Highly diverse-Low abundance methanogenic communities in hypersaline microbial mats of Guerrero Negro B.C.S., assessed through microcosm experiments.

Methanogenic communities of hypersaline microbial mats of Guerrero Negro, Baja California Sur, Mexico, have been recognized to be dominated by methylotrophic methanogens. However, recent studies of environmental samples have evidenced the presence of hydrogenotrophic and methyl-reducing methanogenic members, although at low relative abundances. Physical and geochemical conditions that stimulate the development of these groups in hypersaline environments, remains elusive. Thus, in this study the taxonomic diversity of methanogenic archaea of two sites of Exportadora de Sal S.A was assessed by mcrA gene high throughput sequencing from microcosm experiments with different substrates (both competitive and non-competitive). Results confirmed the dominance of the order Methanosarcinales in all treatments, but an increase in the abundance of Methanomassiliiccocales was also observed, mainly in the treatment without substrate addition. Moreover, incubations supplemented with hydrogen and carbon dioxide, as well as the mixture of hydrogen, carbon dioxide and trimethylamine, managed to stimulate the richness and abundance of other than Methanosarcinales methanogenic archaea. Several OTUs that were not assigned to known methanogens resulted phylogenetically distributed into at least nine orders. Environmental samples revealed a wide diversity of methanogenic archaea of low relative abundance that had not been previously reported for this environment, suggesting that the importance and diversity of methanogens in hypersaline ecosystems may have been overlooked. This work also provided insights into how different taxonomic groups responded to the evaluated incubation conditions.

Rescue of morphological defects in Streptomyces venezuelae by the alkaline volatile compound trimethylamine.

Microorganisms can produce a vast diversity of volatile organic compounds of different chemical classes that are capable of mediating intra- and inter-kingdom interactions. In this study, we showed that the soil-dwelling bacterium Streptomyces venezuelae can produce alkaline volatiles under multiple growth conditions, which we discovered through investigation of the S. venezuelae mutant strain MU-1. Strain MU-1 has a defective morphology and exhibits a bald phenotype due to the lack of aerial mycelia and spores, as confirmed by scanning electron microscopy. Using physical barriers to separate the strains on culture plates, we determined that volatile compounds produced by wild-type S. venezuelae could rescue the phenotype of strain MU-1, and pH analysis of the growth medium indicated that these volatile compounds were alkaline. Ultra-high-performance liquid chromatography, combined with mass spectrometry analysis, showed that wild-type S. venezuelae produced abundant levels of the alkaline volatile trimethylamine (TMA) and the oxide form TMAO; however, the levels of these compounds were much lower in strain MU-1. Notably, exposure to TMA alone could rescue the phenotype of this mutant strain, restoring the production of aerial mycelia and spores. We also showed that the rescue effect by alkaline volatiles is mostly species-specific, suggesting that the volatiles may aid particular mutants or other less-fit variants of closely related species to resume normal physiological status and to compete more effectively in complex communities such as soil. Our study reveals a new and intriguing role for bacterial volatiles, including volatiles that may have toxic effects on other species. IMPORTANCE: Bacterial volatiles have a wide range of biological roles at intra- or inter-kingdom levels. The impact of volatiles has mainly been observed between producing bacteria and recipient bacteria, mostly of different species. In this study, we report that the wild-type, soil-dwelling bacterium Streptomyces venezuelae, which forms aerial hypha and spores as part of its normal developmental cycle, also produces the alkaline volatile compound trimethylamine (TMA) under multiple growth conditions. We showed that the environmental dispersion of TMA produced by S. venezuelae promotes the growth and differentiation of growth-deficient mutants of the same species or other slowly growing Streptomyces bacteria, and thus aids in their survival and their ability to compete in complex environmental communities such as soil. Our novel findings suggest a potentially profound biological role for volatile compounds in the growth and survival of communities of volatile-producing Streptomyces species.

Vitamin D3 supplementation in COVID-19 patients with cardiovascular disease and gut dysbiosis.

BACKGROUND: The COVID-19 pandemic has highlighted the vulnerability of particular patient groups to SARS-CoV-2 infection, including those with cardiovascular diseases, hypertension, and intestinal dysbiosis. COVID-19 affects the gut, suggesting diet and vitamin D3 supplementation may affect disease progression. AIMS: To evaluate levels of Ang II and Ang-(1-7), cytokine profile, and gut microbiota status in patients hospitalized for mild COVID-19 with a history of cardiovascular disease and treated with daily doses of vitamin D3. METHODS: We recruited 50 adult patients. We screened 50 adult patients and accessed pathophysiology study 22, randomized to daily oral doses of 10,000IU vitamin D3 (n=11) or placebo (n=11). Plasma levels of Ang II and Ang-(1-7) were determined by radioimmunoassay, TMA and TMAO were measured by liquid chromatography and interleukins (ILs) 6, 8, 10 and TNF-α by ELISA. RESULTS: The Ang-(1-7)/Ang II ratio, as an indirect measure of ACE2 enzymatic activity, increased in the vitamin D3 group (24±5pg/mL vs. 4.66±2pg/mL, p<0.01). Also, in the vitamin D3-treated, there was a significant decline in inflammatory ILs and an increase in protective markers, such as a substantial reduction in TMAO (5±2µmoles/dL vs. 60±10µmoles/dL, p<0.01). In addition, treated patients experienced less severity of infection, required less intensive care, had fewer days of hospitalization, and a reduced mortality rate. Additionally, improvements in markers of cardiovascular function were seen in the vitamin D3 group, including a tendency for reductions in blood pressure in hypertensive patients. CONCLUSIONS: Vitamin D3 supplementation in patients with COVID-19 and specific conditions is associated with a more favourable prognosis, suggesting therapeutic potential in patients with comorbidities such as cardiovascular disease and gut dysbiosis.

Association of Gut Microbiota-Related Metabolites and Type 2 Diabetes in Two Puerto Rican Cohorts.

(1) Aims: Gut microbiota metabolites may play integral roles in human metabolism and disease progression. However, evidence for associations between metabolites and cardiometabolic risk factors is sparse, especially in high-risk Hispanic populations. We aimed to evaluate the cross-sectional and longitudinal relationships between gut microbiota related metabolites and measures of glycemia, dyslipidemia, adiposity, and incident type 2 diabetes in two Hispanic observational cohorts. (2) Methods: We included data from 670 participants of the Boston Puerto Rican Health Study (BPRHS) and 999 participants of the San Juan Overweight Adult Longitudinal Study (SOALS). Questionnaires and clinical examinations were conducted over 3 years of follow-up for SOALS and 6 years of follow-up for BPRHS. Plasma metabolites, including L-carnitine, betaine, choline, and trimethylamine N-oxide (TMAO), were measured at baseline in both studies. We used multivariable linear models to evaluate the associations between metabolites and cardiometabolic risk factors and multivariable logistic and Poisson regressions to assess associations with prevalent and incident type 2 diabetes, adjusted for potential confounding factors. Cohort-specific analyses were combined using a fixed-effects meta-analysis. (3) Results: Higher plasma betaine was prospectively associated with lower fasting glucose [-0.97 mg/dL (95% CI: -1.59, -0.34), p = 0.002], lower HbA1c [-0.02% (95% CI: -0.04, -0.01), p = 0.01], lower HOMA-IR [-0.14 (95% CI: -0.23, -0.05), p = 0.003], and lower fasting insulin [-0.27 mcU/mL (95% CI: -0.51, -0.03), p = 0.02]. Betaine was also associated with a 22% lower incidence of type 2 diabetes (IRR: 0.78, 95% CI: 0.65, 0.95). L-carnitine was associated with lower fasting glucose [-0.68 mg/dL (95% CI: -1.29, -0.07), p = 0.03] and lower HbA1c at follow-up [-0.03% (95% CI: -0.05, -0.01), p < 0.001], while TMAO was associated with higher fasting glucose [0.83 mg/dL (95% CI: 0.22, 1.44), p = 0.01] and higher triglycerides [3.52 mg/dL (95% CI: 1.83, 5.20), p < 0.0001]. Neither choline nor TMAO were associated with incident type 2 diabetes. (4) Conclusions: Higher plasma betaine showed consistent associations with a lower risk of glycemia, insulinemia, and type 2 diabetes. However, TMAO, a metabolite of betaine, was associated with higher glucose and lipid concentrations. These observations demonstrate the importance of gut microbiota metabolites for human cardiometabolic health.

Potential Association of the Oral Microbiome with Trimethylamine N-Oxide Quantification in Mexican Patients with Myocardial Infarction.

Many attempts have been proposed to evaluate the linkage between the oral-gut-liver axis and the mechanisms related to the diseases' establishment. One of them is the oral microbiota translocation into the bloodstream, liver, and gut, promoting a host dysbiosis and triggering the presence of some metabolites such as trimethylamine N-oxide (TMAO), known as a risk marker for cardiovascular disease, and especially the myocardial infarction (MI). In the present pilot study, the involvement of oral dysbiosis related to the presence of TMAO has been considered an independent component of the standard risk factors (SRs) in the development of MI, which has not been previously described in human cohorts. A positive and significant correlation of TMAO levels with Porphyromonas was identified; likewise, the increase of the genus Peptidiphaga in patients without SRs was observed. We determined that the presence of SRs does not influence the TMAO concentration in these patients. This report is the first study where the relationship between oral dysbiosis and TMAO is specified in the Mexican population. Our findings provide information on the possible contribution of the oral pathogens associated with gut dysbiosis in the development of MI, although further analysis should be performed.

Cytotoxicity evaluation, antibacterial effect, and degree of conversion of QAM-containing adhesives.

The aim of this study was to evaluate the influence of adding quaternary ammonium methacrylates (QAMs) to experimental adhesives by assessing the degree of conversion (DC), cytotoxicity against keratinocytes and fibroblasts, and antibacterial activity against biofilm formation. Two QAMs were added to an experimental adhesive: dimethylaminododecyl methacrylate bromododecane (DMADDM) or dimethylaminododecyl methacrylate bromohexadecane (DMAHDM) at three concentrations each: 1, 2.5, and 5 wt.%. Experimental adhesive without QAMs (control group) and commercially available Transbond XT Primer (3M Unitek, Monrovia, California, USA) were used for comparisons. The adhesives were tested for DC, cytotoxicity against keratinocytes and fibroblasts, and antibacterial activity against biofilm formation. DC, cytotoxicity against fibroblasts, and antibacterial activity were analyzed using one-way ANOVA and Tukey's multiple comparisons. Cytotoxicity against keratinocytes was evaluated using the Kruskal Wallis and Dunn's post-hoc (α = 5%) tests. Transbond showed lower DC as compared to 5% DMAHDM, 1% DMADDM, and 5% DMADDM (p < 0.05). However, all groups presented proper DC when compared to commercial adhesives in the literature. In the evaluation of cytotoxicity against keratinocytes, Transbond induced higher viability than 2.5 wt.% groups (p < 0.05). Against fibroblasts, Transbond induced higher viability as compared to 5 wt.% groups (p < 0.05). DMAHDM at 5 wt.% reduced biofilm formation when compared to all the other groups (p < 0.05). Despite their cytotoxic effect against keratinocytes, gingival fibroblasts showed higher viability. DMAHDM at 5 wt.% decreased Streptococcus mutans viability. The incorporation of DMAHDM at 5 wt.% may be a strategy for reducing the development of white spot lesions.

Trimethylamine N-oxide and kidney diseases: what do we know?

In the human gut, there is a metabolically active microbiome whose metabolic products reach various organs and are used in the physiological activities of the body. When dysbiosis of intestinal microbial homeostasis occurs, pathogenic metabolites may increase and one of them is trimethyl amine-N-oxide (TMAO). TMAO is thought to have a role in the pathogenesis of insulin resistance, diabetes, hyperlipidemia, atherosclerotic heart diseases, and cerebrovascular events. TMAO level is also associated with renal inflammation, fibrosis, acute kidney injury, diabetic kidney disease, and chronic kidney disease. In this review, the effect of TMAO on various kidney diseases is discussed.

Consumption of Fish in Chronic Kidney Disease - A Matter of Depth.

Fish is an excellent source of ω-3 polyunsaturated fatty acids (PUFAs), amino acids, collagen, vitamins, and iodine and its intake is associated with health benefits, mainly reduces risk of cardiovascular mortality. However, recent studies have shown that fish is also an important source of trimethylamine N-oxide (TMAO), a uremic toxin produced by the gut microbiota that promotes an increased risk of cardiovascular diseases. In patients with chronic kidney disease (CKD), TMAO levels are markedly increased due to gut dysbiosis and reduced kidney function. No study has yet evaluated the effects of a fish-rich diet on TMAO plasma levels and cardiovascular outcomes. This review discusses the pros and cons of a fish-rich diet in patients with CKD - a matter of depth.

A Free Fatty Acid Synthetic Agonist Accelerates Wound Healing and Improves Scar Quality in Mice.

BACKGROUND: Impaired wound healing is a health problem around the world, and the search for a novel product to repair wounded skin is a major topic in the field. GW9508 is a synthetic molecule described as a selective agonist of free fatty acid receptors (FFARs) 1 and 4, and there is evidence of its anti-inflammatory effects on several organs of the body. PURPOSE: Here, we aimed to evaluate the effects of topical GW9508 on wound healing in mice. RESEARCH DESIGN: First, we used bioinformatic methods to determine the expression of FFAR1 and FFAR4 mRNA in the skin from a human cell atlas assembled with single-cell transcriptomes. Next, we employed 6-week-old C57BL6J mice with 2 wounds inflicted in the back. The mice were randomly divided into 2 groups, a control group, which received topical vehicle, and a treatment group, which received GW9508, for 12 days. The wound was monitored by photographic documentation every 2 days, and samples were collected at day 6 and 12 post injury for RT-PCR, western blot and histology analyses. RESULTS: FFAR1 and FFAR4 mRNA are expressed in skin cells in similar amounts to those in other tissues. Topical GW9508 accelerated wound healing and decreased gene expression of IL-10 and metalloproteinase 9 on days 6 and 12 post injury. It increased the quantity of Collagen I and improved the organization of collagen fibres. Conclusions: Our results show that GW9508 could be an attractive drug treatment for wounded skin. Future studies need to be performed to assess the impact of GW9508 in chronic wound models.

A red wine intervention does not modify plasma trimethylamine N-oxide but is associated with broad shifts in the plasma metabolome and gut microbiota composition.

BACKGROUND: Gut microbiota profiles are closely related to cardiovascular diseases through mechanisms that include the reported deleterious effects of metabolites, such as trimethylamine N-oxide (TMAO), which have been studied as diagnostic and therapeutic targets. Moderate red wine (RW) consumption is reportedly cardioprotective, possibly by affecting the gut microbiota. OBJECTIVES: To investigate the effects of RW consumption on the gut microbiota, plasma TMAO, and the plasma metabolome in men with documented coronary artery disease (CAD) using a multiomics assessment in a crossover trial. METHODS: We conducted a randomized, crossover, controlled trial involving 42 men (average age, 60 y) with documented CAD comparing 3-wk RW consumption (250 mL/d, 5 d/wk) with an equal period of alcohol abstention, both preceded by a 2-wk washout period. The gut microbiota was analyzed via 16S rRNA high-throughput sequencing. Plasma TMAO was evaluated by LC-MS/MS. The plasma metabolome of 20 randomly selected participants was evaluated by ultra-high-performance LC-MS/MS. The effect of RW consumption was assessed by individual comparisons using paired tests during the abstention and RW periods. RESULTS: Plasma TMAO did not differ between RW intervention and alcohol abstention, and TMAO concentrations showed low intraindividual concordance over time, with an intraclass correlation coefficient of 0.049 during the control period. After RW consumption, there was significant remodeling of the gut microbiota, with a difference in ß diversity and predominance of Parasutterella, Ruminococcaceae, several Bacteroides species, and Prevotella. Plasma metabolomic analysis revealed significant changes in metabolites after RW consumption, consistent with improved redox homeostasis. CONCLUSIONS: Modulation of the gut microbiota may contribute to the putative cardiovascular benefits of moderate RW consumption. The low intraindividual concordance of TMAO presents challenges regarding its role as a cardiovascular risk biomarker at the individual level. This study was registered at clinical trials.gov as NCT03232099.

Brazil nut supplementation does not affect trimethylamine-n-oxide plasma levels in patients with coronary artery disease.

The purposes of this study were to assess the effect of Brazil nut supplementation on trimethylamine N-oxide (TMAO) levels and glutathione peroxidase (GPx) activity in patients with coronary artery disease (CAD). Patients with CAD were randomly assigned to two groups, Brazil nut group (23 patients, 48% male, 62.7 ± 6.8 years, 29.4 ± 5.8 kg/m2 ), which received one Brazil nut per day for 3 months, and the control group (14 patients, 43% male, 63.7 ± 8.7 years, 28.4 ± 4.2 kg/m2 ) who did not receive any supplementation. After 3 months, TMAO levels and their precursors did not change in either group. Although not significant, GPx activity increased by 41% in the Brazil nut group. TMAO levels were negatively associated with total fiber intake (r = -0.385 and p = .02). A 3-month Brazil nut supplementation did not change TMAO levels and GPx activity in CAD patients. PRACTICAL APPLICATIONS: Trimethylamine N-oxide (TMAO) has been associated with oxidative stress and cardiovascular disease risk. Thus, the increase in antioxidants enzymes production could be a promising strategy to reduce TMAO-mediated oxidative stress. In this context, nutritional strategies are well-known as activators of cellular antioxidant responses. As Brazil nuts have a known role in reducing oxidative stress by increasing glutathione peroxidase (GPx) activity (a selenium-dependent antioxidant enzyme), this study hypothesized that Brazil nuts could be a strategy that, via antioxidant capacity, would reduce TMAO plasma levels. Although no changes in TMAO levels and GPx activity can be observed in this study, it is believed that other results can be obtained depending on the dosage used. Thus, this study can open new paths and direct other studies with different doses and treatment times to evaluate the effects of Brazil Nuts on TMAO levels.

Alzheimer's disease and gut microbiota: does trimethylamine N-oxide (TMAO) play a role?

Alzheimer's disease (AD) is a neurodegenerative disease that affects memory and cognitive function. Clinical evidence has put into question our current understanding of AD development, propelling researchers to look into further avenues. Gut microbiota has emerged as a potential player in AD pathophysiology. Lifestyle factors, such as diet, can have negative effects on the gut microbiota and thus host health. A Western-type diet has been highlighted as a risk factor for both gut microbiota alteration as well as AD development. The gut-derived trimethylamine N-oxide (TMAO) has been previously implied in the development of cardiovascular diseases with recent evidence suggesting a plausible role of TMAO in AD development. Therefore, the main goal of the present review is to provide the reader with potential mechanisms of action through which consumption of a Western-type diet could increase AD risk, by acting through microbiota-produced TMAO. Although a link between TMAO and AD is far from definitive, this review will serve as a call for research into this new area of research.

First observation on the predation of a non-arthropod species by a dung beetle species: The case of Canthon chalybaeus and the snail Bulimulus apodemetes.

We described, for the first time, a case of predation of a non-arthropod species by a dung beetle species. Canthon chalybaeus Blanchard, 1843 kills healthy individuals of the terrestrial snail Bulimulus apodemetes (D'Orbigny, 1835) showing an evident pattern of physical aggressiveness in the attacks using the dentate clypeus and the anterior tibiae. The description of this predatory behaviour was complemented with the analysis of the chemical secretions of the pygidial glands of C. chalybaeus, highlighting those main chemical compounds that, due to their potential toxicity, could contribute to death of the snail. We observed a high frequency of predatory interactions reinforcing the idea that predation in dung beetles is not accidental and although it is opportunistic it involves a series of behavioural sophistications that suggest an evolutionary pattern within Deltochilini that should not only be better studied from a behavioural point of view but also phylogenetically.

Can diet modulate trimethylamine N-oxide (TMAO) production? What do we know so far?

BACKGROUND: Trimethylamine N-oxide (TMAO) is a metabolite that has attracted attention due to its positive association with several chronic non-communicable diseases such as insulin resistance, atherosclerotic plaque formation, diabetes, cancer, heart failure, hypertension, chronic kidney disease, liver steatosis, cardiac fibrosis, endothelial injury, neural degeneration and Alzheimer's disease. TMAO production results from the fermentation by the gut microbiota of dietary nutrients such as choline and carnitine, which are transformed to trimethylamine (TMA) and converted into TMAO in the liver by flavin-containing monooxygenase 1 and 3 (FMO1 and FMO3). Considering that TMAO is involved in the development of many chronic diseases, strategies have been found to enhance a healthy gut microbiota. In this context, some studies have shown that nutrients and bioactive compounds from food can modulate the gut microbiota and possibly reduce TMAO production. OBJECTIVE: This review has as main objective to discuss the studies that demonstrated the effects of food on the reduction of this harmful metabolite. METHODS: All relevant articles until November 2020 were included. The articles were searched in Medline through PubMed. RESULTS: Both the food is eaten acutely and chronically, by altering the nature of the gut microbiota, influencing colonic TMA production. Furthermore, hepatic production of TMAO by the flavin monooxygenases in the liver may also be influenced by phenolic compounds present in foods. CONCLUSION: The evidence presented in this review shows that TMAO levels can be reduced by some bioactive compounds. However, it is crucial to notice that there is significant variation among the studies. Further clinical studies should be conducted to evaluate these dietary components' effectiveness, dose, and intervention time on TMAO levels and its precursors.

Trimethylamine N-oxide levels are associated with NASH in obese subjects with type 2 diabetes.

AIMS: Trimethylamine N-oxide (TMAO), choline and betaine serum levels have been associated with metabolic diseases including type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). These associations could be mediated by insulin resistance. However, the relationships among these metabolites, insulin resistance and NAFLD have not been thoroughly investigated. Moreover, it has recently been suggested that TMAO could play a role in NAFLD by altering bile acid metabolism. We examined the association between circulating TMAO, choline and betaine levels and NAFLD in obese subjects. METHODS: Serum TMAO, choline, betaine and bile acid levels were measured in 357 Mexican obese patients with different grades of NAFLD as determined by liver histology. Associations of NAFLD with TMAO, choline and betaine levels were tested. Moreover, association of TMAO levels with non-alcoholic steatohepatitis (NASH) was tested separately in patients with and without T2D. RESULTS: TMAO and choline levels were significantly associated with NAFLD histologic features and NASH risk. While increased serum TMAO levels were significantly associated with NASH in patients with T2D, in non-T2D subjects this association lost significance after adjusting for sex, BMI and HOMA2-IR. Moreover, circulating secondary bile acids were associated both with increased TMAO levels and NASH. CONCLUSIONS: In obese patients, circulating TMAO levels were associated with NASH mainly in the presence of T2D. Functional studies are required to evaluate the role of insulin resistance and T2D in this association, both highly prevalent in NASH patients.

Targeting FFA1 and FFA4 receptors in cancer-induced cachexia.

Free fatty acid (FFA) receptors FFA1 and FFA4 are omega-3 molecular targets in metabolic diseases; however, their function in cancer cachexia remains unraveled. We assessed the role of FFA1 and FFA4 receptors in the mouse model of cachexia induced by Lewis lung carcinoma (LLC) cell implantation. Naturally occurring ligands such as α-linolenic acid (ALA) and docosahexaenoic acid (DHA), the synthetic FFA1/FFA4 agonists GW9508 and TUG891, or the selective FFA1 GW1100 or FFA4 AH7614 antagonists were tested. FFA1 and FFA4 expression and other cachexia-related parameters were evaluated. GW9508 and TUG891 decreased tumor weight in LLC-bearing mice. Regarding cachexia-related end points, ALA, DHA, and the preferential FFA1 agonist GW9508 rescued body weight loss. Skeletal muscle mass was reestablished by ALA treatment, but this was not reflected in the fiber cross-sectional areas (CSA) measurement. Otherwise, TUG891, GW1100, or AH7614 reduced the muscle fiber CSA. Treatments with ALA, GW9508, GW1100, or AH7614 restored white adipose tissue (WAT) depletion. As for inflammatory outcomes, ALA improved anemia, whereas GW9508 reduced splenomegaly. Concerning behavioral impairments, ALA and GW9508 rescued locomotor activity, whereas ALA improved motor coordination. Additionally, DHA improved grip strength. Notably, GW9508 restored abnormal brain glucose metabolism in different brain regions. The GW9508 treatment increased leptin levels, without altering uncoupling protein-1 downregulation in visceral fat. LLC-cachectic mice displayed FFA1 upregulation in subcutaneous fat, but not in visceral fat or gastrocnemius muscle, whereas FFA4 was unaltered. Overall, the present study shed new light on FFA1 and FFA4 receptors' role in metabolic disorders, indicating FFA1 receptor agonism as a promising strategy in mitigating cancer cachexia.

Novel variants in outer protein surface of flavin-containing monooxygenase 3 found in an Argentinian case with impaired capacity for trimethylamine N-oxygenation.

Flavin-containing monooxygenase 3 (FMO3) is a polymorphic drug metabolizing enzyme associated with the genetic disorder trimethylaminuria. We phenotyped a white Argentinian 11-year-old girl by medical sensory evaluation. After pedigree analysis with her brother and parents, this proband showed to harbor a new allele p.(P73L; E158K; E308G) FMO3 in trans configuration with the second new one p.(F140S) FMO3. Recombinant FMO3 proteins of the wild-type and the novel two variants underwent kinetic analyses of their trimethylamine N-oxygenation activities. P73L; E158K; E308G and F140S FMO3 proteins exhibited moderately and severely decreased trimethylamine N-oxygenation capacities (~50% and ~10% of wild-type FMO3, respectively). Amino acids P73 and F140 were located on the outer surface region in a crystallographic structure recently reported of a FMO3 analog. Changes in these positions would indirectly impact on key FAD-binding residues. This is the first report and characterization of a patient of fish odor syndrome caused by genetic aberrations leading to impaired FMO3-dependent N-oxygenation of trimethylamine found in the Argentinian population. We found novel structural determinants of FAD-binding domains, expanding the list of known disease-causing mutations of FMO3. Our results suggest that individuals homozygous for any of these new variants would develop a severe form of this disorder.

Activation of GPR40 induces hypothalamic neurogenesis through p38- and BDNF-dependent mechanisms.

Hypothalamic adult neurogenesis provides the basis for renewal of neurons involved in the regulation of whole-body energy status. In addition to hormones, cytokines and growth factors, components of the diet, particularly fatty acids, have been shown to stimulate hypothalamic neurogenesis; however, the mechanisms behind this action are unknown. Here, we hypothesized that GPR40 (FFAR1), the receptor for medium and long chain unsaturated fatty acids, could mediate at least part of the neurogenic activity in the hypothalamus. We show that a GPR40 ligand increased hypothalamic cell proliferation and survival in adult mice. In postnatal generated neurospheres, acting in synergy with brain-derived neurotrophic factor (BDNF) and interleukin 6, GPR40 activation increased the expression of doublecortin during the early differentiation phase and of the mature neuronal marker, microtubule-associated protein 2 (MAP2), during the late differentiation phase. In Neuro-2a proliferative cell-line GPR40 activation increased BDNF expression and p38 activation. The chemical inhibition of p38 abolished GPR40 effect in inducing neurogenesis markers in neurospheres, whereas BDNF immunoneutralization inhibited GPR40-induced cell proliferation in the hypothalamus of adult mice. Thus, GPR40 acts through p38 and BDNF to induce hypothalamic neurogenesis. This study provides mechanistic advance in the understating of how a fatty acid receptor regulates adult hypothalamic neurogenesis.

Chronic activation of GPR40 does not negatively impact upon BRIN-BD11 pancreatic ß-cell physiology and function.

BACKGROUND: Free fatty acids (FFAs) are known for their dual effects on insulin secretion and pancreatic ß-cell survival. Short-term exposure to FFAs, such as palmitate, increases insulin secretion. On the contrary, long-term exposure to saturated FFAs results in decreased insulin secretion, as well as triggering oxidative stress and endoplasmic reticulum (ER) stress, culminating in cell death. The effects of FFAs can be mediated either via their intracellular oxidation and consequent effects on cellular metabolism or via activation of the membrane receptor GPR40. Both pathways are likely to be activated upon both short- and long-term exposure to FFAs. However, the precise role of GPR40 in ß-cell physiology, especially upon chronic exposure to FFAs, remains unclear. METHODS: We used the GPR40 agonist (GW9508) and antagonist (GW1100) to investigate the impact of chronically modulating GPR40 activity on BRIN-BD11 pancreatic ß-cells physiology and function. RESULTS: We observed that chronic activation of GPR40 did not lead to increased apoptosis, and both proliferation and glucose-induced calcium entry were unchanged compared to control conditions. We also observed no increase in H2O2 or superoxide levels and no increase in the ER stress markers p-eIF2α, CHOP and BIP. As expected, palmitate led to increased H2O2 levels, decreased cell viability and proliferation, as well as decreased metabolism and calcium entry. These changes were not counteracted by the co-treatment of palmitate-exposed cells with the GPR40 antagonist GW1100. CONCLUSIONS: Chronic activation of GPR40 using GW9508 does not negatively impact upon BRIN-BD11 pancreatic ß-cells physiology and function. The GPR40 antagonist GW1100 does not protect against the deleterious effects of chronic palmitate exposure. We conclude that GPR40 is probably not involved in mediating the toxicity associated with chronic palmitate exposure.

Heavy metals detection in river water with cantilever nanobiosensor.

Heavy metals can be highly toxic depending on the dose and the chemical form. In this context, sensing devices such as nanobiosensors have been presented as a promising tool to monitor contaminants at micro and nanoscale. In this work, cantilever nanobiosensors with phosphatase alkaline were developed and applied to detect heavy metals (Pb, Ni, Cd, Zn, Co, and Al) in river water. The nanobiosensor surface was functionalized by the self-assembled monolayers (SAM) technique using 16-mercaptohexadecanoic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N- hydroxysuccinimide (NHS), and phosphatase alkaline enzyme. The sensing layer deposited on the cantilever surface presented a uniform morphology, at nanoscale, with 80 nm of thickness. The nanobiosensor showed a detection limit in the ppb range and high sensitivity, with a stability of fifteen days. The developed cantilever nanobiosensor is a simple tool, suitable for the direct detection of contaminants in river water.