Molecular basis of various forms of maple syrup urine disease in Chilean patients.

BACKGROUND: Maple syrup urine disease (MSUD) is an autosomal recessive inherited metabolic disorder caused by the deficient activity of the branched-chain α-keto acid dehydrogenase (BCKD) enzymatic complex. BCKD is a mitochondrial complex encoded by four genes: BCKDHA, BCKDHB, DBT, and DLD. MSUD is predominantly caused by mutations in the BCKDHA, BCKDHB, and DBT genes which encode the E1α, E1ß, and E2 subunits of the BCKD complex, respectively. The aim of this study was to characterize the genetic basis of MSUD in a cohort of Chilean MSUD patients by identifying point mutations in the BCKDHA, BCKDHB, and DBT genes and to describe their impact on the phenotypic heterogeneity of these patients. METHODS: This manuscript describes a cross-sectional study of 18 MSUD patients carried out using PCR and DNA sequencing. RESULTS: Four novel pathogenic mutations were identified: one in BCKDHA (p.Thr338Ile), two in BCKDHB (p.Gly336Ser e p.Pro240Thr), and one in DBT (p.Gly406Asp). Four additional pathogenic mutations found in this study have been described previously. There were no correlations between the genotype and phenotype of the patients. CONCLUSION: If MSUD is diagnosed earlier, with a newborn screening approach, it might be possible to establish genotype-phenotype relationships more efficiently.

IARS2 regulates proliferation, migration, and angiogenesis of human umbilical vein endothelial cells

SUMMARY OBJECTIVE: In this study, we aimed at investigating the role of isoleucyl-tRNA synthetase in the growth, migration, and angiogenesis of human umbilical vein endothelial cells and the underlying molecular mechanism. METHODS: To assess the role of isoleucyl-tRNA synthetase, we silenced isoleucyl-tRNA synthetase in human umbilical vein endothelial cells using lentiviral 2 specific short hairpin RNAs (short hairpin RNAs 1 and 2) and examined silencing efficiency using real time quantitative polymerase chain reaction and western blot analyses. Short hairpin RNAs 1-isoleucyl-tRNA synthetase had greater knockdown efficiency, it was used in the entire downstream analysis. Short hairpin RNAs 1- isoleucyl-tRNA synthetase silencing effects on cell proliferation, cell colony generation, cell migration, as well as angiogenesis were assessed using cell counting kit-8, colony development, cell migration, and angiogenesis tube formation assays, respectively. RESULTS: Compared to the control group, anti-isoleucyl-tRNA synthetase short hairpin RNAs significantly silenced isoleucyl-tRNA synthetase expression in human umbilical vein endothelial cells, and suppressed their proliferation, migration, and angiogenic capacity. To characterize the underlying mechanism, western blot analyses showed that isoleucyl-tRNA synthetase knockdown suppressed phosphorylation of extracellular-regulated kinase ½ and protein-serine- threonine kinase, as well as expression of vascular endothelial growth factor, GSK-3β, and β-catenin. CONCLUSIONS: We have shown, for the first time, the critical role of isoleucyl-tRNA synthetase in human umbilical vein endothelial cells. Our data show that isoleucyl-tRNA synthetase knockdown suppresses human umbilical vein endothelial cell proliferation, migration, and angiogenesis. We have also shown that isoleucyl-tRNA synthetase knockdown suppresses phosphorylation of extracellular-regulated kinase ½ and protein-serine- threonine kinase, as well as expression of vascular endothelial growth factor, GSK-3β, and β-catenin. Together, these data highlight isoleucyl-tRNA synthetase as a potential antitumor anti-angiogenic target.

Maple syrup urine disease in Brazilian patients: variants and clinical phenotype heterogeneity.

BACKGROUND: Maple syrup urine disease (MSUD) is an autosomal recessive inherited metabolic disease caused by deficient activity of the branched-chain α-keto acid dehydrogenase (BCKD) enzymatic complex. BCKD is a mitochondrial complex encoded by BCKDHA, BCKDHB, DBT, and DLD genes. MSUD is predominantly caused by Variants in BCKDHA, BCKDHB, and DBT genes encoding the E1α, E1ß, and E2 subunits of BCKD complex, respectively. The aim of this study was to characterize the genetic basis of MSUD by identifying the point variants in BCKDHA, BCKDHB, and DBT genes in a cohort of Brazilian MSUD patients and to describe their phenotypic heterogeneity. It is a descriptive cross-sectional study with 21 MSUD patients involving molecular genotyping by Sanger sequencing. RESULTS: Eight new variants predicted as pathogenic were found between 30 variants (damaging and non-damaging) identified in the 21 patients analyzed: one in the BCKDHA gene (p.Tyr120Ter); five in the BCKDHB gene (p.Gly131Val, p.Glu146Glnfs * 13, p.Phe149Cysfs * 9, p.Cys207Phe, and p.Lys211Asn); and two in the DBT gene (p.Glu148Ter and p.Glu417Val). Seventeen pathogenic variants were previously described and five variants showed no pathogenicity according to in silico analysis. CONCLUSION: Given that most of the patients received late diagnoses, the study results do not allow us to state that the molecular features of MSUD variant phenotypes are predictive of clinical severity.

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)

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.

The effect of bcaa on isometric force following endurance exercise in a hot environment / Efeito dos aacr na força isométrica após exercício de endurance em um ambiente quente / Efecto de los aacr en la fuerza isométrica después de ejercicio de endurance en un ambiente cálido

ABSTRACT Introduction: Fatigue due to endurance exercise results from both peripheral and central changes, and may influence subsequent performance during a strength task. The increase in serotonin concentration is one of the central factors associated with endurance exercise-induced fatigue, particularly in hot environments. A nutritional strategy employed to reduce serotonergic activation is supplementation with branched-chain amino acids (BCAA). Objective: To investigate whether BCAA supplementation attenuates the reduction in isometric force caused by prior endurance exercise in a hot environment. Methods: Nine volunteers (aged 25.4 ± 1.2 years) performed a 2-min maximal voluntary isometric contraction (MVCISO) of upper limb muscles before and after an endurance exercise on a cycle ergometer at 40% of the maximal aerobic power. The volunteers underwent three experimental trials: 1) endurance exercise in a temperate environment (23°C and 60% RH); exercise in a hot environment (35°C and 60% RH) with the ingestion of: 2) a placebo solution or 3) a solution containing BCAA 30 mg.kg−1. During the MVCISO test, the isometric force of flexor muscles of the right elbow, core body temperature (TCORE) and heart rate (HR) were measured. Results: Isometric force decreased following endurance exercise in the hot environment, and BCAA administration did not attenuate this reduction. Greater TCORE and HR values were observed following endurance exercise in the heat, compared to pre-exercise values, and supplementation did not interfere with these physiological responses. Conclusion: The reduction in isometric force, caused by previous endurance exercise in a hot environment, was not diminished by supplementation with BCAA. Level of evidence I; Type of study: Therapeutic studies - Investigation of treatment outcomes.

RESUMO Introdução: A fadiga decorrente de um exercício de endurance ocorre devido a alterações tanto periféricas quanto centrais e pode influenciar no desempenho subsequente durante um teste de força. Sabe-se que o aumento da concentração de serotonina é um dos fatores centrais associados à fadiga induzida pelo exercício de endurance, principalmente em ambientes quentes. Uma estratégia nutricional utilizada para diminuir a ativação serotonérgica é a suplementação com aminoácidos de cadeia ramificada (AACR). Objetivo: Investigar se a suplementação com AACR atenua a redução da força isométrica causada pela realização prévia de um exercício de endurance em ambiente quente. Métodos: Nove voluntários (25,4±1,2 anos) realizaram uma contração voluntária máxima isométrica (CVMISO) de membro superior durante 2 min, antes e após um exercício de endurance em um cicloergômetro a 40% da potência máxima aeróbica. Os voluntários foram submetidos a três situações experimentais: 1) exercício de endurance em ambiente temperado (23°C e 60% URA); exercício em ambiente quente (35°C; 60% URA) com ingestão de: 2) solução placebo ou 3) solução contendo 30 mg.kg−1 de AACR. Durante o teste de CVMISO, a força isométrica dos músculos flexores do cotovelo direito, a temperatura corporal interna (TINT) e a frequência cardíaca (FC) foram medidas. Resultados: A força isométrica diminuiu após o exercício de endurance no ambiente quente e a administração de AACR não atenuou essa redução. Valores maiores de TINT e FC foram observados após o exercício de endurance em ambiente quente em relação aos valores do pré-exercício, sendo que a suplementação também não interferiu nessas respostas fisiológicas. Conclusão: A redução da força isométrica, devido à realização prévia de exercício de endurance em ambiente quente, não foi atenuada pela suplementação com AACR. Nível de evidência I; Tipo de estudo: Estudos terapêuticos - Investigação dos resultados do tratamento.

RESUMEN Introducción: La fatiga derivada de un ejercicio de endurance ocurre debido a las alteraciones tanto periféricas como centrales y puede influir en el desempeño subsiguiente durante un test de fuerza. Se sabe que el aumento de la concentración de serotonina es uno de los factores centrales asociados a la fatiga inducida por el ejercicio de endurance, principalmente en ambientes cálidos. Una estrategia nutricional empleada para disminuir la activación serotonérgica es la suplementación con aminoácidos de cadena ramificada (AACR). Objetivo: Investigar si la suplementación con AACR atenúa la reducción de la fuerza isométrica causada por la realización previa de un ejercicio de endurance en ambiente cálido. Métodos: Nueve voluntarios (25,4 + 1,2 años) realizaron una contracción voluntaria máxima isométrica (CVMISO) de dos minutos de miembro superior, antes y después de un ejercicio de endurance en un cicloergómetro a 40% de la potencia máxima aeróbica. Los voluntarios fueron sometidos a tres situaciones experimentales: 1) ejercicio de endurance en ambiente templado (23° C y 60% HR); ejercicio en ambiente cálido (35° C, 60% HR) con ingestión de: 2) solución placebo o 3) solución conteniendo 30 mg.kg−1 de AACR. Durante el test de CVMISO, se midieron la fuerza isométrica de los músculos flexores del codo derecho, la temperatura corporal interna (TINT) y la frecuencia cardíaca (FC). Resultados: La fuerza isométrica disminuyó después del ejercicio de endurance en el ambiente cálido y la administración de AACR no atenuó esa reducción. Se observaron mayores valores de TINT y FC después del ejercicio de endurance en ambiente cálido con relación a los valores del pre ejercicio, siendo que la suplementación tampoco interfirió en estas respuestas fisiológicas. Conclusión: La reducción de la fuerza isométrica, debido a la realización previa de ejercicio de endurance en ambiente cálido, no fue atenuada por la suplementación con AACR. Nivel de evidencia I; Tipo de estudio: Estudios terapéuticos - Investigación de los resultados del tratamiento.

Isolated branched-chain amino acid intake and muscle protein synthesis in humans: a biochemical review / Consumo isolado de aminoácidos de cadeia ramificada e síntese de proteína muscular em humanos: uma revisão bioquímica

ABSTRACT Alongside a proper diet, ergogenic aids with potential direct and/or indirect physical performance enhancing effects are sought after for improved adaptation to physical training. Nutritional ergogenics include diet composition changes and/or dietary supplementation. Branched-chain amino acids valine, leucine and isoleucine are widely popular among products with ergogenic claims. Their major marketing appeal derives from allegations that branched-chain amino acids intake combined with resistance physical exercise stimulates muscle protein synthesis. Evidence supporting the efficacy of branched-chain amino acids alone for muscle hypertrophy in humans is somewhat equivocal. This brief review describes physiological and biochemical mechanisms underpinning the effects of complete protein source and branched-chain amino acid intake on skeletal muscle growth in the postabsorptive and post-exercise state. Evidence in favor of or against potential anabolic effects of isolated branched-chain amino acid intake on muscle protein synthesis in humans is also examined.

RESUMO No treinamento físico, buscam-se, além de uma dieta adequada, recursos ergogênicos que possam maximizar direta e/ou indiretamente o desempenho físico. Entre as categorias de recursos ergogênicos, o nutricional compreende a modulação da composição dietética e/ou uso de suplementação. A comercialização dos suplementos de aminoácidos de cadeia ramificada valina, leucina e isoleucina possui muita popularidade entre aqueles com alegação ergogênica. O principal marketing está na afirmação de que o consumo isolado de aminoácidos de cadeia ramificada associado ao exercício físico resistido estimula a síntese de proteína muscular. As evidências da eficácia da ingestão isolada de aminoácidos de cadeia ramificada para a hipertrofia muscular em humanos parecem equivocadas. Nesta breve revisão, apresentamos a compreensão fisiológica e bioquímica de como a ingestão de uma fonte completa de proteína e de aminoácidos de cadeia ramificada afeta o crescimento do músculo esquelético no estado pós-absortivo e pós-exercício. Mostramos também as evidências que suportam ou não a afirmação dos potenciais efeitos anabólicos na síntese de proteína muscular dos aminoácidos de cadeia ramificada quando consumidos isoladamente em humanos.

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.

Inborn Errors of Ketone Body Metabolism and Transport: An Update for the Clinic and for Clinical Laboratories

Abstract Major progress occurred in understanding inborn errors of ketone body transport and metabolism between the International Congresses on Inborn Errors of Metabolism in Barcelona (2013) and Rio de Janeiro (2017). These conditions impair either ketogenesis (presenting as episodes of hypoketotic hypoglycemia) or ketolysis (presenting as ketoacidotic episodes); for both groups, immediate intravenous glucose administration is the most critical and (mHGGCS, HMGCS2) effective treatment measure. Ketogenesis Deficiencies: New biomarkers were described for mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHGGCS, HMGCS2) deficiency. New patient series refined clinical knowledge of 3-hydroxy-3-methylglutaryl-CoA lyase (HGGCL, HMGCL) deficiency. Although affected humans have not been described, two animal model phenotypes are pertinent: zebrafish deficient in monocarboxylate transporter 7 (MCT7, slc16a6) (decreased ketone body exit from hepatocytes) or mice lacking D-3-hydroxy-n-butyrate dehydrogenase (BDH1, BDH1) (isolated hyperacetoacetatemia; fatty liver). Ketolysis Deficiencies: Monocarboxylate transporter 1 (MCT1, SLC16A1) deficiency is a newly described defect of ketone body transport, joining deficiencies of succinyl-CoA:3-oxoacid CoA transferase (SCOT, OXCT1) and methylacetoacetyl-CoA thiolase (MAT, ACAT1). Some heterozygotes for MCT1 or SCOT deficiency develop ketoacidosis.

Suppression of Endogenous Glucose Production by Isoleucine and Valine and Impact of Diet Composition.

Leucine has been shown to acutely inhibit hepatic glucose production in rodents by a mechanism requiring its metabolism to acetyl-CoA in the mediobasal hypothalamus (MBH). In the early stages, all branched-chain amino acids (BCAA) are metabolized by a shared set of enzymes to produce a ketoacid, which is later metabolized to acetyl-CoA. Consequently, isoleucine and valine may also modulate glucose metabolism. To examine this possibility we performed intrahypothalamic infusions of isoleucine or valine in rats and assessed whole body glucose kinetics under basal conditions and during euglycemic pancreatic clamps. Furthermore, because high fat diet (HFD) consumption is known to interfere with central glucoregulation, we also asked whether the action of BCAAs was affected by HFD. We fed rats a lard-rich diet for a short interval and examined their response to central leucine. The results showed that both isoleucine and valine individually lowered blood glucose by decreasing liver glucose production. Furthermore, the action of the BCAA leucine was markedly attenuated by HFD feeding. We conclude that all three BCAAs centrally modulate glucose metabolism in the liver and that their action is disrupted by HFD-induced insulin resistance.

Biochemical Characterization of Branched Chain Amino Acids Uptake in Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, it alternates among vertebrate and invertebrate hosts. Metabolic flexibility is a main biochemical characteristic of this parasite, which is able to obtain energy by oxidizing a variety of nutrients that can be transported from the extracellular medium. Moreover, several of these metabolites, more specifically amino acids, have a variety of functions beyond being sources of energy. Branched chain amino acids (BCAA), beyond their role in ATP production, are involved in sterol biosynthesis; for example, leucine is involved as a negative regulator of the parasite differentiation process occurring in the insect midgut. BCAA are essential metabolites in most nonphotosynthetic eukaryotes, including trypanosomes. In view of this, the metabolism of BCAA in T. cruzi depends mainly on their transport into the cell. In this work, we kinetically characterized the BCAA transport in T. cruzi epimastigotes. Our data point to BCAA as being transported by a single saturable transport system able to recognize leucine, isoleucine and valine. In view of this, we used leucine to further characterize this system. The transport increased linearly with temperature from 10 to 45 °C, allowing the calculation of an activation energy of 51.30 kJ/mol. Leucine uptake was an active process depending on ATP production and a H(+) gradient, but not on a Na(+) or K(+) gradient at the cytoplasmic membrane level.

Vitamin D and L-Isoleucine Promote Antimicrobial Peptide hBD-2 Production in Peripheral Blood Mononuclear Cells from Elderly Individuals.

Elderly individuals are susceptible to develop infectious diseases; promoting innate immunity to prevent infections is a key issue. Human ß-defensin-2 (hBD-2) is an antimicrobial peptide with antimicrobial and immunomodulatory properties. L-isoleucine and vitamin D are important molecules that induce hBD-2. The Aim of this study was to determine the use L-isoleucine and Vitamin D to induce hBD-2 in cells from healthy elderly individuals and elderly individuals with recurrent infections. We explored three groups: young adults (n = 20) used as control group, elderly adults (n = 18) and elderly with recurrent infections (n = 11). PBMCs (peripheral blood mononuclear cells) were isolated from the different groups and then were treated with L-isoleucine or vitamin D3. hBD-2 concentration was assessed with a sandwich enzyme Immunosorbent assay by triplicate. Using the vehicle as a mock control. Our results showed that a percentage of the individuals responded to the treatments producing hBD-2 (p < 0.05). These results showed that both molecules induced hBD-2 in elderly individuals and can be potentially used as prophylactic therapy to decrease infection diseases rates in this vulnerable group.

Exome sequencing identifies RDH12 compound heterozygous mutations in a family with severe retinitis pigmentosa.

OBJECTIVE: Retinitis pigmentosa (RP) is the most prevalent type of inherited retinal degeneration and one of the commonest causes of genetically determined visual dysfunction worldwide. To date, approximately 35 genes have been associated with nonsyndromic autosomal recessive RP (arRP), however the small contribution of each gene to the total prevalence of arRP and the lack of a clear genotype-phenotype correlation complicate the genetic analysis in affected patients. Next generation sequencing technologies are powerful and cost-effective methods for detecting causative mutations in both sporadic and familial RP cases. METHODS: A Mexican family with 5 members affected from arRP was studied. All patients underwent a complete ophthalmologic examination. Molecular methods included genome-wide SNP homozygosity mapping, exome sequencing analysis, and Sanger-sequencing confirmation of causal mutations. RESULTS: No regions of shared homozygosity among affected subjects were identified. Exome sequencing in a single patient allowed the detection of two missense mutations in the RDH12 gene: a c.446T>C transition predicting a novel p.L149P substitution, and a c.295C>A transversion predicting a previously reported p.L99I replacement. Sanger sequencing confirmed that all affected subjects carried both RDH12 mutations. CONCLUSIONS: This study adds to the molecular spectrum of RDH12-related retinopathy and offers an additional example of the power of exome sequencing in the diagnosis of recessively inherited retinal degenerations.

Effect of branched-chain amino acids, valine, isoleucine and leucine on the biosythesis of bitespiramycin 4"-O-acylspiramycins

Bitespiramycin, a group of 4"-O-acylated spiramycins with 4"-O-isovalerylspiramycins as the major components, was produced by recombinantspiramycin-producing strain Streptomyces spiramyceticus harboring a 4"-O-acyltransferase gene. The experiment was initially performed in synthetic medium with 0.5 g l-1 Valine, Isoleucine or Leucine feeding at 36 h cultivation. When valine was fed, the biological titer of bitespiramycin was 45.3 percent higher than that of the control group, but the relative content of total isovalerylspiramycin components decreased by 22.5 percent. In the case of ilecine, the biological titer of bitespiramycin and the total isovalerylspiramycins alone were 85 percent and 72.1 percent of the control group, respectively. In contrast, the relative content of other acylated spiramycins increased by 54.41 percent. However, leucine feeding increased the relative content of total isovalerylspiramycins by 41.9 percent while the biological titer of bitespiramycin was nearly equal to that of the control group. The improvement effect of leucine on the biosynthesis of isovalerylspiramycins was further confirmed by feeding of 2.0 g l-1 leucine to the culture with complex medium. After batch feeding with a total amount of 2.0 g l-1 leucine to the culture from 70 h to 90 h, the biological titer of bitespiramycin was almost unreduced, and the final relative content of total isovalerylspiramycins increased from 31.1 percent to 46.9 percent.

Effect of branched-chain amino acids, valine, isoleucine and leucine on the biosythesis of bitespiramycin 4"-O-acylspiramycins.

Bitespiramycin, a group of 4"-O-acylated spiramycins with 4"-O-isovalerylspiramycins as the major components, was produced by recombinant spiramycin-producing strain Streptomyces spiramyceticus harboring a 4"-O-acyltransferase gene. The experiment was initially performed in synthetic medium with 0.5 g l(-1) Valine, Isoleucine or Leucine feeding at 36 h cultivation. When valine was fed, the biological titer of bitespiramycin was 45.3% higher than that of the control group, but the relative content of total isovalerylspiramycin components decreased by 22.5%. In the case of ilecine, the biological titer of bitespiramycin and the total isovalerylspiramycins alone were 85% and 72.1% of the control group, respectively. In contrast, the relative content of other acylated spiramycins increased by 54.41%. However, leucine feeding increased the relative content of total isovalerylspiramycins by 41.9% while the biological titer of bitespiramycin was nearly equal to that of the control group. The improvement effect of leucine on the biosynthesis of isovalerylspiramycins was further confirmed by feeding of 2.0 g l(-1) leucine to the culture with complex medium. After batch feeding with a total amount of 2.0 g l(-1) leucine to the culture from 70 h to 90 h, the biological titer of bitespiramycin was almost unreduced, and the final relative content of total isovalerylspiramycins increased from 31.1% to 46.9%.

Effect of branched-chain amino acids, valine, isoleucine and leucine on the biosythesis of bitespiramycin 4"-O-acylspiramycins

Bitespiramycin, a group of 4"-O-acylated spiramycins with 4"-O-isovalerylspiramycins as the major components, was produced by recombinantspiramycin-producing strain Streptomyces spiramyceticus harboring a 4"-O-acyltransferase gene. The experiment was initially performed in synthetic medium with 0.5 g l-1 Valine, Isoleucine or Leucine feeding at 36 h cultivation. When valine was fed, the biological titer of bitespiramycin was 45.3% higher than that of the control group, but the relative content of total isovalerylspiramycin components decreased by 22.5%. In the case of ilecine, the biological titer of bitespiramycin and the total isovalerylspiramycins alone were 85% and 72.1% of the control group, respectively. In contrast, the relative content of other acylated spiramycins increased by 54.41%. However, leucine feeding increased the relative content of total isovalerylspiramycins by 41.9% while the biological titer of bitespiramycin was nearly equal to that of the control group. The improvement effect of leucine on the biosynthesis of isovalerylspiramycins was further confirmed by feeding of 2.0 g l-1 leucine to the culture with complex medium. After batch feeding with a total amount of 2.0 g l-1 leucine to the culture from 70 h to 90 h, the biological titer of bitespiramycin was almost unreduced, and the final relative content of total isovalerylspiramycins increased from 31.1% to 46.9%.