FTO Gene Polymorphisms at the Crossroads of Metabolic Pathways of Obesity and Epigenetic Influences.

In this review, we summarize the current state of knowledge on the fat mass and obesity-associated (FTO) gene and its role in obesity. The FTO-encoded protein is involved in multiple molecular pathways contributing to obesity as well as other metabolic complexities. This review emphasizes the epigenetic influence on the FTO gene as a new approach in the treatment and management of obesity. Several known substances have a positive effect on reducing FTO expression. Depending on which variant of the single nucleotide polymorphism (SNP) is present, the profile and level of gene expression changes. Implementation of environmental change measures could lead to reduced phenotypic manifestation of FTO expression. Treating obesity through FTO gene regulation will have to include various complex signal pathways in which FTO takes part. Identification of FTO gene polymorphisms may be useful for the development of individual obesity management strategies, including the recommendation of taking certain foods and supplements.

Association of Vitamins D, B9 and B12 with Obesity-Related Diseases and Oral Microbiota Composition in Obese Women in Croatia.

Research background: Oral microbiota has become an important factor in obesity, but its association with obesity-related diseases and serum 25-hydroxy vitamin D [25(OH)D] and B complex amounts is still uncertain. The main aim of the paper is to determine the variation in oral microbiota composition as a response to the vitamin status and obesity-related diseases in obese females from Croatia. We hypothesized that the prevalence of probiotic or pathogenic bacteria in the oral cavity of obese women in Croatia depends on the amounts of vitamin B9 (folic acid), B12 and 25(OH)D in serum and/or hypertension, diabetes and prediabetes diagnosis. Experimental approach: To test the defined research hypothesis, female individuals with body mass index (BMI)≥30 kg/m2 (N=70) were recruited to participate in this study. Obese women were divided into groups according to BMI value, diagnosis of obesity-related diseases and amount of micronutrient in blood. For the quantitative determination of folic acid, vitamin B12 and 25(OH)D in serum, an electrochemiluminescence protein binding assay (ECLIA) was performed. Microorganisms isolated from the saliva of obese women were analyzed by MALDI-TOF mass spectrometer. Results and conclusions: The presented results do not support the hypothesis that the prevalence of probiotic or pathogenic bacteria in the oral cavity of obese women in Croatia depends on the amount of micronutrients. On the other hand, hypertension and diabetes/prediabetes favour the growth of oral pathogens, specifically increased levels of Candida sp. Novelty and scientific contribution: To the best of our knowledge, this is the first study showing the relationship between obesity, micronutrient amount, oral microbiota composition, and the incidence of obesity-related disease. We included only obese women from Croatia, so it is regionally specific. Also, we have shown that oral microbiota composition is not connected with micronutrient deficiencies but only with obesity-related diseases.

PHYSICO chemical properties and toxicological effect of landfill groundwaters and leachates.

Waste landfills represent a global problem, which is more pronounced in developing countries because of the lack of resources to implement procedures that include separation and waste processing. The aim of this research was to analyze leachate and ground waters samples at the site, upstream and downstream from the landfill during different year seasons on a registered non-hazardous waste dump and to conduct physico-chemical and biological assays to determine potential risk for the ecosystem. Potential cytotoxic, prooxidative and mutagenic effects of leachates and water samples were evaluated on human laryngeal cell line (HEp2). Leachates collected at landfill site caused genotoxic effect and had a higher pH, chemical oxygen demand (COD), biochemical oxygen demand (BOD) and elevated concentrations of phosphorus, chloride, nitrogen compounds and sulphate. Genotoxicity of the leachate was increased in samples collected in dry and warm period of the year. These results are in accordance to the physico-chemical analysis which revealed that during summer period, because of intense degradation process at high temperatures increased concentrations of different chemicals can be found in leachate. Groundwater collected downstream and upstream from landfill did not show statistically significant (geno)toxic effect, irrespective of the sampling season. Chemical analysis revealed that all compounds in groundwater were below permitted values. Purification process at landfill is effective and compounds that reach groundwater do not represent a toxicological threat.

Higher prevalence of FTO gene risk genotypes AA rs9939609, CC rs1421085, and GG rs17817449 and saliva containing Staphylococcus aureus in obese women in Croatia.

Obesity is composed of multifunctional interactions of eating habits, behaviors, microbiota, genetics, and other unknown factors. We hypothesize that correlations occur between the fat mass and obesity-associated single nucleotide polymorphisms (FTO SNPs), the composition of microorganisms in the saliva, and life habits in obese women from Zagreb County. Our results of the analysis of 3 FTO SNPs showed a statistically significant positive correlation among the frequencies of the high-risk genotypes AA rs9939609 (P = .0367), CC rs1421085 (P = .0367), and GG rs17817449 (P = .0065) of the FTO gene in obese cases. Interestingly, 39.13% of obese women were triple homozygous for all 3 risk alleles. Furthermore, the composition of the oral microbiota in the obese group showed a higher occurrence of a major human pathogen, bacterium Staphylococcus aureus, but a significantly low presence of bacteria Streptococcus oralis, Streptococcus mitis, and Serratia ureilytica compared with the control group. The investigation also revealed that obese women prefer to consume candy and snacks and more meat and meat-derived products, sleep less than 6 hours per day, and had higher hypertension in comparison with the control group. These results support the hypothesis that female obesity is strongly related to all 3 variants of the FTO gene and perhaps a specific composition of microbiota in saliva due to dietary habits. Considering the bimodal distribution of the SNPs and bacterial content of saliva in obese women taken together are factors to consider for risk of obesity.

Characterization of a S-adenosyl-l-methionine (SAM)-accumulating strain of Scheffersomyces stipitis.

S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in greater SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism.

Sub-emetic toxicity of Bacillus cereus toxin cereulide on cultured human enterocyte-like Caco-2 cells.

Cereulide (CER) intoxication occurs at relatively high doses of 8 µg/kg body weight. Recent research demonstrated a wide prevalence of low concentrations of CER in rice and pasta dishes. However, the impact of exposure to low doses of CER has not been studied before. In this research, we investigated the effect of low concentrations of CER on the behavior of intestinal cells using the Caco-2 cell line. The MTT (mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and the SRB (sulforhodamine B) reactions were used to measure the mitochondrial activity and cellular protein content, respectively. Both assays showed that differentiated Caco-2 cells were sensitive to low concentrations of CER (in a MTT reaction of 1 ng/mL after three days of treatment; in an SRB reaction of 0.125 ng/mL after three days of treatment). Cell counts revealed that cells were released from the differentiated monolayer at 0.5 ng/mL of CER. Additionally, 0.5 and 2 ng/mL of CER increased the lactate presence in the cell culture medium. Proteomic data showed that CER at a concentration of 1 ng/mL led to a significant decrease in energy managing and H2O2 detoxification proteins and to an increase in cell death markers. This is amongst the first reports to describe the influence of sub-emetic concentrations of CER on a differentiated intestinal monolayer model showing that low doses may induce an altered enterocyte metabolism and membrane integrity.

The effect of starvation stress on Lactobacillus brevis L62 protein profile determined by de novo sequencing in positive and negative mass spectrometry ion mode.

RATIONALE: We describe a novel negative chemically activated fragmentation/positive chemically activated fragmentation (CAF-/CAF+) technique for protein identification. The technique was used to investigate Lactobacillus brevis adaptation to nutrient deprivation. METHODS: The CAF-/CAF+ method enables de novo sequencing of derivate peptides with negative and positive ion mode matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS). Peptide sequences obtained from MS/MS spectra were matched against the National Center for Biotechnology Information (NCBI) non-redundant (nr) database and confirmed by the mass spectrometry data of elucidated peptide mass sequences derived from the annotated genome. This improved protein identification method highlighted 36 differentially expressed proteins in the proteome of L. brevis after 75 days of starvation. RESULTS: The results revealed the key differences in the metabolic pathways that are responsible for the survival of L. brevis in a hostile environment. Proteomics analysis demonstrated that numerous proteins engaged in glucose and amino-acid catabolizing pathways, glycerolipid metabolizing pathways, and stress-response mechanisms are differentially expressed after long-term starvation. Amino acid and proteomics analysis indicated that starved L. brevis metabolized arginine, glycine, and histidine from dead cells as alternative nutrient sources. The production of lactic acid also varied between the parent cells and the starved cells. CONCLUSIONS: Differentially expressed proteins identified exclusively by peptide sequence reading provided promising results for CAF-/CAF+ implementation in a standard proteomics workflow (e.g., biomarker and mutation discovery and biotyping). The practical performance of a reliable de novo sequencing technique in routine proteomics analysis is emphasized in this article.

Characterization of Lactobacillus brevis L62 strain, highly tolerant to copper ions.

Lactic acid bacteria (LAB) as starter culture in food industry must be suitable for large-scale industrial production and possess the ability to survive in unfavorable processes and storage conditions. Approaches taken to address these problems include the selection of stress-resistant strains. In food industry, LAB are often exposed to metal ions induced stress. The interactions between LAB and metal ions are very poorly investigated. Because of that, the influence of non-toxic, toxic and antioxidant metal ions (Zn, Cu, and Mn) on growth, acid production, metal ions binding capacity of wild and adapted species of Leuconostoc mesenteroides L3, Lactobacillus brevis L62 and Lactobacillus plantarum L73 were investigated. The proteomic approach was applied to clarify how the LAB cells, especially the adapted ones, protect themselves and tolerate high concentrations of toxic metal ions. Results have shown that Zn and Mn addition into MRS medium in the investigated concentrations did not have effect on the bacterial growth and acid production, while copper ions were highly toxic, especially in static conditions. Leuc. mesenteroides L3 was the most efficient in Zn binding processes among the chosen LAB species, while L. plantarum L73 accumulated the highest concentration of Mn. L. brevis L62 was the most copper resistant species. Adaptation had a positive effect on growth and acid production of all species in the presence of copper. However, the adapted species incorporated less metal ions than the wild species. The exception was adapted L. brevis L62 that accumulated high concentration of copper ions in static conditions. The obtained results showed that L. brevis L62 is highly tolerant to copper ions, which allows its use as starter culture in fermentative processes in media with high concentration of copper ions.

Obesity: genome and environment interactions.

Obesity has become one of the major threats for public health in industrialised world among adults, but also among adolescents and children. It is influenced by the interaction of genes, nutrition, environment, and lifestyle. Environmental and lifestyle risk factors include foetal and lifelong environment, nutrient quality, chemical and microbial exposure, and psychical stress, all of which are important contributing influences. Removing or limiting chemical and pharmaceutical obesogens from human environment could make a difference in the growing epidemic of obesity. Additionally, nutrigenomics describes how modifications in individual diets can improve health and prevent chronic diseases, as well as obesity, by understanding the effects of a genetic profile in the interaction between food and increase in body weight. Furthermore, individual genetic variations in genome represent an individual's predisposition for obesity. Therefore, the use of individual genetic information, avoiding obesogens, and a healthy lifestyle could help to improve the management of obesity and maintain a healthy weight.

Genotoxicity of tryptophol in a battery of short-term assays on human white blood cells in vitro.

The genotoxic effects of tryptophol (indole-3-ethanol), an aromatic alcohol and known secondary metabolite of the opportunistic yeast Candida albicans and other Candida spp., were studied using a battery of short-term assays on human white blood cells in vitro. The concentration range of tryptophol tested was 0.25 mM to 2.00 mM. Lymphocyte viability and induction of apoptosis/necrosis were studied by simultaneous use of a fluorescent assay with ethidium bromide and acridine orange. Levels of primary DNA damage and dynamics of DNA repair were evaluated using the alkaline comet assay while the levels and nature of residual DNA damage were assessed by the analysis of structural chromosome aberrations, the sister chromatid exchange test and the cytokinesis-block micronucleus assay. The results obtained suggest cytotoxic, cytostatic and genotoxic effects of the tryptophol treatment in vitro that were mainly dose-dependent. The type and the extent of DNA lesions detected in tryptophol-treated samples indicate the possibility that observed damage is mediated by highly reactive aldehyde metabolite and/or free radicals produced by treatment. The results show that mortality of lymphocytes in tryptophol-treated samples was primarily caused by apoptosis. The generation of additional DNA strand breaks and cytogenetic consequences (chromosome aberrations, sister chromatid exchanges and micronuclei), as observed in this study, sustain the possibility that tryptophol toxicity is mediated by the formation of DNA cross-links and aldehyde-protein adducts. In conclusion, this preliminary study elucidates only a part of tryptophol toxicity to human cells. Because current evidence is not sufficient to obtain information relevant for human risk assessment, further in vitro and in vivo studies are essential in order to clarify remaining issues, especially to elucidate the exact mechanisms and nature of the damage produced following treatment as well to estimate possible interindividual variability in genotoxic responses to the chemical.

The growth advantage in stationary-phase (GASP) phenomenon in mixed cultures of enterobacteria.

Growth advantage in stationary phase (GASP) is the term used to describe the ability of mutants with an increased fitness from 10-day-old enterobacteria culture to out-compete 1-day-old cells of the same initial strain during a prolonged stationary phase, although the aged cells are introduced as a minority. We studied this bacterial trait in mixed cultures of two enterobacterial species, Escherichia coli and Salmonella enterica, wild type in addition to derived mutants from both strains that contain chromosomal-encoded resistance to either nalidixic acid or streptomycin. The strong GASP phenotype was obtained in mixed cultures with the aged mutant strains, but not when the isogenic antibiotic-sensitive strains were used. This phenomenon was associated with chromosomal rearrangements in 10-day-old bacterial antibiotic-resistant mutated cells.