Leucine Metabolism Research Articles

Newborn screening for isovaleric acidemia in Quanzhou, China

BackgroundIsovaleric acidemia (IVA) is a rare autosomal recessive disorder of leucine metabolism caused by a defective isovaleryl-CoA dehydrogenase (IVD) gene. Reports of IVA diagnoses following newborn screening (NBS) in the Chinese population are few. MethodsWe investigated the biochemical, clinical, and molecular profiles of 5 patients with IVA in China. The estimated incidence of IVA in Quanzhou, China is 1 in 1:84,469. ResultsInitial NBS revealed mild to markedly increased isovalerylcarnitine (C5) concentrations in all 5 patients, and differential diagnosis revealed increased urinary isovaleryglycine concentrations in 2 patients. One patient presented with acute neonatal symptoms, whereas the other 4 remained asymptomatic. Eight distinct IVD gene variants were identified. The most common variant was c.1208A > G (p.Y403C), with an allele frequency of 30%. Five variants were previously unreported, namely, c.499A > G (p.M167V), c.640A > G (p.T214A), c.740G > A (p.G247E), c.832G > C (p.V278L), and c.1195G > C (p.D399H). Different in silico prediction analyses suggested that these previously unreported missense variants are pathogenic. Protein modelling analyses also showed that these missense variants may cause structural damage and dysfunction in IVD. ConclusionsPatients with IVA may have C5 concentrations approaching the cut-off values, highlighting the need for stringent recall criteria and second-tier tests to improve screening performance.

Abstract B50: HnRNP A3 regulates the nuclear EGFR localization and cancer progression

Abstract Aberrant subcellular translocation and distribution of epidermal growth factor receptor (EGFR) represents a major yet underappreciated cancer development mechanism in non-small cell lung cancer (NSCLC). In this study, we investigated the subcellular interactome of EGFR by free labeling approaches combined with LC-MS/MS, aiming to understand the associated protein networks involved in the tumorigenesis of NSCLC. A total of 54, 77, and 63 EGFR-interacting proteins were identified specifically in the cytosol, mitochondria, and nucleus fractions, respectively. Pathway-wise analyses of these proteins using the KEGG database revealed that the proteins interacting with cytosolic and nuclear EGFR proteins are involved in pathways of ribosome and spliceosome, respectively. The EGFR-interacting proteins in mitochondria are involved in the metabolism of propanoate, fatty acid, valine, leucine, and isoleucine. One of the nuclear EGFR-interacting proteins, hnRNP A3, was shown in this work to modulate the accumulation of nuclear EGFR. Downregulation of hnRNP A3 reduced the nuclear EGFR accumulation and was accompanied with reduced tumor growth ability both in vitro and in vivo. These results suggest that variations in the subcellular translocation and distribution of EGFR within NSCLC cells could affect tumor progression. Citation Format: Chi-Yuan Chen, Yu-Han Lin, Tong-Hong Wang, Chih-Ching Wu. HnRNP A3 regulates the nuclear EGFR localization and cancer progression [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B50.

Leucine and branched-chain amino acid metabolism contribute to the growth of bone sarcomas by regulating AMPK and mTORC1 signaling.

Osteosarcoma and chondrosarcoma are sarcomas of the bone and the cartilage that are primarily treated by surgical intervention combined with high toxicity chemotherapy. In search of alternative metabolic approaches to address the challenges in treating bone sarcomas, we assessed the growth dependence of these cancers on leucine, one of the branched-chain amino acids (BCAAs), and BCAA metabolism. Tumor biopsies from bone sarcoma patients revealed differential expression of BCAA metabolic enzymes. The cytosolic branched-chain aminotransferase (BCATc) that is commonly overexpressed in cancer cells, was down-regulated in chondrosarcoma (SW1353) in contrast with osteosarcoma (143B) cells that expressed both BCATc and its mitochondrial isoform BCATm. Treating SW1353 cells with gabapentin, a selective inhibitor of BCATc, further revealed that these cells failed to respond to gabapentin. Application of the structural analog of leucine, N-acetyl-leucine amide (NALA) to disrupt leucine uptake, indicated that all bone sarcoma cells used leucine to support their energy metabolism and biosynthetic demands. This was evident from the increased activity of the energy sensor AMP-activated protein kinase (AMPK), down-regulation of complex 1 of the mammalian target of rapamycin (mTORC1), and reduced cell viability in response to NALA. The observed changes were most profound in the 143B cells, which appeared highly dependent on cytosolic and mitochondrial BCAA metabolism. This study thus demonstrates that bone sarcomas rely on leucine and BCAA metabolism for energy and growth; however, the differential expression of BCAA enzymes and the presence of other carbon sources may dictate how efficiently these cancer cells take advantage of BCAA metabolism.

Leucine Supplementation: A Novel Strategy for Modulating Lipid Metabolism and Energy Homeostasis.

Lipid metabolism is an important and complex biochemical process involved in the storage of energy and maintenance of normal biological functions. Leucine, a branched amino acid, has anti-obesity effects on glucose tolerance, lipid metabolism, and insulin sensitivity. Leucine also modulates mitochondrial dysfunction, representing a new strategy to target aging, neurodegenerative disease, obesity, diabetes, and cardiovascular disease. Although various studies have been carried out, much uncertainty still exists and further studies are required to fully elucidate the relationship between leucine and lipid metabolism. This review offers an up-to-date report on leucine, as key roles in both lipid metabolism and energy homeostasis in vivo and in vitro by acceleration of fatty acid oxidation, lipolysis, activation of the adenosine 5′-monophosphate-activated protein kinase (AMPK)–silent information regulator of transcription 1 (SIRT1)–proliferator-activated receptor γ coactivator-1α (PGC-1α) pathway, synthesis, and/or secretion of adipokines and stability of the gut microbiota.

Energy metabolism profile of the effects of amino acid treatment on skeletal muscle cells: Leucine inhibits glycolysis of myotubes.

Amino acids are not only components of proteins, but also can be metabolized to energy substances or be used as signaling molecules. However, basic knowledge of the relationship between amino acid treatment and energy metabolism is still insufficient. The aims of this study was to profile the effects of essential amino acid and alanine treatment on the energy metabolism of both myoblasts and myotubes and to contribute to the understanding of the basic relationship between amino acid treatment and energy metabolism of skeletal muscle cell. We profiled whether amino acid (essential amino acids and alanine) treatment can affect the energy metabolism (glycolysis, mitochondrial respiration) of cultured skeletal muscle cells. C2C12 myoblasts and differentiated myotubes were treated with 5 mM each amino acid for 1 h, then the energy metabolism was measured by using extracellular flux analyzer. Although not all of the amino acid treatments could affect the energy metabolism of C2C12 myoblasts, leucine, isoleucine, lysine, phenylalanine, and histidine decreased the extracellular acidification rate, an indirect indicator of glycolysis, in differentiated myotubes without alteration of oxygen consumption rate, an indirect indicator of mitochondrial respiration. By glycolysis stress test, we found that leucine treatment inhibited glycolysis of myotubes when the substrate of glycolysis is sufficient in cultured media. The inhibitory effect of glycolysis by leucine was not canceled by rapamycin (an inhibitor for mTOR). But, 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (an inhibitor for branched-chain α ketoacid dehydrogenase complex kinase) increased branched-chain amino acid catabolism, which decreased the glycolysis of myotubes. Findings from the present study complemented the basic knowledge of amino acid treatment on the energy metabolism of cultured skeletal muscle cells and suggested the inhibitory effects of glycolysis by branched-chain amino acid catabolism.

Effect of chemical ingredients B and C in Kansui stir-baked with vinegar on fecal metabolomics in rats

To reveal the toxic mechanism of Kansui stir-baked with vinegar(VEK), conducta comparative study on the metabolites of fecal samples of rats before and after being treated with chemical constituents group B and C(VEKB/VEKC) extracted from VEK by metabolomics approach. The fecal samples of each group were analyzed using ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry(UFLC-Q-TOF-MS). Then the data was processed by principal component analysis(PCA) and partial least square discriminant analysis(OPLS-DA) to screen and identify biomarkers relating to the toxicity of VEK. Besides, t-test was adopted for univariate statistical analysis, so as to study the changes of these biomarkers in drug groups before and after being treated with VEKB/VEKC and explore the effect of VEKB/VEKC on the metabolism of rat feces. Furthermore, the toxic mechanism of VEKB/VEKC was explored based on the results of the metabolic pathway analysis. The results displayed that compared with control group, the metabolism of fecal samples of VEKB and VEKC treated groups show obvious changes, and the VEKB treated group show more significant changes. A total of 16 potential biomarkers and 5 metabolic pathways relating to the toxicity of VEK were found and identified. And the toxicity of VEK might be associated with the disorder of such metabolic pathways as tryptophan metabolism, primary bile acid biosynthesis, amino sugar and nucleotide sugar metabolism, purine metabolism, and degradation of valine, leucine and isoleucine. This study provides a scientific basis for the clinical safety application of VEK.

Unexpected differences in the pharmacokinetics of N-acetyl-DL-leucine enantiomers after oral dosing and their clinical relevance.

The enantiomers of many chiral drugs not only exhibit different pharmacological effects in regard to targets that dictate therapeutic and toxic effects, but are also handled differently in the body due to pharmacokinetic effects. We investigated the pharmacokinetics of the enantiomers of N-acetyl-leucine after administration of the racemate (N-acetyl-DL-leucine) or purified, pharmacologically active L-enantiomer (N-acetyl-L-leucine). The results suggest that during chronic administration of the racemate, the D-enantiomer would accumulate, which could have negative effects. Compounds were administered orally to mice. Plasma and tissue samples were collected at predetermined time points (0.25 to 8 h), quantified with liquid chromatography/mass spectrometry, and pharmacokinetic constants were calculated using a noncompartmental model. When administered as the racemate, both the maximum plasma concentration (Cmax) and the area under the plasma drug concentration over time curve (AUC) were much greater for the D-enantiomer relative to the L-enantiomer. When administered as the L-enantiomer, the dose proportionality was greater than unity compared to the racemate, suggesting saturable processes affecting uptake and/or metabolism. Elimination (ke and T1/2) was similar for both enantiomers. These results are most readily explained by inhibition of uptake at an intestinal carrier of the L-enantiomer by the D-enantiomer, and by first-pass metabolism of the L-, but not D-enantiomer, likely by deacetylation. In brain and muscle, N-acetyl-L-leucine levels were lower than N-acetyl-D-leucine, consistent with rapid conversion into L-leucine and utilization by normal leucine metabolism. In summary, the enantiomers of N-acetyl-leucine exhibit large, unexpected differences in pharmacokinetics due to both unique handling and/or inhibition of uptake and metabolism of the L-enantiomer by the D-enantiomer. Taken together, these results have clinical implications supporting the use of N-acetyl-L-leucine instead of the racemate or N-acetyl-D-leucine, and support the research and development of only N-acetyl-L-leucine.

PM2.5 exposure perturbs lung microbiome and its metabolic profile in mice

Fine particulate matter (PM2.5) have become a major public health concern because of their adverse effects on health. Lungs are considered the primary organ affected by PM2.5. In order to understand the mechanism underlying PM2.5-induced lung injury, 16S rRNA gene sequencing, and liquid chromatography-mass spectrometry (LC-MS) metabolomics analysis were conducted to investigate the impact of PM2.5 exposure on lung microbiome and its metabolic profile. Mice were exposed to PM2.5 through intratracheal instillation and a lung injury model was established. 16S rRNA gene sequencing indicated that PM2.5 exposure significantly altered the richness, evenness, and composition of the lung microbiome. Metabolomics profiling showed that the levels of lung metabolites were perturbed after PM2.5 exposure. The altered metabolites mainly belonged to metabolic pathways, such as the citrate cycle, glyoxylate and dicarboxylate metabolism, pyruvate metabolism, purine and pyrimidine metabolism, and valine, leucine, and isoleucine metabolism. The altered lung microbiota showed significant correlations with lung metabolites. The levels of fumaric acid negatively correlated with the relative abundance of Ruminococcaceae, Enterobacteriaceae, and Pseudomonadaceae. These results revealed that PM2.5 exposure not only significantly altered the lung microbiome composition but also perturbed a number of metabolites involved in diverse metabolic pathways. This study improves our understanding of the mechanism of lung injury after PM2.5 exposure.

Abstract P1-06-03: Breast cancers exhibit diverse metabolic cross-feeding behaviors associated with differing long-term survival

Abstract Introduction Intrinsic breast cancer subtypes (e.g., Normal-like, Luminal A/B, HER2-enriched, Triple Negative) vary in their responsiveness to neoadjuvant chemotherapy (NACT) and are associated with differing long-term prognosis. Expression dis-regulation of critical regulatory pathways partially accounts for variance in cancer phenotype responsiveness which has allowed development of targeted therapies to improve prognosis for some subtypes. Expression dis-regulation also plays a role in regulating cancer metabolism—the Warburg Effect being the most notable example. Metabolic dis-regulation opens avenues for cancer cells to exploit environmental niches arising in the tumor microenvironment (TME) (e.g., lactate cross-feeding). Focusing on the role TME plays in defining cancer behavior, we undertook a theoretical investigation to uncover how metabolic cross-feeding affects subtype behavior. Methods An in silico analysis of cross-feeding variability arising from metabolic differences in Luminal A/B (LA, LB), HER2-enriched (HER2+) and Triple Negative (TN) breast cancers was performed. Community models of the TME were created by coupling metabolic models of adipocytes, glandular, and cancer tissues derived from a comprehensive reconstruction of human metabolism (Pornputtapong, Nookaew, Jensen, Database, 2015). A parsimonious flux balance analysis technique was used to elucidate the consumption, production, and cross-feeding of metabolites by the three tissues (Lewis et al. Mol. Syst. Biol. 2010). Constraints were applied to the model to limit the uptake of 701 metabolites to levels measured in the blood (Wishart et al., Nucleic Acids Res. 2018). In total, 1222 community models were generated by personalizing cancer models using expression levels of metabolic enzyme transcripts for breast cancer patients in the TCGA BRCA study (The Cancer Genome Atlas Network, Nature, 2019). Results Previously characterized gene expression features were recapitulated via metabolic consumption/production in the models: TNBC/HER2+ had higher asparagine, glutamine (Glu), tryptophan, phenylalanine, consumption than LA/LB (van Geldermolsen et al. Oncogone 2016), Glu was cross-fed from cancer to fat in TNBC (Cao et al. BMC Cancer, 2014), TNBC produced methionine and proline (Kanaan et al. Can. Gen. Prot. 2014), and TNBC/HER2+ cancers consumed high density lipoprotein produced by cancer (Balabum et al. Cancer Met. 2017). The community models also revealed novel findings. TNBC/HER2+ relied on glucose as the primary energy source, while LA/LB relied on amino acids alanine, glycine and Glu. TNBC/HER2+ produced high levels of lactate which was consumed by adipose tissues. Uniquely, glycine produced by cancer was consumed by fat in TNBC. Several environmental niches were induced in the healthy tissue by the presence of the cancer; for example, ornithine was predicted to be cross-fed from fatty to glandular tissues in TNBC/HER2+ cancers, and glutamate and inosine from glandular to fatty tissues in HER2+ cancers. To identify metabolic differences that could explain variability in survival, hierarchical clustering was performed resulting in 13 distinct metabolic categories. As one illustrative example, two clusters enriched in LA/LB cancers had different 10-year survival rates. The poorer prognosis cluster lacked valine, leucine, isoleucine biosynthesis, and arginine, proline and pyruvate metabolism, suggesting resource starvation. Since many NACT therapies act by killing proliferating cells and pathological complete response is correlated to better prognosis, we posit that such starving tumors will be less susceptible to treatment. Conclusion While preliminary, these results suggest that metabolic pathway usage can lead to difference in growth, cross-feeding and potentially drug efficacy. Citation Format: Joseph R Peterson, John A Cole Jr., Tyler M. Earnest, Michael J. Hallock, Tushar Pandey, Katherine Ritchie, Eduardo Braun. Breast cancers exhibit diverse metabolic cross-feeding behaviors associated with differing long-term survival [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-06-03.

Metabolic Profiling of the Cerebrospinal Fluid in Pediatric Epilepsy.

To characterize metabolic profiles within the central nervous system in epilepsy, we performed gas chromatography-tandem mass spectrometry (GC-MS/MS)-based metabolome analysis of the cerebrospinal fluid (CSF) in pediatric patients with and without epilepsy. The CSF samples obtained from 64 patients were analyzed by GC-MS/MS. Multivariate analyses were performed for two age groups, 0-5 years of age and 6-17 years of age, to elucidate the effects of epilepsy and antiepileptic drugs on the metabolites. In patients aged 0-5 years (22 patients with epilepsy, 13 without epilepsy), epilepsy patients had reduced 2-ketoglutaric acid and elevated pyridoxamine and tyrosine. In patients aged 6-17 years (12 with epilepsy, 17 without epilepsy), epilepsy patients had reduced 1,5-anhydroglucitol. Valproic acid was associated with elevated 2-aminobutyric acid, 2-ketoisocaproic acid, 4-hydroxyproline, acetylglycine, methionine, N-acetylserine, and serine. Reduced energy metabolism and alteration of vitamin B6 metabolism may play a role in epilepsy in young children. The roles of 1,5-anhydroglucitol in epilepsy in older children and in levetiracetam and zonisamide treatment remain to be explained. Valproic acid influenced the levels of amino acids and related metabolites involved in the metabolism of serine, methionine, and leucine.

A tRNA synthase in metabolic control

Signal Transduction Leucyl-tRNA synthetase 1 (LARS1), which covalently couples leucine to its cognate transfer RNAs (tRNAs), appears to have broader roles in the control of leucine metabolism. The enzyme also serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which regulates protein synthesis, metabolism, autophagy, and cell growth. Yoon et al. show that in cells deprived of glucose, LARS1 is phosphorylated by Unc-51 like autophagy activating kinase 1 (see the Perspective by Lehman and Abraham). This phosphorylation decreases leucine binding to LARS1 and, in turn, should decrease translation, reduce activation of mTORC1, and perhaps free up leucine for use in the generation of adenosine triphosphate in glucose-starved cells. Science , this issue p. [205][1]; see also p. [146][2] [1]: /lookup/doi/10.1126/science.aau2753 [2]: /lookup/doi/10.1126/science.aba2335

Glucose-dependent control of leucine metabolism by leucyl-tRNA synthetase 1

Despite the importance of glucose and amino acids for energy metabolism, interactions between the two nutrients are not well understood. We provide evidence for a role of leucyl-tRNA synthetase 1 (LARS1) in glucose-dependent control of leucine usage. Upon glucose starvation, LARS1 was phosphorylated by Unc-51 like autophagy activating kinase 1 (ULK1) at the residues crucial for leucine binding. The phosphorylated LARS1 showed decreased leucine binding, which may inhibit protein synthesis and help save energy. Leucine that is not used for anabolic processes may be available for catabolic pathway energy generation. The LARS1-mediated changes in leucine utilization might help support cell survival under glucose deprivation. Thus, depending on glucose availability, LARS1 may help regulate whether leucine is used for protein synthesis or energy production.

Tryptophan, glutamine, leucine, and micronutrient supplementation improves environmental enteropathy in Zambian adults: a randomized controlled trial

ABSTRACTBackgroundEnvironmental enteropathy (EE) refers to villus blunting, reduced absorption, and microbial translocation in children and adults in tropical or deprived residential areas. In previous work we observed an effect of micronutrients on villus height (VH).ObjectiveWe aimed to determine, in a randomized controlled trial, if amino acid (AA) or multiple micronutrient (MM) supplementation can improve intestinal structure or barrier dysfunction in Zambian adults with EE.MethodsAA (tryptophan, leucine, and glutamine) and/or MM supplements were given for 16 wk in a 2 × 2 factorial comparison against placebo. Primary outcomes were changes in VH, in vivo small intestinal barrier dysfunction assessed by confocal laser endomicroscopy (CLE), and mechanistic (or mammalian) target of rapamycin complex 1 (MTORC1) nutrient responsiveness in lamina propria CD4+ lymphocytes.ResultsOver 16 wk AA, but not MM, supplementation increased VH by 16% (34.5 μm) compared with placebo (P = 0.04). Fluorescein leak, measured by CLE, improved only in those allocated to both AA and MM supplementation. No effect was seen on MTORC1 activation, but posttreatment MTORC1 and VH were correlated (ρ = 0.51; P = 0.001), and change in MTORC1 was correlated with change in VH in the placebo group (ρ = 0.63; P = 0.03). In secondary analyses no effect was observed on biomarkers of microbial translocation. Metabolomic analyses suggest alterations in a number of microbial- and host-derived metabolites including the leucine metabolite β-hydroxy-β-methylbutyrate, which was increased by AA supplementation and correlated with VH.ConclusionsIn this phase 2 trial, AA supplementation protected against a decline in VH over the supplementation period, and improved barrier function when combined with micronutrients. Leucine and MTORC1 metabolism may be involved in the mechanism of effect. This trial was registered at www.pactr.org as PACTR201505001104412.

Implications of Gene Inheritance Patterns on the Heterosis of Abdominal Fat Deposition in Chickens.

Heterosis, a phenomenon characterized by the superior performance of hybrid individuals relative to their parents, has been widely utilized in livestock and crop breeding, while the underlying genetic basis remains elusive in chickens. Here, we performed a reciprocal crossing experiment with broiler and layer chickens and conducted RNA sequencing on liver tissues for reciprocal crosses and their parental lines to identify inheritance patterns of gene expression. Our results showed that heterosis of the abdominal fat percentage was 69.28%–154.71% in reciprocal crosses. Over-dominant genes of reciprocal crosses were significantly enriched in three biological pathways, namely, butanoate metabolism, the synthesis and degradation of ketone bodies, and valine, leucine, and isoleucine degradation. Among these shared over-dominant genes, we found that a lipid-related gene, HMGCL, was enriched in these pathways. Furthermore, we validated this gene as over-dominant using qRT-PCR. Although no shared significant pathway was detected in the high-parent dominant genes of reciprocal crosses, high-parent dominant gene expression was the major gene inheritance pattern in reciprocal crosses and we could not exclude the effect of high-parent dominant genes. These findings suggest that non-additive genes play important roles in the heterosis of important traits in chickens and have important implications regarding our understanding of heterosis.

Simulated Weightlessness Perturbs the Intestinal Metabolomic Profile of Rats.

Recently, disorders of intestinal homeostasis in the space environment have been extensively demonstrated. Accumulating evidence have suggested microgravity and simulated weightlessness could induce dysbiosis of intestinal microbiota, which may contribute to the bowel symptoms during spaceflight. However, the specific responses of intestinal metabolome under simulated weightlessness and its relationship with the intestinal microbiome and immune characteristics remain largely unknown. In the current study, 20 adult Sprague-Dawley (SD) rats were randomly divided into the control group and the simulated weightlessness group using a hindlimb unloading model. The metabolomic profiling of cecal contents from eight rats of each group was investigated by gas chromatography-time of flight/mass spectrometry. The significantly different metabolites, biomarkers, and related pathways were identified. Multivariate analysis, such as principal component analysis and orthogonal projections to latent structures-discriminant analysis, demonstrated an obvious separation between the control group and the simulated weightlessness group. Significantly different metabolites, such as xylose, sinapinic acid, indolelactate, and digalacturonic acid, were identified, which participate in mainly pyrimidine metabolism, pentose and glucuronate interconversions, and valine, leucine and isoleucine metabolism. Cytidine-5′-monophosphate, 4-hydroxypyridine, and phloretic acid were determined as pivotal biomarkers under simulated weightlessness. Moreover, the significantly different metabolites were remarkably correlated with dysbiosis of the intestinal microbiota and disturbance of immunological characteristics induced by simulated weightlessness. These metabolic features provide crucial candidates for therapeutic targets for metabolic disorders under weightlessness.

Eight novel mutations detected from eight Chinese patients with isovaleric acidemia

BackgroundIsovaleric acidemia (IVA), a rare autosomal recessive disorder in leucine metabolism caused by defected IVD gene, is characterized by episodes of acute metabolic crisis and psychomotor development retardation. This study aimed to determine the clinical, biochemical, and mutation spectrum of patients with IVA from mainland China. MethodsEight patients (three boys and five girls) from eight unrelated families were collected, IVD gene mutations and phenotypes were examined. ResultsThe patients were admitted because of vomiting, feeding difficulty, psychomotor retardation and “dirty sock” odor. Elevated blood isovaleryl (C5)-carnitine and urine isovalerylglycine were detected from all our patients. Fourteen mutations of the IVD gene were detected, eight of them are novel, c.145C>T (p.Q49Ter), c.359G>A (p.R120Q), c.424C>T (p.R142C), c.458T>C (p.L153P), c.466-1G>T, c.676_677insA (p.T226Nfs*13), c.1039G>A (p.A347T) and c.1076A>G (p.D359G). With this study, a total of 34 alleles were studied in the Chinese population. c.1208A>G (p.Y403C), the common mutation in Taiwan, accounts for 9/34 alleles (7 in previous reports and 2 in this study). ConclusionsWe described eight novel mutations detected from eight unrelated Chinese patients and provided evidence to support that the p.Y403C is the hotspot mutation in this population.

Erwinia amylovora Auxotrophic Mutant Exometabolomics and Virulence on Apples.

The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic E. amylovora mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type E. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents.IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic E. amylovora auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

Comparison of Bacterial Populations in the Ceca of Swine at Two Different Stages and their Functional Annotations.

The microbial composition in the cecum of pig influences host health, immunity, nutrient digestion, and feeding requirements significantly. Advancements in metagenome sequencing technologies such as 16S rRNAs have made it possible to explore cecum microbial population. In this study, we performed a comparative analysis of cecum microbiota of crossbred Korean native pigs at two different growth stages (stage L = 10 weeks, and stage LD = 26 weeks) using 16S rRNA sequencing technology. Our results revealed remarkable differences in microbial composition, α and β diversity, and differential abundance between the two stages. Phylum composition analysis with respect to SILVA132 database showed Firmicutes to be present at 51.87% and 48.76% in stages L and LD, respectively. Similarly, Bacteroidetes were present at 37.28% and 45.98% in L and LD, respectively. The genera Prevotella, Anaerovibrio, Succinivibrio, Megasphaera were differentially enriched in stage L, whereas Clostridium, Terrisporobacter, Rikenellaceae were enriched in stage LD. Functional annotation of microbiome by level-three KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that glycine, serine, threonine, valine, leucine, isoleucine arginine, proline, and tryptophan metabolism were differentially enriched in stage L, whereas alanine, aspartate, glutamate, cysteine, methionine, phenylalanine, tyrosine, and tryptophan biosynthesis metabolism were differentially enriched in stage LD. Through machine-learning approaches such as LEfSe (linear discriminant analysis effect size), random forest, and Pearson’s correlation, we found pathways such as amino acid metabolism, transport systems, and genetic regulation of metabolism are commonly enriched in both stages. Our findings suggest that the bacterial compositions in cecum content of pigs are heavily involved in their nutrient digestion process. This study may help to meet the demand of human food and can play significant roles in medicinal application.

Novel Metabolites Are Associated With Augmentation Index and Pulse Wave Velocity: Findings From the Bogalusa Heart Study.

Metabolomics study may help identify novel mechanisms underlying arterial stiffening. We performed untargeted metabolomics profiling among 1,239 participants of the Bogalusa Heart Study. After quality control, 1,202 metabolites were evaluated for associations with augmentation index (AI) and pulse wave velocity (PWV), using multivariate linear regression adjusting for age, sex, race, education, smoking, drinking, body weight, body height, physical activity, and estimated glomerular filtration rate. Heart rate, blood pressure and antihypertensive medication usage, lipids, and fasting glucose were sequentially adjusted in the sensitivity analyses for significant metabolites. Weighted correlation network analysis was applied to build metabolite networks. Six novel metabolites were negatively associated with AI, of which, 3-methyl-2-oxobutyrate had the lowest P value and the largest effect size (β = -6.67, P = 5.99 × 10-6). Heart rate contributed to a large proportion (25%-58%) of the association for each metabolite. Twenty-one novel metabolites were identified for PWV, of which, fructose (β = 0.61, P = 6.18 × 10-10) was most significant, and histidine had the largest effect size (β = -1.09, P = 2.51 × 10-7). Blood pressure played a major contribution (9%-54%) to the association for each metabolite. Furthermore, 16 metabolites were associated with arterial stiffness independent of traditional risk factors. Network analysis identified 2 modules associated with both AI and PWV (P < 8.00 × 10-4). One was composed of metabolites from the glycerolipids synthesis and recycling pathway, and the other was involved in valine, leucine, and isoleucine metabolism. One module related to sphingomyelin metabolism was associated with PWV only (P = 0.002). This study has identified novel and important metabolites and metabolic networks associated with arterial stiffness.

Isovaleric Acidemia in a 5 Years of Boy: A Case Report

Isovaleric acidemia (IVA) is an autosomal recessive disease of leucine metabolism due to deficiency of isovaleryl-CoA dehydrogenase (IVD). In this case report a five years old boy was admitted to National Institute of Neurosciences and Hospital, Dhaka Bangladesh with the history of fever, cough, vomiting, diarrhea, stupor and extreme sleepiness for 3 days. Second degree consanguinity was documented between the parents. Neurological examination revealed exaggerated reflexes and ankle clonus was present bilaterally. EEG and MRI of brain had normal results. Urine organic acid analysis by gas chromatography-mass spectrometry showed slight increment in concentration of 3 hydroxyisovaleric acid and an elevated concentration of isovalerylglycine. Tandem mass spectrometry of acylcarnitines in dried blood spots showed elevated C5-carnitine isovalerylcarnitine (10.3umol/l). Based on history, clinical examination and laboratory results, a diagnosis of isovaleric academia was ascertained. Patient was treated accordingly with rehydration fluid, correction of metabolic acidosis, antibiotic and supportive care&#x0D; Journal of Current and Advance Medical Research 2019;6(1):64-66