Normal vision and development in mice with low functional expression of Kir7.1 in heterozygosis for a blindness-producing mutation inactivating the channel.

K+ channel Kir7.1 expressed at the apical membrane of the retinal pigment epithelium (RPE) plays an essential role in retinal function. An isoleucine-to-threonine mutation at position 120 of the protein is responsible for blindness-causing vitreo-retinal dystrophy. We have studied the molecular mechanism of action of Kir7.1-I120T in vitro by heterologous expression and in vivo in CRISPR-generated knockin mice. Full-size Kir7.1-I120T reaches the plasma membrane but lacks any activity. Analysis of Kir7.1 and the I120T mutant in mixed transfection experiments, and that of tandem tetrameric constructs made by combining wild type (WT) and mutant protomers, leads us to conclude that they do not form heterotetramers in vitro. Homozygous I120T/I120T mice show cleft palate and tracheomalacia and do not survive beyond P0, whereas heterozygous WT/I120T develop normally. Membrane conductance of RPE cells isolated from WT/WT and heterozygous WT/I120T mice is dominated by Kir7.1 current. Using Rb+ as a charge carrier, we demonstrate that the Kir7.1 current of WT/I120T RPE cells corresponds to approximately 50% of that in cells from WT/WT animals, in direct proportion to WT gene dosage. This suggests a lack of compensatory effects or interference from the mutated allele product, an interpretation consistent with results obtained using WT/- hemizygous mouse. Electroretinography and behavioral tests also show normal vision in WT/I120T animals. The hypomorphic ion channel phenotype of heterozygous Kir7.1-I120T mutants is therefore compatible with normal development and retinal function. The lack of detrimental effect of this degree of functional deficit might explain the recessive nature of Kir7.1 mutations causing human eye disease.NEW & NOTEWORTHY Human retinal pigment epithelium K+ channel Kir7.1 is affected by generally recessive mutations leading to blindness. We investigate one such mutation, isoleucine-to-threonine at position 120, both in vitro and in vivo in knockin mice. The mutated channel is inactive and in heterozygosis gives a hypomorphic phenotype with normal retinal function. Mutant channels do not interfere with wild-type Kir7.1 channels which are expressed concomitantly without hindrance, providing an explanation for the recessive nature of the disease.

Biomarkers for Duchenne muscular dystrophy progression: impact of age in the mdx tongue spared muscle.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy without an effective treatment, caused by mutations in the DMD gene, leading to the absence of dystrophin. DMD results in muscle weakness, loss of ambulation, and death at an early age. Metabolomics studies in mdx mice, the most used model for DMD, reveal changes in metabolites associated with muscle degeneration and aging. In DMD, the tongue muscles exhibit unique behavior, initially showing partial protection against inflammation but later experiencing fibrosis and loss of muscle fibers. Certain metabolites and proteins, like TNF-α and TGF-ß, are potential biomarkers for dystrophic muscle characterization. METHODS: To investigate disease progression and aging, we utilized young (1 month old) and old (21-25 months old) mdx and wild-type tongue muscles. Metabolite changes were analyzed using 1H nuclear magnetic resonance, while TNF-α and TGF-ß were assessed using Western blotting to examine inflammation and fibrosis. Morphometric analysis was conducted to assess the extent of myofiber damage between groups. RESULTS: The histological analysis of the mid-belly tongue showed no differences between groups. No differences were found between the concentrations of metabolites from wild-type or mdx whole tongues of the same age. The metabolites alanine, methionine, and 3-methylhistidine were higher, and taurine and glycerol were lower in young tongues in both wild type and mdx (p < 0.001). The metabolites glycine (p < 0.001) and glutamic acid (p = 0.0018) were different only in the mdx groups, being higher in young mdx mice. Acetic acid, phosphocreatine, isoleucine, succinic acid, creatine, and the proteins TNF-α and TGF-ß had no difference in the analysis between groups (p > 0.05). CONCLUSIONS: Surprisingly, histological, metabolite, and protein analysis reveal that the tongue of old mdx remains partially spared from the severe myonecrosis observed in other muscles. The metabolites alanine, methionine, 3-methylhistidine, taurine, and glycerol may be effective for specific assessments, although their use for disease progression monitoring should be cautious due to age-related changes in the tongue muscle. Acetic acid, phosphocreatine, isoleucine, succinate, creatine, TNF-α, and TGF-ß do not vary with aging and remain constant in spared muscles, suggesting their potential as specific biomarkers for DMD progression independent of aging.

Light effects on Lasiodiplodia theobromae metabolome cultured in vitro.

INTRODUCTION: The present work identified and compared intracellular metabolites and metabolic networks in mycelial cultures of Lasiodiplodia theobromae grown under 12 natural light and 24 hours' dark using a 1 H NMR-based metabolomics approach. MATERIALS AND METHODS: Fungal cultures were grown in potato dextrose media, and metabolites were extracted by sonication with sodium phosphate-buffered saline (pH = 6.0, 10% D2O, 0.1 mM TSP) from mycelium samples collected every week over four weeks. RESULTS: Multivariate analyses revealed that the light exposure group showed a positive correlation within beta-hydroxybutyrate, acetoacetate, acetone, betaine, choline, glycerol, and phosphocholine. On the other hand, phenyl acetate, leucine, isoleucine, valine, and tyrosine were positively correlated with dark conditions. Light favored the oxidative degradation of valine, leucine, and isoleucine, leading to the accumulation of choline, phosphocholine, betaine, and ketone bodies (ketogenesis). Ketogenesis, gluconeogenesis, and the biosynthesis of choline, phosphocholine, and betaine, were considered discriminatory routes for light conditions. The light-sensing pathways were interlinked with fungal development, as verified by the increased production of mycelia biomass without fruiting bodies and stress signaling, as demonstrated by the increased production of pigments.

Branched chain amino acids catabolism as a source of new drug targets in pathogenic protists.

Leucine, isoleucine, and valine, collectively termed Branched Chain Amino Acids (BCAA), are hydrophobic amino acids (AAs) and are essential for most eukaryotes since in these organisms they cannot be biosynthesized and must be supplied by the diet. These AAs are structurally relevant for muscle cells and, of course, important for the protein synthesis process. The metabolism of BCAA and its participation in different biological processes in mammals have been relatively well described. However, for other organisms as pathogenic parasites, the literature is really scarce. Here we review the BCAA catabolism, compile evidence on their relevance for pathogenic eukaryotes with special emphasis on kinetoplastids and highlight unique aspects of this underrated pathway.

Late evening snack and oral amino acid capsules improved respiratory quotient and Fischer ratio in patients with alcoholic liver cirrhosis.

INTRODUCTION AND OBJECTIVES: Appropriate nutritional support may improve energy metabolism in alcoholic liver cirrhosis (ALC) patients. We explored the effect of a late evening snack (LES) and oral amino acid (OAA) capsules on energy metabolism and the Fischer ratio in ALC. PATIENTS AND METHODS: Ninety-one ALC patients were enrolled and randomly divided into three groups: 31 patients in the LES and OAA group, 32 in the LES group, and 28 controls. Respiratory quotient (RQ), carbohydrate oxidation rate (CHO%), fat oxidation rate (FAT%), serum isoleucine and the Fischer ratio were measured at baseline and at months 1, 3, and 6 of follow-up. RESULTS: The RQ in the LES and OAA group was 0.79 ± 0.06, 0.80 ± 0.04, 0.82 ± 0.04, and 0.82 ± 0.04 at baseline and at months 1, 3, and 6 of follow-up, respectively. These values were significantly higher than those in the LES group (P < 0.05). The RQ in the LES group was significantly higher than that in the control group at month 1 and month 6 (P < 0.05). CHO% in the LES and OAA group was significantly increased and FAT% was significantly decreased at month 3 of follow-up (P < 0.05). In the LES and OAA group, serum isoleucine and the Fischer ratio were markedly increased compared with the LES group and control group (P < 0.05). CONCLUSIONS: LES can significantly increase the RQ in ALC. LES and OAA were more effective than LES alone in improving serum isoleucine and the Fischer ratio.

The Aging Process: A Metabolomics Perspective.

Aging process is characterized by a progressive decline of several organic, physiological, and metabolic functions whose precise mechanism remains unclear. Metabolomics allows the identification of several metabolites and may contribute to clarifying the aging-regulated metabolic pathways. We aimed to investigate aging-related serum metabolic changes using a metabolomics approach. Fasting blood serum samples from 138 apparently healthy individuals (20−70 years old, 56% men) were analyzed by Proton Nuclear Magnetic Resonance spectroscopy (1H NMR) and Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS), and for clinical markers. Associations of the metabolic profile with age were explored via Correlations (r); Metabolite Set Enrichment Analysis; Multiple Linear Regression; and Aging Metabolism Breakpoint. The age increase was positively correlated (0.212 ≤ r ≤ 0.370, p < 0.05) with the clinical markers (total cholesterol, HDL, LDL, VLDL, triacylglyceride, and glucose levels); negatively correlated (−0.285 ≤ r ≤ −0.214, p < 0.05) with tryptophan, 3-hydroxyisobutyrate, asparagine, isoleucine, leucine, and valine levels, but positively (0.237 ≤ r ≤ 0.269, p < 0.05) with aspartate and ornithine levels. These metabolites resulted in three enriched pathways: valine, leucine, and isoleucine degradation, urea cycle, and ammonia recycling. Additionally, serum metabolic levels of 3-hydroxyisobutyrate, isoleucine, aspartate, and ornithine explained 27.3% of the age variation, with the aging metabolism breakpoint occurring after the third decade of life. These results indicate that the aging process is potentially associated with reduced serum branched-chain amino acid levels (especially after the third decade of life) and progressively increased levels of serum metabolites indicative of the urea cycle.

Prediction of semen analysis parameter improvement after varicocoelectomy using 1 H NMR-based metabonomics assays.

BACKGROUND: Varicocoele is the most common correctable cause of male infertility; however, predicting varicocoelectomy outcomes is difficult. "Omics" techniques have been increasingly used to develop new diagnostic and prognostics tools for several male infertility causes, and could be applied to study varicocoele. OBJECTIVES: The objective is to create metabolomics models capable of segregating men who improved semen analysis (SA) parameters or achieved natural pregnancy after microsurgical varicocoelectomy (MV) from those who did not, using hydrogen-1 nuclear magnetic resonance (1 H NMR) spectra of seminal plasma of pre-operative samples. MATERIAL AND METHODS: We recruited 29 infertile men with palpable varicocoele. 1 H NMR spectra of seminal plasma were obtained from pre-operative samples and used to create metabonomics models. Improvement was defined as an increase in the total motile progressive sperm count (TMC) of the post-operative SA when compared to the baseline, and pregnancy was assessed for 24 months after MV. RESULTS: Using linear discriminant analysis (LDA), we created a model that discriminated the men who improved SA from those who did not with accuracy of 93.1%. Another model segregated men who achieved natural pregnancy from men who did not. We identified seven metabolites that were important for group segregation: caprylate, isoleucine, N-acetyltyrosine, carnitine, N-acetylcarnitine, creatine, and threonine. DISCUSSION: We described the use of metabonomics model to predict with high accuracy the outcomes of MV in infertile men with varicocoele. The most important metabolites for group segregation are involved in energy metabolism and oxidative stress response, highlighting the pivotal role of these mechanisms in the pathophysiology of varicocoele. CONCLUSIONS: 1 H NMR spectroscopy of seminal plasma can be used in conjunction with multivariate statistical tools to create metabonomics models useful to segregate men with varicocoele based on the reproductive outcomes of MV. These models may help counseling infertile men with varicocoele regarding their prognosis after surgery.

Integration of proteomic and metabolomic analyses: New insights for mapping informal workers exposed to potentially toxic elements.

Occupational exposure to potentially toxic elements (PTEs) is a concerning reality of informal workers engaged in the jewelry production chain that can lead to adverse health effects. In this study, untargeted proteomic and metabolomic analyses were employed to assess the impact of these exposures on informal workers' exposome in Limeira city, São Paulo state, Brazil. PTE levels (Cr, Mn, Ni, Cu, Zn, As, Cd, Sn, Sb, Hg, and Pb) were determined in blood, proteomic analyses were performed for saliva samples (n = 26), and metabolomic analyses in plasma (n = 145) using ultra-high performance liquid chromatography (UHPLC) coupled with quadrupole-time-of-flight (Q-TOF) mass spectrometry. Blood PTE levels of workers, controls, and their family members were determined by inductively coupled plasma-mass spectrometry (ICP-MS). High concentration levels of Sn and Cu were detected in welders' blood (p < 0.001). Statistical analyses were performed using MetaboAnalyst 4.0. The results showed that 26 proteins were upregulated, and 14 proteins downregulated on the welder group, and thirty of these proteins were also correlated with blood Pb, Cu, Sb, and Sn blood levels in the welder group (p < 0.05). Using gene ontology analysis of these 40 proteins revealed the biological processes related to the upregulated proteins were translational initiation, SRP-dependent co-translational protein targeting to membrane, and viral transcription. A Metabolome-Wide Association Study (MWAS) was performed to search for associations between blood metabolites and exposure groups. A pathway enrichment analysis of significant features from the MWAS was then conducted with Mummichog. A total of 73 metabolomic compounds and 40 proteins up or down-regulated in welders were used to perform a multi-omics analysis, disclosing seven metabolic pathways potentially disturbed by the informal work: valine leucine and isoleucine biosynthesis, valine leucine and isoleucine degradation, arginine and proline metabolism, ABC transporters, central carbon metabolism in cancer, arachidonic acid metabolism and cysteine and methionine metabolism. The majority of the proteins found to be statistically up or downregulated in welders also correlated with at least one blood PTE level, providing insights into the biological responses to PTE exposures in the informal work exposure scenario. These findings shed new light on the effects of occupational activity on workers' exposome, underscoring the harmful effects of PTE.

Deficiency in the Essential Amino Acids l-Isoleucine, l-Leucine and l-Histidine and Clinical Measures as Predictors of Moderate Depression in Elderly Women: A Discriminant Analysis Study.

Increases in depression are common in some elderly women. Elderly women often show moderate depressive symptoms, while others display minimal depressive symptoms. These discrepancies have produced contradictory and inconclusive outcomes, which have not been explained entirely by deficits in neurotransmitter precursors. Deficiency in some amino acids have been implicated in major depression, but its role in non-clinical elderly women is not well known. An analysis of essential amino acids, depression and the use of discriminant analysis can help to clarify the variation in depressive symptoms exhibited by some elderly women. The aim was to investigate the relationship of essential amino acids with affective, cognitive and comorbidity measures in elderly women without major depression nor severe mood disorders or psychosis, specifically thirty-six with moderate depressive symptoms and seventy-one with minimal depressive symptoms. The plasma concentrations of nineteen amino acids, Beck Depression Inventory (BDI) scores, Geriatric Depression Scale (GDS) scores, global cognitive scores and comorbidities were submitted to stepwise discriminant analysis to identify predictor variables. Seven predictors arose as important for belong to the group based on amino acid concentrations, with the moderate depressive symptoms group characterized by higher BDI, GDS and cognitive scores; fewer comorbidities; and lower levels of l-histidine, l-isoleucine and l-leucine. These findings suggest that elderly women classified as having moderate depressive symptoms displayed a deficiency in essential amino acids involved in metabolism, protein synthesis, inflammation and neurotransmission.

Sex/Gender Modifies the Association Between the MC4R p.Ile269Asn Mutation and Type 2 Diabetes in the Mexican Population.

CONTEXT: Studies in mice and humans suggest that melanocortin-4 receptor (MC4R) deficiency affects body weight in a sex-/gender-dependent manner. However, similar evidence for type 2 diabetes (T2D) is scarce. OBJECTIVE AND DESIGN: We investigated whether sex/gender modifies the association between the loss-of-function MC4R p.Ile269Asn mutation and T2D in 6929 Mexican adults (3175 T2D cases and 3754 normal glucose tolerance [NGT] controls). The 2003 American Diabetes Association criteria were used to define NGT and T2D. The MC4R p.Ile269Asn mutation was genotyped in all participants using TaqMan technology. RESULTS: The MC4R p.Ile269Asn mutation was associated with T2D in 6929 Mexican adults (Ncontrols = 3754, Ncases = 3175, odds ratio [OR] = 2.00, 95% confidence interval [CI], 1.35-2.97; P = 5.7 × 10-4). The MC4R p.Ile269Asn mutation had a frequency of 0.86 and 1.05% in women with NGT and T2D, and 0.78 and 1.32% in men with NGT and T2D, respectively. We identified a significant interaction between the MC4R p.Ile269Asn mutation and sex/gender on T2D risk (P = 0.049). Although a strong association between the mutation and T2D was observed in men (Ncontrols = 2418, Ncases = 1807, OR = 2.63, 95% CI, 1.62-4.28, P = 9.3 × 10-5), results were not significant in women (Ncontrols = 1336, Ncases = 1368, OR = 1.16, 95% CI, 0.60-2.26, P = 0.65). Further adjustment for body mass index in the logistic regression model did not alter the sex-/gender-specific pattern of association (men: OR = 2.22, 95% CI, 1.34-3.67, P = 0.0019; women: OR = 1.02, 95% CI, 0.51-2.02, P = 0.95). CONCLUSION: This is the first report of a male-specific association between the MC4R p.Ile269Asn loss-of-function mutation and T2D in the Mexican population.

Molecular dynamic simulations, GIAO-NMR and TD-DFT spectroscopy analyze for zwitterionic isoleucine (ILE)N , 1 ≤ N ≤ 6, in water solution.

In this article, we investigate the effects of the isoleucine (ILE)N amino acid chain growth, N = 1.0.6, the ILE conformational effect as well as the solvent presence on the electrical and magnetic spectroscopic properties when these compounds are in aqueous solution. Computational molecular dynamics simulations were performed to include the solvent medium and generate uncorrelated configurations involving solute-solvent structures. The charge point model for solvent was used to obtain the results for quantum mechanical calculation, in special DFT calculations, for (ILE)N structures. Our results for the magnetic shielding constant obtained via GIAO-DFT-NMR calculations show that there is evidence of a magnetic behavior that characterizes the number of peptide bonds and, therefore, how the N isoleucine polypeptide chain is composed. TD-DFT results also show an absorption band shift to larger wavelengths indicating a dependence on N growth.

A Novel Mutation in the FSH Receptor (I423T) Affecting Receptor Activation and Leading to Primary Ovarian Failure.

CONTEXT: Follicle-stimulating hormone (FSH) plays an essential role in gonadal function. Loss-of-function mutations in the follicle-stimulating hormone receptor (FSHR) are an infrequent cause of primary ovarian failure. OBJECTIVE: To analyze the molecular physiopathogenesis of a novel mutation in the FSHR identified in a woman with primary ovarian failure, employing in vitro and in silico approaches, and to compare the features of this dysfunctional receptor with those shown by the trafficking-defective D408Y FSHR mutant. METHODS: Sanger sequencing of the FSHR cDNA was applied to identify the novel mutation. FSH-stimulated cyclic adenosine monophosphate (cAMP) production, ERK1/2 phosphorylation, and desensitization were tested in HEK293 cells. Receptor expression was analyzed by immunoblotting, receptor-binding assays, and flow cytometry. Molecular dynamics simulations were performed to determine the in silico behavior of the mutant FSHRs. RESULTS: A novel missense mutation (I423T) in the second transmembrane domain of the FSHR was identified in a woman with normal pubertal development but primary amenorrhea. The I423T mutation slightly impaired plasma membrane expression of the mature form of the receptor and severely impacted on cAMP/protein kinase A signaling but much less on ß-arrestin-dependent ERK1/2 phosphorylation. Meanwhile, the D408Y mutation severely affected membrane expression, with most of the FSH receptor located intracellularly, and both signal readouts tested. Molecular dynamics simulations revealed important functional disruptions in both mutant FSHRs, mainly the loss of interhelical connectivity in the D408Y FSHR. CONCLUSIONS: Concurrently, these data indicate that conformational differences during the inactive and active states account for the distinct expression levels, differential signaling, and phenotypic expression of the I423T and D408Y mutant FSHRs.

High leucine levels affecting valine and isoleucine recommendations in low-protein diets for broiler chickens.

Four experiments were conducted to estimate the optimal standardized ileal digestible (SID) level of branched-chain amino acids in low-protein diets during the starter, grower, and finisher periods, using the response surface methodology, and to study their effects on performance and mRNA expression of genes involved in the mechanistic target of rapamycin (mTOR) pathway of broiler chickens from 8 to 21 D of age. In experiments 1, 2, and 3, a total of 1,500 Cobb male broiler chickens were assigned to 15 diets of a central composite rotatable design (CCD) of response surface methodology containing 5 levels of SID Leu, Val, and Ile with 5 replicate pens of 20 birds each. A 3-factor, 5-level CCD platform was used to fit the second-order polynomial equation of broiler performance. In experiment 4, a total of 540 8-day-old Cobb male broiler chickens were distributed in a completely randomized 2 x 3 x 3 factorial arrangement with 2 SID Leu levels (1.28 or 1.83%), 3 SID Val levels (0.65, 0.90, or 1.20%), and 3 SID Ile levels (0.54, 0.79, or 1.09%) for a total of 18 treatments with 5 replicate cages of 6 birds each. High Leu levels impaired (P < 0.05) gain:feed when birds were fed marginal Val or Ile diets. However, gain:feed was restored when both Val and Ile were supplemented to reach adequate or high levels. High Leu levels increased (P < 0.05) mRNA expression of S6K1 and eEF2 genes only in birds fed high Ile levels. Dietary SID Leu, Val, and Ile levels required for gain:feed optimization in low-protein diets were estimated at 1.37, 0.94, and 0.87% during the starter period; 1.23, 0.82, and 0.75% during the grower period; and 1.15, 0.77, and 0.70% during the finisher phase, respectively. Higher Val and Ile levels are required to optimize the effect of Leu supplementation on mRNA expression of mTOR pathway genes in the pectoralis major muscle of broilers from day 1 to 21 after hatch.

Validation of the T86I mutation in the gyrA gene as a highly reliable real time PCR target to detect Fluoroquinolone-resistant Campylobacter jejuni.

BACKGROUND: Campylobacter jejuni is a leading cause of bacterial diarrhea worldwide, and increasing rates of fluoroquinolone (FQ) resistance in C. jejuni are a major public health concern. The rapid detection and tracking of FQ resistance are critical needs in developing countries, as these antimicrobials are widely used against C. jejuni infections. Detection of point mutations at T86I in the gyrA gene by real-time polymerase chain reaction (RT-PCR) is a rapid detection tool that may improve FQ resistance tracking. METHODS: C. jejuni isolates obtained from children with diarrhea in Peru were tested by RT-PCR to detect point mutations at T86I in gyrA. Further confirmation was performed by sequencing of the gyrA gene. RESULTS: We detected point mutations at T86I in the gyrA gene in 100% (141/141) of C. jejuni clinical isolates that were previously confirmed as ciprofloxacin-resistant by E-test. No mutations were detected at T86I in gyrA in any ciprofloxacin-sensitive isolates. CONCLUSIONS: Detection of T86I mutations in C. jejuni is a rapid, sensitive, and specific method to identify fluoroquinolone resistance in Peru. This detection approach could be broadly employed in epidemiologic surveillance, therefore reducing time and cost in regions with limited resources.

Association between Dietary Habits and Physical Function in Brazilian and Italian Older Women.

The present study investigated and compared the patterns of dietary protein intake and physical function in Brazilian and Italian older women. Seventy-five Brazilian older women were recruited in a community senior center. Fifty-three age-matched Italian older women were selected from participants of the Longevity check-up (Lookup) 7+ study. In both samples, physical performance was evaluated by isometric handgrip strength (IHG) and five-time sit-to-stand (5 × STS) tests, while diet was assessed through 24-h recall. Results indicated that Brazilian women had a higher intake of plant-based protein (52.7% vs. 30.5% kcal), while Italian women consumed greater amounts of animal-derived protein (29.7% vs. 41.5% kcal). In Brazilian women, the binary logistic regression analysis indicated that body weight-adjusted protein consumption was associated with IHG adjusted by body mass index and with 5 × STS performance. In the Italian sample, the intake of isoleucine, leucine, and valine was significantly associated with 5 × STS performance. Our findings indicate that Brazilian and Italian community-dwelling older women show different patterns of protein intake, with higher consumption of plant-based protein in the Brazilian sample and higher ingestion of animal-derived protein in the Italian subgroup. These dietary patterns may differentially impact the relationship between physical function and protein intake observed in Brazilian and Italian older women.

Branched-Chain amino acids intake is negatively related to body adiposity in individuals at cardiometabolic risk / Consumo de aminoácidos de cadeia ramificada está negativamente relacionado com adiposidade corporal em indivíduos com risco cardiometabólico

ABSTRACT Objective To assess the relationship between branched-chain amino acids intake in the current diet and the metabolic and body adiposity markers in a population at cardiovascular risk. Methods This is a cross-sectional study with 282 adults and elderly people from the Cardiovascular Health Care Program of the Universidade Federal de Viçosa. Sociodemographic, anthropometric and body composition data, as well as metabolic biomarkers were collected using standardized protocols. Dietary intake of branched amino acids was assessed using a 24-hour recall. Results Individuals with a higher branched-chain amino acids intake (≥2.6g/day, median value) had lower body fat (29.6 vs 32.2%; p=0.019), and higher serum ferritin (113.2 vs. 60.1mg/dL; p=0.006) and uric acid concentrations (4.4 vs. 4.0; p=0.023). In addition, a lower prevalence of overweight and excessive abdominal fat (p<0.05) was found in the individuals with higher branched-chain amino acids intake. They also had a higher daily intake of fiber, copper, zinc, magnesium, and iron, as well as a lower intake of total lipids. Conclusion In the present study, the intake of branched amino acids is negatively related to total and central adiposity, but more studies are needed to fully elucidate this possible relationship. (Brazilian Registry of Clinical Trials, code RBR-5n4y2g).

RESUMO Objetivo Avaliar a relação entre o consumo de aminoácidos de cadeia ramificada na dieta atual e os marcadores de adiposidade metabólica e corporal em uma população com perfil de elevado risco cardiovascular. Métodos Trata-se de um estudo transversal com 282 adultos e idosos do Programa de Atenção Cardiovascular da Universidade Federal de Viçosa. Dados sociodemográficos, antropométricos e de composição corporal, além de biomarcadores metabólicos, foram coletados utilizando protocolos padronizados. O consumo alimentar de aminoácidos ramificados foi avaliado através de um recordatório de 24 horas. Resultados Indivíduos com maior consumo de aminoácidos de cadeia ramificada (≥2,6g/dia, valor da mediana) apresentaram menores valores de gordura corporal (29,6 vs 32,2%; p=0,019) e maiores valores de séricos de ferritina (113,2 vs. 60,1mg/dL; p=0,006) e ácido úrico (4,4 vs. 4,0; p=0,023). Além disso, foi encontrada uma menor prevalência de sobrepeso e excesso de gordura abdominal (p<0,05) nos indivíduos com maior consumo de aminoácidos de cadeia ramificada. Eles também apresentaram um maior consumo diário de fibra, cobre, zinco, magnésio e ferro, além de um menor consumo de lipídios totais. Conclusão No presente estudo, o consumo de aminoácidos ramificados está negativamente relacionado à adiposidade total e central, porém mais estudos são necessários para elucidar completamente essa possível relação. (Registro Brasileiro de Ensaios Clínicos, código RBR-5n4y2g)

Pleiotropic effects of alpha-SNAP M105I mutation on oocyte biology: ultrastructural and cellular changes that adversely affect female fertility in mice.

After sperm-oocyte fusion, cortical granules (CGs) located in oocyte cortex undergo exocytosis and their content is released into the perivitelline space to avoid polyspermy. Thus, cortical granule exocytosis (CGE) is a key process for fertilization success. We have demonstrated that alpha-SNAP -and its functional partner NSF- mediate fusion of CGs with the plasma membrane in mouse oocytes. Here, we examined at cellular and ultrastructural level oocytes from hyh (hydrocephalus with hop gait) mice, which present a missense mutation in the Napa gene that results in the substitution of methionine for isoleucine at position 105 (M105I) of alpha-SNAP. Mutated alpha-SNAP was mislocalized in hyh oocytes while NSF expression increased during oocyte maturation. Staining of CGs showed that 9.8% of hyh oocytes had abnormal localization of CGs and oval shape. Functional tests showed that CGE was impaired in hyh oocytes. Interestingly, in vitro fertilization assays showed a decreased fertilization rate for hyh oocytes. Furthermore, fertilized hyh oocytes presented an increased polyspermy rate compared to wild type ones. At ultrastructural level, hyh oocytes showed small mitochondria and a striking accumulation and secretion of degradative structures. Our findings demonstrate the negative effects of alpha-SNAP M105 mutation on oocyte biology and further confirm the relevance of alpha-SNAP in female fertility.

Factorial models to estimate isoleucine requirements for broilers.

The objective of this work was to determine the efficiency of utilization (EU) and produce factorial models for optimal isoleucine (Ile) intake. Six dose-response trials were carried out, three for males and three for females, with 640 Ross 308 in each studied phase. The initial (1-14 days), grower (15-28 days) and finisher (29-42 days) phases were evaluated to cover the growing phase of the broiler chicken. In total, eight treatments were randomly distributed to four replicates of 20 birds each. The treatments consisted of seven crescent levels of Ile and one counter proof to ensure that Ile was the first limiting amino acid in the diet. Dilution technique was applied to produce the levels of Ile and keep the amino acid ratio with lysine. The EU was determined to account for whole body or partitioned for feather-free body (Bff) and feather. Two distinct factorial models were adjusted, M1 and M2. The M2 model was evaluated for one or two EU, being denominated as M2 and M3. When the efficiency was partitioned, the values of 53% and 69% for feather and Bff were determined. The optimal Ile intake estimated for each model were of 275, 908, 1,412 mg of Ile/bird/day (M1); 258, 829, 1,321 mg of Ile/bird/day (M2); and 284, 835, 1,288 mg of Ile/bird/day (M3) for initial, grower and finisher phases respectively. The EU partitioned for feather-free body and feather reduced the biased of the model M3. Overall, higher values of Ile intake are estimated when model M1 is used, which may be the difference in account for body weight gain (M1) or only protein gain (M2 and M3) to estimate the amount of amino acid required for broiler.

High pressure Raman scattering of DL­isoleucine crystals and DFT calculations.

DL­isoleucine single crystals were grown by the slow evaporation method at ambient temperature. Their vibrational properties were studied at ambient temperature as a function of pressure by Raman scattering. At ambient conditions the mode assignment was done in terms of the Potential Energy Distribution (PED) through density functional theory calculations. Both nitrogen and neon were used as pressure transmitting media. The pressure-dependent investigation shows modifications in the Raman spectra recorded between 30 and 3200 cm-1 that were interpreted as phase transitions undergone by the crystal between 1.3 and 1.9 GPa and between 3.6 and 5.1 GPa. Finally, stress was simulated on the unit cell of the crystal from ambient up to 5.0 GPa.

Structural analysis of the woodland strawberry COI1-JAZ1 co-receptor for the plant hormone jasmonoyl-isoleucine.

The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates fundamental plant processes. Fragaria vesca, the woodland strawberry, is a model plant for the Rosaceae family, in which the JA-Ile perception is poorly understood at the molecular level. JA-Ile promotes binding of JAZ repressor to COI1 protein in Arabidopsis to activate jasmonate (JA)-dependent responses. The aim of this work was to understand the molecular basis of the interaction between the F. vesca COI1 (FvCOI1) and JAZ1 (FvJAZ1) promoted by JA-Ile using a computational approach. Multiple sequence alignments and phylogenetic analyses of amino acid sequences were performed for FvCOI1, FvJAZ1 and their ortholog sequences. 3D structures for FvCOI1 and FvJAZ1 proteins were built by methods of homology modeling, using AtCOI1-JA-Ile-AtJAZ1 as template and then they were further refined and validated by molecular dynamics (MD) simulation. A molecular docking approach along with MDS analysis were used to gain insights into the interaction between a putative degron-like sequence present in FvJAZ1 with the FvCOI1-JA-Ile complex. FvCOI1 and FvJAZ1 showed high and moderate sequence identity, respectively, with the corresponding ortholog proteins from other plant species including apple, grape, tomato and Arabidopsis. Moreover, the FvJAZ1 has a variant C-terminal IPMQRK sequence instead of the canonical LPIARR degron sequence located in the Jas domain of AtJAZ1. The MD simulation results showed that the FvCOI1-JA-Ile-FvJAZ1 complex was stable, and the IPMQRK peptide of FvJAZ1 directly interacted with FvCOI1 and JA-Ile. The present research provides novel insight into the molecular interactions among key JA-signaling components in the model plant F. vesca, being few examples of characterized JA-Ile receptors at a structural level in plants.