects on myelin integrity in the brain. Some intestinal microbes possess the genes to produce vitamin K in the form of menaquinone (MK). MK appears in higher concentration in tissues, such as the brain, particularly MK4, than the dietary form of phylloquinone (PK). Lower PK concentrations have been reported in patients with Alzheimer disease while higher serum PK concentrations have been positively associated with verbal episodic memory. Despite knowledge of the importance of vitamin K for various health parameters, few studies have measured MK concentration and biosynthesis by gut commensals. OBJECTIVE: The aim of the current study was to investigate the relation between genes involved in gut-microbiota derived MK, concentrations of MK isoforms, and cognitive function. METHODS: Shotgun metagenomic sequencing of the gut microbiome of 74 elderly individuals with different cognitive ability levels was performed. From this, gene counts for microbial MK biosynthesis were determined. Associations between clusters of individuals, grouped based on a similar presence and prevalence of MK biosynthesis genes, and cognitive ability were investigated. Fecal MK concentrations were quantified by HPLC to investigate correlations with subject clusters. RESULTS: Separation of subject groups defined by banded quantification of the genetic potential of their microbiome to biosynthesize MK was associated with significant differences in cognitive ability [assessed using the Mini-Mental State Examination (MMSE)]. Three MK isoforms were found to be positively associated with MMSE, along with the identification of key components of the MK pathway that drive this association. Although the causality and direction of these associations remain unknown, these findings justify further studies. CONCLUSIONS: This study provides evidence that although total concentrations of MK did not covary with cognition, certain MK isoforms synthesized by the gut microbiome, particularly the longer chains, are positively associated with cognition.

APOE genotype influences the gut microbiome structure and function in humans and mice: relevance for Alzheimer's disease pathophysiology.

Apolipoprotein E (APOE) genotype is the strongest prevalent genetic risk factor for Alzheimer's disease (AD). Numerous studies have provided insights into the pathologic mechanisms. However, a comprehensive understanding of the impact of APOE genotype on microflora speciation and metabolism is completely lacking. In this study, we investigated the association between APOE genotype and the gut microbiome composition in human and APOE-targeted replacement (TR) transgenic mice. Fecal microbiota amplicon sequencing from matched individuals with different APOE genotypes revealed no significant differences in overall microbiota diversity in group-aggregated human APOE genotypes. However, several bacterial taxa showed significantly different relative abundance between APOE genotypes. Notably, we detected an association of Prevotellaceae and Ruminococcaceae and several butyrate-producing genera abundances with APOE genotypes. These findings were confirmed by comparing the gut microbiota of APOE-TR mice. Furthermore, metabolomic analysis of murine fecal water detected significant differences in microbe-associated amino acids and short-chain fatty acids between APOE genotypes. Together, these findings indicate that APOE genotype is associated with specific gut microbiome profiles in both humans and APOE-TR mice. This suggests that the gut microbiome is worth further investigation as a potential target to mitigate the deleterious impact of the APOE4 allele on cognitive decline and the prevention of AD.-Tran, T. T. T., Corsini, S., Kellingray, L., Hegarty, C., Le Gall, G., Narbad, A., Müller, M., Tejera, N., O'Toole, P. W., Minihane, A.-M., Vauzour, D. APOE genotype influences the gut microbiome structure and function in humans and mice: relevance for Alzheimer's disease pathophysiology.

Prebiotic supplementation in frail older people affects specific gut microbiota taxa but not global diversity.

BACKGROUND: There are complex interactions between aging, frailty, diet, and the gut microbiota; modulation of the gut microbiota by diet could lead to healthier aging. The purpose of this study was to test the effect of diets differing in sugar, fat, and fiber content upon the gut microbiota of mice humanized with microbiota from healthy or frail older people. We also performed a 6-month dietary fiber supplementation in three human cohorts representing three distinct life-stages. METHODS: Mice were colonized with human microbiota and then underwent an 8-week dietary intervention with either a high-fiber/low-fat diet typical of elderly community dwellers or a low-fiber/high-fat diet typical of long-stay residential care subjects. A cross-over design was used where the diets were switched after 4 weeks to the other diet type to identify responsive taxa and innate immunity changes. In the human intervention, the subjects supplemented their normal diet with a mix of five prebiotics (wheat dextrin, resistant starch, polydextrose, soluble corn fiber, and galactooligo-saccharide) at 10 g/day combined total, for healthy subjects and 20 g/day for frail subjects, or placebo (10 g/day maltodextrin) for 26 weeks. The gut microbiota was profiled and immune responses were assayed by T cell markers in mice, and serum cytokines in humans. RESULTS: Humanized mice maintained gut microbiota types reflecting the respective healthy or frail human donor. Changes in abundance of specific taxa occurred with the diet switch. In mice with the community type microbiota, the observed differences reflected compositions previously associated with higher frailty. The dominance of Prevotella present initially in community inoculated mice was replaced by Bacteroides, Alistipes, and Oscillibacter. Frail type microbiota showed a differential effect on innate immune markers in both conventional and germ-free mice, but a moderate number of taxonomic changes occurring upon diet switch with an increase in abundance of Parabacteroides, Blautia, Clostridium cluster IV, and Phascolarctobacterium. In the human intervention, prebiotic supplementation did not drive any global changes in alpha- or beta-diversity, but the abundance of certain bacterial taxa, particularly Ruminococcaceae (Clostridium cluster IV), Parabacteroides, Phascolarctobacterium, increased, and levels of the chemokine CXCL11 were significantly lower in the frail elderly group, but increased during the wash-out period. CONCLUSIONS: Switching to a nutritionally poorer diet has a profound effect on the microbiota in mouse models, with changes in the gut microbiota from healthy donors reflecting previously observed differences between elderly frail and non-frail individuals. However, the frailty-associated gut microbiota did not reciprocally switch to a younger healthy-subject like state, and supplementation with prebiotics was associated with fewer detected effects in humans than diet adjustment in animal models.

Biochemical Function, Molecular Structure and Evolution of an Atypical Thioredoxin Reductase from Desulfovibrio vulgaris.

Thioredoxin reductase (TR) regulates the intracellular redox environment by reducing thioredoxin (Trx). In anaerobes, recent findings indicate that the Trx redox network is implicated in the global redox regulation of metabolism but also actively participates in protecting cells against O2. In the anaerobe Desulfovibrio vulgaris Hildenborough (DvH), there is an intriguing redundancy of the Trx system which includes a classical system using NADPH as electron source, a non-canonical system using NADH and an isolated TR (DvTRi). The functionality of DvTRi was questioned due to its lack of reactivity with DvTrxs. Structural analysis shows that DvTRi is a NAD(P)H-independent TR but its reducer needs still to be identified. Moreover, DvTRi reduced by an artificial electron source is able to reduce in turn DvTrx1 and complexation experiments demonstrate a direct interaction between DvTRi and DvTrx1. The deletion mutant tri exhibits a higher sensitivity to disulfide stress and the gene tri is upregulated by O2 exposure. Having DvTRi in addition to DvTR1 as electron source for reducing DvTrx1 must be an asset to combat oxidative stress. Large-scale phylogenomics analyses show that TRi homologs are confined within the anaerobes. All TRi proteins displayed a conserved TQ/NGK motif instead of the HRRD motif, which is selective for the binding of the 2'-phosphate group of NADPH. The evolutionary history of TRs indicates that tr1 is the common gene ancestor in prokaryotes, affected by both gene duplications and horizontal gene events, therefore leading to the appearance of TRi through subfunctionalization over the evolutionary time.

Comparative Genome Analysis Provides Insights into Both the Lifestyle of Acidithiobacillus ferrivorans Strain CF27 and the Chimeric Nature of the Iron-Oxidizing Acidithiobacilli Genomes.

The iron-oxidizing species Acidithiobacillus ferrivorans is one of few acidophiles able to oxidize ferrous iron and reduced inorganic sulfur compounds at low temperatures (<10°C). To complete the genome of At. ferrivorans strain CF27, new sequences were generated, and an update assembly and functional annotation were undertaken, followed by a comparative analysis with other Acidithiobacillus species whose genomes are publically available. The At. ferrivorans CF27 genome comprises a 3,409,655 bp chromosome and a 46,453 bp plasmid. At. ferrivorans CF27 possesses genes allowing its adaptation to cold, metal(loid)-rich environments, as well as others that enable it to sense environmental changes, allowing At. ferrivorans CF27 to escape hostile conditions and to move toward favorable locations. Interestingly, the genome of At. ferrivorans CF27 exhibits a large number of genomic islands (mostly containing genes of unknown function), suggesting that a large number of genes has been acquired by horizontal gene transfer over time. Furthermore, several genes specific to At. ferrivorans CF27 have been identified that could be responsible for the phenotypic differences of this strain compared to other Acidithiobacillus species. Most genes located inside At. ferrivorans CF27-specific gene clusters which have been analyzed were expressed by both ferrous iron-grown and sulfur-attached cells, indicating that they are not pseudogenes and may play a role in both situations. Analysis of the taxonomic composition of genomes of the Acidithiobacillia infers that they are chimeric in nature, supporting the premise that they belong to a particular taxonomic class, distinct to other proteobacterial subgroups.

A Comprehensive tRNA Genomic Survey Unravels the Evolutionary History of tRNA Arrays in Prokaryotes.

Considering the importance of tRNAs in the translation machinery, scant attention has been paid to tRNA array units defined as genomic regions containing at least 20 tRNA genes with a minimal tRNA gene density of two tRNA genes per kilobase. Our analysis of Acidithiobacillus ferrivorans CF27 and Acidithiobacillus ferrooxidans ATCC 23270(T) genomes showed that both display a tRNA array unit with syntenic conservation which mainly contributed to the tRNA gene redundancy in these two organisms. Our investigations into the occurrence and distribution of tRNA array units revealed that 1) this tRNA organization is limited to few phyla and mainly found in Gram-positive bacteria; and 2) the presence of tRNA arrays favors the redundancy of tRNA genes, in particular those encoding the core tRNA isoacceptors. Finally, comparative array organization revealed that tRNA arrays were acquired through horizontal gene transfer (from Firmicutes or unknown donor), before being subjected to tRNA rearrangements, deletions, and duplications. In Bacilli, the most parsimonious evolutionary history involved two common ancestors and the acquisition of their arrays arose late in evolution, in the genera branches. Functional roles of the array units in organism lifestyle, selective genetic advantage and translation efficiency, as well as the evolutionary advantages of organisms harboring them were proposed. Our study offers new insight into the structural organization and evolution of tRNA arrays in prokaryotic organisms.