Lifestyle-Driven Variations in Nutrimiromic MicroRNA Expression Patterns across and beyond Genders.

The importance of diet and lifestyle in maintaining overall health has long been recognised. MicroRNAs (miRNAs) have emerged as key players in the intricate interplay between health and disease. This study, including 305 participants, examined the role of miRNAs from capillary blood as indicators of individual physiological characteristics, diet, and lifestyle influences. Key findings include specific miRNAs associated with inflammatory processes and dietary patterns. Notably, miR-155 was associated with subjects with metabolic diseases and upregulated in age. Additionally, the study revealed diet-related miRNA expressions: high consumption of vegetables, fruits, and whole grains correlated with increased levels of miR-let-7a and miR-328, both implicated in anti-inflammatory pathways, and decreased expression of pro-inflammatory miR-21. In the context of smoking, we found a significant decrease in miRNA-142, known for its downregulation in lung cancer. We observed a sex-biased expression of various miRNAs with significant upregulation of miR-151a in females and a higher expression of miR-155 in ageing females, representing a possible mechanism for the increased susceptibility to autoimmune diseases. In conclusion, the study underscores the significant influence of lifestyle, nutrition, and sex on miRNA profiles. Circulating miRNAs demonstrate significant potential as biomarkers in personalized medicine, highlighting their utility in tailoring healthcare to individual needs.

MiR-10a, miR-15a, let-7a, and let-7g expression as stress-relevant biomarkers to assess acute or chronic psychological stress and mental health in human capillary blood.

BACKGROUND: Psychological stress, as an important cofactor in the development of many acute and chronic diseases, is crucial for general health or well-being, and improved markers are needed to distinguish situations of progressive pathological development, such as depression, anxiety, or burnout, to be recognized at an early stage. Epigenetic biomarkers play an important role in the early detection and treatment of complex diseases such as cancer, and metabolic or mental disorders. Therefore, this study aimed to identify so-called miRNAs, which would be suitable as stress-related biomarkers. METHODS AND RESULTS: In this study, 173 participants (36.4% males, and 63.6% females) were interviewed about stress, stress-related diseases, lifestyle, and diet to assess their acute and chronic psychological stress status. Using qPCR analysis, 13 different miRNAs (miR-10a-5p, miR-15a-5p, miR-16-5p, miR-19b-3p, miR-26b-5p, miR-29c-3p, miR-106b-5p, miR-126-3p, miR-142-3p, let-7a-5p, let-7g-5p, miR-21-5p, and miR-877-5p) were analyzed in dried capillary blood samples. Four miRNAs were identified, miR-10a-5p, miR-15a-5p, let-7a-5p, and let-7g-5p (p < 0.05), which could be used as possible candidates for measuring pathological forms of acute or chronic stress. Let-7a-5p, let-7g-5p, and miR-15a-5p (p < 0.05) were also significantly higher in subjects with at least one stress-related disease. Further, correlations were identified between let-7a-5p and meat consumption (p < 0.05) and between miR-15a-5p and coffee consumption (p < 0.05). CONCLUSION: The examination of these four miRNAs as biomarkers using a minimally invasive method offers the possibility of detecting health problems at an early stage and counteracting them to maintain general and mental health.

Sex-Specific miRNA Differences in Liquid Biopsies from Subjects with Solid Tumors and Healthy Controls.

Dysregulation of epigenetic mechanisms has been recognized to play a crucial role in cancer development, but these mechanisms vary between sexes. Therefore, we focused on sex-specific differences in the context of cancer-based data from a recent study. A total of 12 cell-free DNA methylation targets in CpG-rich promoter regions and 48 miRNAs were analyzed by qPCR in plasma samples from 8 female and 7 male healthy controls as well as 48 female and 80 male subjects with solid tumors of the bladder, brain, colorectal region (CRC), lung, stomach, pancreas, and liver. Due to the small sample size in some groups and/or the non-balanced distribution of men and women, sex-specific differences were evaluated statistically only in healthy subjects, CRC, stomach or pancreas cancer patients, and all cancer subjects combined (n female/male-8/7, 14/14, 8/15, 6/6, 48/80, respectively). Several miRNAs with opposing expressions between the sexes were observed for healthy subjects (miR-17-5p, miR-26b-5p); CRC patients (miR-186-5p, miR-22-3p, miR-22-5p, miR-25-3p, miR-92a-3p, miR-16-5p); stomach cancer patients (miR-133a-3p, miR-22-5p); and all cancer patients combined (miR-126-3p, miR-21-5p, miR-92a-3p, miR-183-5p). Moreover, sex-specific correlations that were dependent on cancer stage were observed in women (miR-27a-3p) and men (miR-17-5p, miR-20a-5p). Our results indicate the complex and distinct role of epigenetic regulation, particularly miRNAs, depending not only on the health status but also on the sex of the patient. The same miRNAs could have diverse effects in different tissues and opposing effects between the biological sexes, which should be considered in biomarker research.

MiRNA-based "fitness score" to assess the individual response to diet, metabolism, and exercise.

Background: Regular, especially sustained exercise plays an important role in the prevention and treatment of multiple chronic diseases. Some of the underlying molecular and cellular mechanisms behind the adaptive response to physical activity are still unclear, but recent findings suggest a possible role of epigenetic mechanisms, especially miRNAs, in the progression and management of exercise-related changes. Due to the combination of the analysis of epigenetic biomarkers (miRNAs), the intake of food and supplements, and genetic dispositions, a "fitness score" was evaluated to assess the individual response to nutrition, exercise, and metabolic influence. Methods: In response to a 12-week sports intervention, we analyzed genetic and epigenetic biomarkers in capillary blood from 61 sedentary, healthy participants (66.1% females, 33.9% males, mean age 33 years), including Line-1 methylation, three SNPs, and ten miRNAs using HRM and qPCR analysis. These biomarkers were also analyzed in a healthy, age- and sex-matched control group (n, 20) without intervention. Food frequency intake, including dietary supplement intake, and general health questionnaires were surveyed under the supervision of trained staff. Results: Exercise training decreased the expression of miR-20a-5p, -22-5p, and -505-3p (p < 0.02) and improved the "fitness score," which estimates eight different lifestyle factors to assess, nutrition, inflammation, cardiovascular fitness, injury risk, regeneration, muscle and hydration status, as well as stress level. In addition, we were able to determine correlations between individual miRNAs, miR-20a-5p, -22-5p, and -101-3p (p < 0.04), and the genetic predisposition for endurance and/or strength and obesity risk (ACE, ACTN3, and FTO), as well as between miRNAs and the body composition (p < 0.05). MiR-19b-3p and -101-3p correlated with the intake of B vitamins. Further, miR-19b-3p correlated with magnesium and miR-378a-3p with iron intake (p < 0.05). Conclusions: In summary, our results indicate that a combined analysis of several biomarkers (miRNAs) can provide information about an individual's training adaptions/fitness, body composition, nutritional needs, and possible recovery. In contrast to most studies using muscle biopsies, we were able to show that these biomarkers can also be measured using a minimally invasive method.

PGC-1α Methylation, miR-23a, and miR-30e Expression as Biomarkers for Exercise- and Diet-Induced Mitochondrial Biogenesis in Capillary Blood from Healthy Individuals: A Single-Arm Intervention.

Healthy mitochondria and their epigenetic control are essential to maintaining health, extending life expectancy, and improving cardiovascular performance. Strategies to maintain functional mitochondria during aging include training; cardiovascular exercise has been suggested as the best method, but strength training has also been identified as essential to health and healthy aging. We therefore investigated the effects of concurrent exercise training and dietary habits on epigenetic mechanisms involved in mitochondrial (mt) functions and biogenesis. We analyzed epigenetic biomarkers that directly target the key regulator of mitochondrial biogenesis, PGC-1α, and mtDNA content. Thirty-six healthy, sedentary participants completed a 12-week concurrent training program. Before and after the intervention, dried blood spot samples and data on eating habits, lifestyle, and body composition were collected. MiR-23a, miR-30e expression, and mtDNA content were analyzed using real-time quantitative polymerase chain reaction (qPCR) analysis. PGC-1α methylation was analyzed using bisulfite pyrosequencing. MiR-23a, miR-30e expression, and PGC-1α methylation decreased after the intervention (p < 0.05). PGC-1α methylation increased with the consumption of red and processed meat, and mtDNA content increased with the ingestion of cruciferous vegetables (p < 0.05). Our results indicate that concurrent training could improve mitochondrial biogenesis and functions by altering the epigenetic regulation. These alterations can also be detected outside of the skeletal muscle and could potentially affect athletic performance.

Comprehensive Approach to Distinguish Patients with Solid Tumors from Healthy Controls by Combining Androgen Receptor Mutation p.H875Y with Cell-Free DNA Methylation and Circulating miRNAs.

Liquid biopsy-based tests emerge progressively as an important tool for cancer diagnostics and management. Currently, researchers focus on a single biomarker type and one tumor entity. This study aimed to create a multi-analyte liquid biopsy test for the simultaneous detection of several solid cancers. For this purpose, we analyzed cell-free DNA (cfDNA) mutations and methylation, as well as circulating miRNAs (miRNAs) in plasma samples from 97 patients with cancer (20 bladder, 9 brain, 30 breast, 28 colorectal, 29 lung, 19 ovarian, 12 pancreas, 27 prostate, 23 stomach) and 15 healthy controls via real-time qPCR. Androgen receptor p.H875Y mutation (AR) was detected for the first time in bladder, lung, stomach, ovarian, brain, and pancreas cancer, all together in 51.3% of all cancer samples and in none of the healthy controls. A discriminant function model, comprising cfDNA mutations (COSM10758, COSM18561), cfDNA methylation markers (MLH1, MDR1, GATA5, SFN) and miRNAs (miR-17-5p, miR-20a-5p, miR-21-5p, miR-26a-5p, miR-27a-3p, miR-29c-3p, miR-92a-3p, miR-101-3p, miR-133a-3p, miR-148b-3p, miR-155-5p, miR-195-5p) could further classify healthy and tumor samples with 95.4% accuracy, 97.9% sensitivity, 80% specificity. This multi-analyte liquid biopsy-based test may help improve the simultaneous detection of several cancer types and underlines the importance of combining genetic and epigenetic biomarkers.

Five Days Periodic Fasting Elevates Levels of Longevity Related Christensenella and Sirtuin Expression in Humans.

Periodic fasting (PF) is an increasingly popular approach that assists in the management of metabolic and inflammatory diseases as well as in preventing mechanisms involved in aging. However, little is known about the effects of fasting on gut microbiota and its impact on the epigenetic regulation of metabolically relevant enzymes, especially sirtuins (SIRTs). We analyzed the effect of periodic fasting on the human gut microbiota, SIRTs expression, and mitochondrial content in 51 males and females. The participants fasted under supervision for five consecutive days following the Buchinger fasting guidelines. Ketogenesis, selected mRNAs, miRNAs, mitochondrial (mt) DNA, and gut composition were analyzed before and after PF. PF triggered a significant switch in metabolism, as indicated by the increase in ß-hydroxybutyrate (BHB) and pyruvate dehydrogenase kinase isoform 4 (PDK4) expression in the capillary blood. MtDNA, SIRT1, SIRT3, and miRlet7b-5p expression in blood cells were elevated, whereas SIRT6 and miR125b-5p were not affected. Following fasting, gut microbiota diversity increased, and a statistically significant correlation between SIRT1 gene expression and the abundance of Prevotella and Lactobacillus was detected. The abundance of longevity related Christensenella species increased after fasting and inversely correlated with age as well as body mass index (BMI). Thus, this represents the first study that showing that fasting not only changes the composition of the gut microbiota, making it more diverse, but also affects SIRT expression in humans.

Nutraceutical Approaches of Autophagy and Neuroinflammation in Alzheimer's Disease: A Systematic Review.

Aging and the emergence of age-associated illnesses are one of the major challenges of our present society. Alzheimer's disease (AD) is closely associated with aging and is defined by increasing memory loss and severe dementia. Currently, there are no therapy options available that halt AD progression. This work investigates three hallmarks of the disease (autophagy, neuroinflammation, and senescence) and systematically analyzes if there is a beneficial effect from three substances derived from food sources, the so called "nutraceuticals" epigallocatechin gallate, fisetin, and spermidine, on these hallmarks. The results imply a positive outlook for the reviewed substances to qualify as a novel treatment option for AD. A combination of nutraceutical substances and other preventive measures could have significant clinical impact in a multi-layered therapy approach to counter AD.

Epigallocatechin Gallate Effectively Affects Senescence and Anti-SASP via SIRT3 in 3T3-L1 Preadipocytes in Comparison with Other Bioactive Substances.

AIM: We investigated different bioactive compounds including epigallocatechin gallate (EGCG), anthocyanidin, resveratrol, phloretin, spermidine, butyrate, and ß-hydroxybutyrate with regard to their effect on SIRT3 via NRF2 and modulation of the proinflammatory senescence-associated secretory phenotype (SASP) in senescence induced 3T3-L1 preadipocytes. METHODS: For induction of senescence, 3T3-L1 preadipocytes were incubated with bromodeoxyuridine (BrdU) for 8 days. Cell cycle inhibition was observed, and ß-galactosidase activity was measured. After BrdU treatment, cells were treated with different bioactive compounds in various concentrations for 96 h. ELISA was used for determining proinflammatory cytokine IL6 in SASP cells. RESULTS: CDKN1a increased significantly after BrdU incubation compared to untreated control (p < 0.01). All secondary plant ingredients used for treatment, but not anthocyanidin 50 µM, decrease CDKN1a expression (p < 0.05), whereas most endogenous substances did not attenuate CDKN1a. IL6 secretion positively correlated with CDKN1a (p < 0.01), whereas EGCG could diminish both, IL6 and CDKN1a with the strongest effect (p < 0.01). Although NRF2 positively correlated with SIRT3 activation (p < 0.05), only resveratrol (p < 0.01) and anthocyanidin (p < 0.05) could activate NRF2 significantly. Solely anthocyanidin 50 µM (p < 0.05) and 100 µM (p < 0.01) and EGCG 50 µM (p < 0.01) could increase SIRT3 expression. Activation of SIRT3 with EGCG correlated with lowered IL6 secretion significantly (p < 0.05) but not with anthocyanidin. CONCLUSION: Accumulation of senescent cells in adipose tissue plays an important role in obesity and age-related diseases. SIRT3, located in the mitochondria, can regulate ROS via different pathways. Thus, targeting SIRT3 activating compounds such as EGCG may delay senescence of cells and senescence induced inflammatory processes.

Identification of Clostridium spp. derived from a sheep and cattle slaughterhouse by matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rDNA sequencing.

Clostridia are widespread and some of them are serious human pathogens. Identification of Clostridium spp. is important for managing microbiological risks in the food industry. Samples derived from sheep and cattle carcasses from a slaughterhouse in Iran were analyzed by MALDI-TOF MS using direct transfer and extended direct transfer sample preparation methods and 16S rDNA sequencing. MALDI-TOF MS could identify ten species in 224 out of 240 Clostridium isolates. In comparison to the 16S rDNA sequencing, correct identification rate of the Clostridium spp. at the species level by MALDI-TOF MS technique was 93.3%. 16 isolates were not identified by MALDI-TOF MS but 16s rDNA sequencing identified them as C. estertheticum, C. frigidicarnis, and C. gasigenes species. The most frequently identified Clostridium species were: C. sporogenes (13%), C. cadaveris (12.5%), C. cochlearium (12%) and C. perfringens (10%). Extended direct transfer method [2.26 ± 0.18 log (score)] in comparison to direct transfer method [2.15 ± 0.23 log (score)] improved Clostridium spp. IDENTIFICATION: Using a cut-off score of 1.7 was sufficient for accurate identification of Clostridium species. MALDI-TOF MS identification scores for Clostridium spp. decreased with longer incubation time. Clostridium species predominantly were isolated from carcasses after skinning and evisceration steps in the slaughterhouse. MALDI-TOF MS could be an accurate way to identify Clostridium species. Moreover, continuous improvement of the database and MALDI-TOF MS instrument enhance its performance in food control laboratories.

Bacterial Diversity in Traditional Doogh in Comparison to Industrial Doogh.

Forty-four samples of traditional Doogh and yoghurt were collected from 13 regions of 4 provinces in west of Iran (13 area) and analyzed using molecular methods including PCR, denaturing gradient gel electrophoresis (DGGE) of 16S rDNA, and sequencing. Moreover, collected samples as well as samples from industrially Doogh were analyzed with quantitative real-time PCR (RT-PCR). Analyzed 16S rRNA gene sequences of Doogh samples could be allocated to the presence of Lactobacillus spp. The typical yoghurt starter culture bacteria included four different Lactobacillus species with possible probiotic properties, L. acidophilus, L. helveticus, L. kefiranofaciens, and L. amylovorus. DGGE of traditional Doogh and yoghurt and RT-PCR of traditional Doogh and yoghurt and also industrial Doogh samples demonstrated that traditional Doogh and yoghurt show a higher abundance of total bacteria and lactobacilli and a higher bacterial diversity, respectively. Considering diversity and higher probiotic bacteria content in traditional Doogh, consumers' healthiness in tribes and villages could be promoted with these indigenous products.

Gut Microbiota of Obese, Type 2 Diabetic Individuals is Enriched in Faecalibacterium prausnitzii, Akkermansia muciniphila and Peptostreptococcus anaerobius after Weight Loss.

BACKGROUND: Beside the influence of nutritional habits and reduced physical activity, metabolic syndrome is associated with alterations in the structure of gut microbiota influencing the inflammatory immune responses. Gut microbiota and microbial metabolic activities are known to affect the lipid and glucose metabolism, satiety and chronic low-grade inflammation in the metabolic syndrome. The aim of the study was to identify genera or even species affecting host metabolism in obesity and type 2 diabetes beside the common used indicator: Firmicutes/ Bacteroidetes ratio. METHODS: Differences in gut microbiota were investigated in three groups of subjects over a four month intervention period: type 2 diabetics under GLP1-Agonist therapy, obese individuals without established insulin resistance, both receiving nutritional counseling concerning weight reduction, and a lean control group. Collection of fecal samples was accomplished at two time points, before treatment, and after four months of treatment. For identification of bacteria at species-level we used 454 high-throughput sequencing and fragment length polymorphism analysis based on IS-pro (Intergenic-Spacer-profiling). Five bacterial species, two bacterial genera, total bacterial abundance, and the Firmicutes/Bacteroidetes ratio were determined. RESULTS: Type 2 diabetics showed a higher Firmicutes/Bacteroidetes ratio even with an increase to the second time point (p=0.07). The abundance of B. thetaiotaomicron remained unaffected, whereas B. vulgatus significantly increased in type 2 diabetics (p=0.07) over the study period. Either Alistipes spp. showed an increase in type 2 diabetics between the time points (p=0.06). The abundance of F. prausnitzii (p=0.03) and A. muciniphila (p=0.03) also increased in type 2 diabetics over study period. In addition, the concentration of P. anaerobius (p=0.03) was significantly higher in type 2 diabetics after intervention compared to lean and obese controls. CONCLUSION: Our results clearly show a difference in the gut bacterial composition in type 2 diabetics compared to lean controls or obesity. Therefore, the ratio of Fimicutes/Bacteroidetes might only be an indicator, but a detailed view at species level is even more important in regard to distinction of their functions.

Interleukin-6 CpG Methylation and Body Weight Correlate Differently in Type 2 Diabetes Patients Compared to Obese and Lean Controls.

BACKGROUND/AIMS: Diabetes mellitus type 2 (DMT2) is accompanied by systemic low-grade inflammation with elevated levels of interleukin-6 (IL-6), which is encoded by a gene (IL-6) previously shown to be regulated by DNA methylation. We investigated seven CpG sites in IL-6 in individuals with DMT2, obese individuals and lean controls. Further, the DMT2 group received the glucagon-like peptide 1 agonist liraglutide. METHODS: Blood samples were taken at the beginning of the study and after 4 months. The DNA methylation was assessed using pyrosequencing. RESULTS: Methylation levels at the CpG sites -664, -628 and +13 at the first sampling time point (T1) and at -666 and -664 at the second sampling time point (T2) correlated negatively with initial body weight in the DMT2 group. We found positive correlations for the obese and the lean control group. In the obese group, CpG +27 methylation at T1 correlated with initial body weight (r = 0.685; p = 0.014). In the lean group, CpG -664 at T1 (r = 0.874; p = 0.005) and CpG -628 at T2 (r = 0.632; p = 0.050) correlated with initial body weight. CONCLUSION: These findings are an informative basis for further studies to elucidate epigenetic mechanisms underlying DMT2. Additionally, our results might provide starting points for the development of biomarkers for prevention and therapy strategies.

Increased gut microbiota diversity and abundance of Faecalibacterium prausnitzii and Akkermansia after fasting: a pilot study.

BACKGROUND: An impaired gut microbiota has been reported as an important factor in the pathogenesis of obesity. Weight reduction has already been mentioned to improve gut microbial subpopulations involved in inflammatory processes, though other subpopulations still need further investigation. Thus, weight reduction in the context of a fasting program together with a probiotic intervention may improve the abundance and diversity of gut microbiota. METHODS: In this pilot study, overweight people underwent a fasting program with laxative treatment for 1 week followed by a 6 week intervention with a probiotic formula. Gut microbiota were analyzed on the basis of 16s rDNA with a quantitative real time polymerase chain reaction. Additionally, a food frequency questionnaire with questions about nutritional behavior, lifestyle, and physical activity was administered before and after the intervention. RESULTS: We observed an increase in microbial diversity over the study period. No significant changes in abundance of total bacteria, or of Bacteroidetes, Prevotella, Clostridium cluster XIVa, or Clostridium cluster IV were found, although Faecalibacterium prausnitzii showed an increase over the study period. In addition, Akkermanisa and Bifidobacteria increased in abundance due to intervention. The inflammation-associated gut microbes Enterobacteria and Lactobacilli increased during the first week and then declined by the end of the intervention. Two-thirds of the study participants harbored Archaea. No significant improvements of eating habits were reported, although physical activity improved due to the intervention. CONCLUSIONS: Our results show that caloric restriction affects gut microbiota by proliferating mucin-degrading microbial subpopulations. An additional intervention with a probiotic formula increased probiotic-administered gut microbial populations.

Abundance and diversity of GI microbiota rather than IgG4 levels correlate with abdominal inconvenience and gut permeability in consumers claiming food intolerances.

Food intolerances are an increasing global health problem. Interactions between genetics and environmental changes such as microbial- and stress factors remain poorly understood. Whereas the analyses of IgE mediated allergic responses is based on solid concepts, the roles of microbiota, gut permeability, and IgG antibodies remain widely unclear and are under fierce discussion for scientific relevance. The present pilot study analyzes forty participants, under consultation of nutritional health professionals, for gastrointestinal discomfort and claimed food intolerances. Food frequency questionnaire addresses nutrition, lifestyle and present discomfort. Feces samples are analyzed for dominant microbiota using 16S rDNA based methods and the fecal marker Calprotectin. Blood samples are analyzed for IgG4 levels. The total microbial abundance significantly correlates with claimed discomfort (R=-0.37; p=0.02). The abundance and diversity of microbiota significantly correlates with low Calprotectin values (R=-0.35; p=0.01) and with higher abundance of Faecalibacterium prausnitzii (R=0.78; p<0.01) and Akkermansia (R=0.82; p<0.01). Participants with low discomfort show enhanced Clostridium Cluster XIVa (p=0.008). An increased diversity is also correlating with reduced antibodies against IgG4 of egg white (R=0.68; p<0.01). Data suggest an interaction of low gut permeability and reduced inflammation with an established microbial equilibrium. Self-reported abdominal inconvenience of participants relates mainly to characteristics of microbiota and gut permeability. Anti-inflammatory effects of Faecalibacterium prausnitzii or Lactobacilli and gut barrier functions of Akkermansia may have a key role in food intolerances. The role of IgG4 linking food immune responses with intolerances remains unclear.

Effects of short chain fatty acid producing bacteria on epigenetic regulation of FFAR3 in type 2 diabetes and obesity.

The human gut microbiota and microbial influences on lipid and glucose metabolism, satiety, and chronic low-grade inflammation are known to be involved in metabolic syndrome. Fermentation end products, especially short chain fatty acids, are believed to engage the epigenetic regulation of inflammatory reactions via FFARs (free fatty acid receptor) and other short chain fatty acid receptors. We studied a potential interaction of the microbiota with epigenetic regulation in obese and type 2 diabetes patients compared to a lean control group over a four month intervention period. Intervention comprised a GLP-1 agonist (glucagon-like peptide 1) for type 2 diabetics and nutritional counseling for both intervention groups. Microbiota was analyzed for abundance, butyryl-CoA:acetate CoA-transferase gene and for diversity by polymerase chain reaction and 454 high-throughput sequencing. Epigenetic methylation of the promoter region of FFAR3 and LINE1 (long interspersed nuclear element 1) was analyzed using bisulfite conversion and pyrosequencing. The diversity of the microbiota as well as the abundance of Faecalibacterium prausnitzii were significantly lower in obese and type 2 diabetic patients compared to lean individuals. Results from Clostridium cluster IV and Clostridium cluster XIVa showed a decreasing trend in type 2 diabetics in comparison to the butyryl-CoA:acetate CoA-transferase gene and according to melt curve analysis. During intervention no significant changes were observed in either intervention group. The analysis of five CpGs in the promoter region of FFAR3 showed a significant lower methylation in obese and type 2 diabetics with an increase in obese patients over the intervention period. These results disclosed a significant correlation between a higher body mass index and lower methylation of FFAR3. LINE-1, a marker of global methylation, indicated no significant differences between the three groups or the time points, although methylation of type 2 diabetics tended to increase over time. Our results provide evidence that a different composition of gut microbiota in obesity and type 2 diabetes affect the epigenetic regulation of genes. Interactions between the microbiota and epigenetic regulation may involve not only short chain fatty acids binding to FFARs. Therefore dietary interventions influencing microbial composition may be considered as an option in the engagement against metabolic syndrome.

Changes in human fecal microbiota due to chemotherapy analyzed by TaqMan-PCR, 454 sequencing and PCR-DGGE fingerprinting.

BACKGROUND: We investigated whether chemotherapy with the presence or absence of antibiotics against different kinds of cancer changed the gastrointestinal microbiota. METHODOLOGY/PRINCIPAL FINDINGS: Feces of 17 ambulant patients receiving chemotherapy with or without concomitant antibiotics were analyzed before and after the chemotherapy cycle at four time points in comparison to 17 gender-, age- and lifestyle-matched healthy controls. We targeted 16S rRNA genes of all bacteria, Bacteroides, bifidobacteria, Clostridium cluster IV and XIVa as well as C. difficile with TaqMan qPCR, denaturing gradient gel electrophoresis (DGGE) fingerprinting and high-throughput sequencing. After a significant drop in the abundance of microbiota (p = 0.037) following a single treatment the microbiota recovered within a few days. The chemotherapeutical treatment marginally affected the Bacteroides while the Clostridium cluster IV and XIVa were significantly more sensitive to chemotherapy and antibiotic treatment. DGGE fingerprinting showed decreased diversity of Clostridium cluster IV and XIVa in response to chemotherapy with cluster IV diversity being particularly affected by antibiotics. The occurrence of C. difficile in three out of seventeen subjects was accompanied by a decrease in the genera Bifidobacterium, Lactobacillus, Veillonella and Faecalibacterium prausnitzii. Enterococcus faecium increased following chemotherapy. CONCLUSIONS/SIGNIFICANCE: Despite high individual variations, these results suggest that the observed changes in the human gut microbiota may favor colonization with C. difficile and Enterococcus faecium. Perturbed microbiota may be a target for specific mitigation with safe pre- and probiotics.

Quantification of butyryl CoA:acetate CoA-transferase genes reveals different butyrate production capacity in individuals according to diet and age.

The gastrointestinal microbiota produces short-chain fatty acids, especially butyrate, which affect colonic health, immune function and epigenetic regulation. To assess the effects of nutrition and aging on the production of butyrate, the butyryl-CoA:acetate CoA-transferase gene and population shifts of Clostridium clusters lV and XlVa, the main butyrate producers, were analysed. Faecal samples of young healthy omnivores (24 ± 2.5 years), vegetarians (26 ± 5 years) and elderly (86 ± 8 years) omnivores were evaluated. Diet and lifestyle were assessed in questionnaire-based interviews. The elderly had significantly fewer copies of the butyryl-CoA:acetate CoA-transferase gene than young omnivores (P=0.014), while vegetarians showed the highest number of copies (P=0.048). The thermal denaturation of the butyryl-CoA:acetate CoA-transferase gene variant melting curve related to Roseburia/Eubacterium rectale spp. was significantly more variable in the vegetarians than in the elderly. The Clostridium cluster XIVa was more abundant in vegetarians (P=0.049) and in omnivores (P<0.01) than in the elderly group. Gastrointestinal microbiota of the elderly is characterized by decreased butyrate production capacity, reflecting increased risk of degenerative diseases. These results suggest that the butyryl-CoA:acetate CoA-transferase gene is a valuable marker for gastrointestinal microbiota function.

Characterization of bacteria, clostridia and Bacteroides in faeces of vegetarians using qPCR and PCR-DGGE fingerprinting.

BACKGROUND/AIMS: This study aimed to investigate the quantitative and qualitative changes of bacteria, Bacteroides, Bifidobacterium and Clostridium cluster IV in faecal microbiota associated with a vegetarian diet. METHODS: Bacterial abundances were measured in faecal samples of 15 vegetarians and 14 omnivores using quantitative PCR. Diversity was assessed with PCR-DGGE fingerprinting, principal component analysis (PCA) and Shannon diversity index. RESULTS: Vegetarians had a 12% higher abundance of bacterial DNA than omnivores, a tendency for less Clostridium cluster IV (31.86 +/- 17.00%; 36.64 +/- 14.22%) and higher abundance of Bacteroides (23.93 +/- 10.35%; 21.26 +/- 8.05%), which were not significant due to high interindividual variations. PCA suggested a grouping of bacteria and members of Clostridium cluster IV. Two bands appeared significantly more frequently in omnivores than in vegetarians (p < 0.005 and p < 0.022). One was identified as Faecalibacterium sp. and the other was 97.9% similar to the uncultured gut bacteriumDQ793301. CONCLUSIONS: A vegetarian diet affects the intestinal microbiota, especially by decreasing the amount and changing the diversity of Clostridium cluster IV. It remains to be determined how these shifts might affect the host metabolism and disease risks.