Argininosuccinate Lyase Research Articles

Variants associated with urea cycle disorders in Japanese patients: Nationwide study and literature review.

Urea cycle disorders (UCDs) are inherited metabolic diseases that lead to hyperammonemia with variable clinical manifestations. Using data from a nationwide study, we investigated the onset time, gene variants, clinical manifestations, and treatment of patients with UCDs in Japan. Of the 229 patients with UCDs diagnosed and/or treated between January 2000 and March 2018, identified gene variants and clinical information were available for 102 patients, including 62 patients with ornithine transcarbamylase (OTC) deficiency, 18 patients with carbamoyl phosphate synthetase 1 (CPS1) deficiency, 16 patients with argininosuccinate synthetase (ASS) deficiency, and 6 patients with argininosuccinate lyase (ASL) deficiency. A total of 13, 10, 4, and 5 variants in the OTC, CPS1, ASS, and ASL genes were respectively identified as novel variants, which were neither registered in ClinVar databases nor previously reported. The onset time and severity in patients with UCD could be predicted based on the identified gene variants in each patient from this nationwide study and previous studies. This genetic information may help in predicting the long-term outcome and determining specific treatment strategies such as liver transplantation in patients with UCDs.

The Effects of Quercetin on the Gene Expression of Arginine Metabolism Key Enzymes in Human Embryonic Kidney 293 Cells

Background: Arginine metabolism is an important factor involved in tumorigenesis, progression, and survival of tumor cells. Besides, other metabolites produced in the arginine metabolism process, such as polyamines, nitric oxide, argininosuccinate, and agmatine, play key roles in different stages of tumor development. On the other hand, herbal metabolites are widely used to treat cancer. One of these herbal flavonoids is quercetin. Methods: In this study, according to MTT assay data, two concentrations of quercetin flavonoid were selected (57.5 and 115 µM) to treat human embryonic kidney 293 (HEK293) cells. Then RNA was extracted from the cells and used as a template for cDNA synthesis. Using real-time PCR, the expression of key enzymes involved in arginine metabolism was evaluated, including arginase 2 (Arg2), ornithine carbamoyl transferase (OTC), agmatinase (AGMAT), arginase 1 (Arg1), nitric oxide synthase 1 (nNOS), arginine decarboxylase (ADC), ornithine decarboxylase 1 (ODC), ornithine carbamoyl transferase (OCT), spermidine synthase (SRM), spermine synthase (SMS), argininosuccinate synthase 1 (ASS1), and argininosuccinate lyase (ASL). The Student t-test was used to analyze the data considering a P value of < 0.05 as the significance level. Results: Our results indicated significant changes in the expression of arginine metabolism enzymes after quercetin exposure, confirming a role for quercetin plant flavonoid in regulating arginine metabolism in HEK293 cells. Conclusions: Quercetin could alter the gene expression of the key enzymes involved in arginine metabolism. This was the first study investigating the effects of quercetin on arginine metabolism in HEK293 cells.

Nitric oxide and bone: The phoenix rises again.

The involvement of nitric oxide (NO) in preventing bone loss has long been hypothesized, but despite decades of research the mechanisms remain obscure. In this issue of the JCI, Jin et al. explored NO deficiency using human cell and mouse models that lacked argininosuccinate lyase (ASL), the enzyme involved in synthesizing arginine and NO production. Osteoblasts that did not express ASL produced less NO and failed to differentiate. Notably, in the context of Asl deficiency, heterozygous deletion of caveolin 1, which normally inhibits NO synthesis, restored NO production, osteoblast differentiation, glycolysis, and bone mass. These experiments suggest that ASL regulates arginine synthesis in osteoblasts, which leads to enhanced NO production and increased glucose metabolism. After a period when research slowed, these studies, like the legendary phoenix, renew the exploration of NO in bone biology, and provide exciting translational potential.

Physical, cognitive, and social status of patients with urea cycle disorders in Japan

Urea cycle disorders (UCDs) are inherited metabolic diseases that lead to hyperammonemia. Severe hyperammonemia adversely affects the brain. Therefore, we conducted a nationwide study between January 2000 and March 2018 to understand the present status of UCD patients in Japan regarding diagnosis, treatments, and outcomes. A total of 229 patients with UCDs (126 patients: ornithine transcarbamylase deficiency [OTCD]; 33: carbamoyl phosphate synthetase 1 deficiency [CPS1D]; 48: argininosuccinate synthetase deficiency [ASSD]; 14: argininosuccinate lyase deficiency [ASLD]; and 8: arginase 1 deficiency [ARG1D]) were enrolled in the present study. Although growth impairment is common in patients with UCDs, we discovered that Japanese patients with UCDs were only slightly shorter than the mean height of the general adult population in Japan. Patients with neonatal-onset UCDs are more likely to experience difficulty finding employment and a spouse; however, some patients with late-onset UCDs were employed and married. Additionally, intellectual and developmental disabilities, such as attention deficit hyperactivity disorder (ADHD) and autism, hinder patients with UCDs from achieving a healthy social life. Moreover, we identified that it is vital for patients with UCDs presenting with mild to moderate intellectual disabilities to receive social support. Therefore, we believe the more robust social support system for patients with UCDs may enable them to actively participate in society.

Effects of Thermal Conditioning on Changes in Hepatic and Muscular Tissue Associated With Reduced Heat Production and Body Temperature in Young Chickens.

Effects of increased summer temperatures on poultry production are becoming more pronounced due to global warming, so it is important to consider approaches that might reduce heat stress in chickens. Thermal conditioning in chickens in the neonatal period can improve thermotolerance and reduce body temperature increases when birds are exposed to high ambient temperature later in life. The objective of this study was to investigate physiological and molecular changes associated with heat production and hence body temperature regulation under high ambient temperatures in thermally conditioned chicks. Three-day-old broiler chicks (Chunky) were thermally conditioned by exposure to a high ambient temperature (40°C) for 12 h while control chicks were kept at 30°C. Four days after the treatment, both groups were exposed to 40°C for 15 or 90 min. The increase in rectal temperature during 90 min of exposure to a high ambient temperature was less in thermally conditioned than control chicks. At 15-min of re-exposure treatment, gene expression for uncoupling protein and carnitine palmitoyletransferase 1, key molecules in thermogenesis and fatty acid oxidation, were significantly higher in pectoral muscle of control chicks but not conditioned chicks. Hepatic argininosuccinate synthase (ASS) decreased and hepatic argininosuccinate lyase (ASL) increased after reexposure to a high temperature. The concentrations of hepatic arginosuccinic acid, and ASS and ASL expression, were upregulated in conditioned chicks compared with the control chicks, indicating activity of the urea cycle could be enhanced to trap more energy to reduce heat production in conditioned chicks. These results suggest thermal conditioning can reduce the increase in heat production in muscles of chickens that occurs in high ambient temperatures to promote sensible heat loss. Conditioning may also promote energy trapping process in the liver by altering the heat production system, resulting in an alleviation of the excessive rise of body temperature.

Beclin‐1‐mediated activation of autophagy improves proximal and distal urea cycle disorders

Urea cycle disorders (UCD) are inherited defects in clearance of waste nitrogen with high morbidity and mortality. Novel and more effective therapies for UCD are needed. Studies in mice with constitutive activation of autophagy unravelled Beclin‐1 as druggable candidate for therapy of hyperammonemia. Next, we investigated efficacy of cell‐penetrating autophagy‐inducing Tat‐Beclin‐1 (TB‐1) peptide for therapy of the two most common UCD, namely ornithine transcarbamylase (OTC) and argininosuccinate lyase (ASL) deficiencies. TB‐1 reduced urinary orotic acid and improved survival under protein‐rich diet in spf‐ash mice, a model of OTC deficiency (proximal UCD). In AslNeo/Neo mice, a model of ASL deficiency (distal UCD), TB‐1 increased ureagenesis, reduced argininosuccinate, and improved survival. Moreover, it alleviated hepatocellular injury and decreased both cytoplasmic and nuclear glycogen accumulation in AslNeo/Neo mice. In conclusion, Beclin‐1‐dependent activation of autophagy improved biochemical and clinical phenotypes of proximal and distal defects of the urea cycle.

Arginine recycling in endothelial cells is regulated BY HSP90 and the ubiquitin proteasome system

Despite the saturating concentrations of intracellular l-arginine, nitric oxide (NO) production in endothelial cells (EC) can be stimulated by exogenous arginine. This phenomenon, termed the “arginine paradox” led to the discovery of an arginine recycling pathway in which l-citrulline is recycled to l-arginine by utilizing two important urea cycle enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Prior work has shown that ASL is present in a NO synthetic complex containing hsp90 and endothelial NO synthase (eNOS). However, it is unclear whether hsp90 forms functional complexes with ASS and ASL and if it is involved regulating their activity. Thus, elucidating the role of hsp90 in the arginine recycling pathway was the goal of this study. Our data indicate that both ASS and ASL are chaperoned by hsp90. Inhibiting hsp90 activity with geldanamycin (GA), decreased the activity of both ASS and ASL and decreased cellular l-arginine levels in bovine aortic endothelial cells (BAEC). hsp90 inhibition led to a time-dependent decrease in ASS and ASL protein, despite no changes in mRNA levels. We further linked this protein loss to a proteasome dependent degradation of ASS and ASL via the E3 ubiquitin ligase, C-terminus of Hsc70-interacting protein (CHIP) and the heat shock protein, hsp70. Transient over-expression of CHIP was sufficient to stimulate ASS and ASL degradation while the over-expression of CHIP mutant proteins identified both TPR- and U-box-domain as essential for ASS and ASL degradation. This study provides a novel insight into the molecular regulation l-arginine recycling in EC and implicates the proteasome pathway as a possible therapeutic target to stimulate NO signaling.

The effects of NO on the urea cycle pathway in short-term intermittent hypobaric hypoxia in rats

Short-term hypoxic states can influence the health and life activities of lowlanders who travel shortly to high altitudes, in transitory situations, such as surgical ischemia-reperfusion (to one or several organs), and in some sporting activities, such as parachuting and extreme skiing, mountain rescue teams, regular commercial flight crews, in which the subject may not even notice the hypoxia. NO is an integral part of the human physiological response to hypoxia. Until recently, the urea cycle (UC) was only considered as an important mechanism for neutralizing ammonia. We are the first to reveal an interrelation in hypoxic states between the activities of NO-synthase and UC enzymes in male rats' liver, kidney and brain. In the presented work, we have shown that during short-term intermittent hypobaric hypoxia (IHH) all enzymes of UC play an important role in the maintenance of NO quantity. The results allow thinking that kidney and brain argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) and liver ASS and ASL can be different isoenzymes. It is worth mentioning that the results have revealed new sides of l-arginine metabolism in a hypoxic state in male rats.

Proteomic profiling of biomarkers by MALDI-TOF mass spectrometry for the diagnosis of tracheobronchial stenosis after tracheobronchial tuberculosis.

Tracheobronchial tuberculosis (TB) leads to airway stenosis, irreversible airway damage and even death. The present study aimed to identify biomarkers for the diagnosis of tracheobronchial stenosis (TBS) secondary to tracheobronchial TB. A cohort was recruited, including patients with TBS after tracheobronchial TB, TBS after tracheal intubation or tracheotomy (TIT) and no stenosis of early-stage lung cancer,. Proteomic profiling was performed to gain insight into the mechanisms of the pathological processes. Differentially expressed proteins in the serum and bronchial alveolar lavage fluid (BALF) from patients were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Subsequently, ELISA was performed to validate the changes of protein levels in an additional cohort. MALDI-TOF MS revealed that 8 peptides in the serum, including myeloid-associated differentiation marker, keratin type I cytoskeletal 18, fibrinogen α-chain, angiotensinogen (AGT), apolipoprotein A-I (APOAI), clusterin and two uncharacterized peptides, and nine peptides in BALF, including argininosuccinate lyase, APOAI, AGT and five uncharacterized peptides, were differentially expressed (molecular-weight range, 1,000-10,000 Da) in the TB group compared with the TIT group. The ELISA results indicated that the changes in the protein levels had a similar trend as those identified by proteomic profiling. In conclusion, the present study identified proteins that may serve as potential biomarkers and provide novel insight into the molecular mechanisms underlying TBS after tracheobronchial TB.

Use of Auxotrophic Markers for Targeted Gene Insertions in Trichoderma reesei.

In this chapter, we describe a routinely used strategy for targeted gene insertions in Trichoderma reesei using auxotrophic markers. Generally, targeted gene integrations are advantageous over random, ectopic integration, because the copy number and locus of integration are controlled, abolishing the risk of pleiotropic effects. The use of auxotrophic markers allows a direct, cheap, and easy method for selection. The first step is the construction of recipient strains in a NHEJ-deficient strain. We routinely use deletion strains of pyr4, encoding for the orotidine 5'-phosphate decarboxylase (EC 4.1.1.23) and/or asl1, encoding for the argininosuccinate lyase (EC 4.3.2.1). In the second step, the gene of interest is inserted together with the marker gene. Here we describe the necessary strategy for the construction of the recipient strains and insertion constructs, a PEG-mediated transformation protocol, and a protocol for genetic confirmation of the gene insertion.

Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells.

With our recent success in developing a recombinant human arginase drug against broad-spectrum cancer cell lines, we have explored the potential of a recombinant Bacillus caldovelox arginase mutant (BCA-M) for human cervical cancer treatment. Our studies demonstrated that BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) expression. Drug susceptibilities correlate well with the expressions of major urea cycle genes and completeness of L-arginine regeneration pathways. With the expressions of ASS and ASL genes conferring resistance to L-arginine deiminase (ADI) which is undergoing Phase III clinical trial, BCA-M offers the advantage of a broader spectrum of susceptible cancer cells. Mechanistic studies showed that BCA-M inhibited the growth of human cervical cancer cells by inducing apoptosis and cell cycle arrest at S and/or G2/M phases. Our results also displayed that autophagy served as a protective mechanism, while the growth inhibitory effects of BCA-M could be enhanced synergistically by its combination to the autophagy inhibitor, chloroquine (CQ), on human cervical cancer cells.

Enzymes Involved in the Biosynthesis of Arginine from Ornithine in Maritime Pine (Pinus pinaster Ait.).

The amino acids arginine and ornithine are the precursors of a wide range of nitrogenous compounds in all living organisms. The metabolic conversion of ornithine into arginine is catalyzed by the sequential activities of the enzymes ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASSY) and argininosuccinate lyase (ASL). Because of their roles in the urea cycle, these enzymes have been purified and extensively studied in a variety of animal models. However, the available information about their molecular characteristics, kinetic and regulatory properties is relatively limited in plants. In conifers, arginine plays a crucial role as a main constituent of N-rich storage proteins in seeds and serves as the main source of nitrogen for the germinating embryo. In this work, recombinant PpOTC, PpASSY and PpASL enzymes from maritime pine (Pinus pinaster Ait.) were produced in Escherichia coli to enable study of their molecular and kinetics properties. The results reported here provide a molecular basis for the regulation of arginine and ornithine metabolism at the enzymatic level, suggesting that the reaction catalyzed by OTC is a regulatory target in the homeostasis of ornithine pools that can be either used for the biosynthesis of arginine in plastids or other nitrogenous compounds in the cytosol.

Multi-omics analysis reveals that co-exposure to phthalates and metals disturbs urea cycle and choline metabolism

This study aimed to evaluate the response of HepaRG cells after co-exposure to phthalates and heavy metals, using a high-dimensional biology paradigm (HDB). Liver is the main metabolism site for the majority of xenobiotics. For this reason, the HepaRG cell line was used as an in vitro model, and cells were exposed to two characteristic mixtures of phthalates and heavy metals containing phthalates (DEHP, DiNP, BBzP) and metals (lead, methylmercury, total mercury) in a concentration-dependent manner. The applied chemical mixtures were selected as the most abundant pollutants in the REPRO_PL and PHIME cohorts, which were studied using the exposome-wide approach in the frame of the EU project HEALS. These studies investigated the environmental causation of neurodevelopmental disorders in neonates and across Europe. The INTEGRA computational platform was used for the calculation of the effective concentrations of the chemicals in the liver through extrapolation from human biomonitoring data and this dose (and a ten-times higher one) was applied to the hepatocyte model. Multi-omics analysis was performed to reveal the genes, proteins, and metabolites affected by the exposure to these chemical mixtures. By extension, we could detect the perturbed metabolic pathways. The generated data were analyzed using advanced bioinformatic tools following the HEALS connectivity paradigm for multi-omics pathway analysis. Co-mapped transcriptomics and proteomics data showed that co-exposure to phthalates and heavy metals leads to perturbations of the urea cycle due to differential expression levels of arginase-1 and -2, argininosuccinate synthase, carbamoyl-phosphate synthase, ornithine carbamoyltransferase, and argininosuccinate lyase. Joint pathway analysis of proteomics and metabolomics data revealed that the detected proteins and metabolites, choline phosphate cytidylyltransferase A, phospholipase D3, group XIIA secretory phospholipase A2, α-phosphatidylcholine, and the a 1,2-diacyl-sn-glycero-3-phosphocholine, are responsible for the homeostasis of the metabolic pathways phosphatidylcholine biosynthesis I, and phospholipases metabolism. The urea, phosphatidylcholine biosynthesis I and phospholipase metabolic pathways are of particular interest since they have been identified also in human samples from the REPRO_PL and PHIME cohorts using untargeted metabolomics analysis and have been associated with impaired psychomotor development in children at the age of two. In conclusion, this study provides the mechanistic evidence that co-exposure to phthalates and metals disturb biochemical processes related to mitochondrial respiration during critical developmental stages, which are clinically linked to neurodevelopmental perturbations.

Determination of amino acid profile for argininosuccinic aciduria disorder using High-Performance Liquid Chromatography with fluorescence detection.

Argininosuccinic aciduria is an autosomal, recessive amino acid disorder that is caused by a deficiency of the argininosuccinate lyase enzyme. Citrulline is the most significant marker to detect this disorder. We used the High-performance liquid chromatography with fluorescence detection with 450 nm emission and 330 nm excitation wavelengths, 15 mmol/L potassium dihydrogen phosphate and 5 mmol/L dipotassium hydrogen phosphate as Mobile Phase A, and 50 mL water, 250 mL acetonitrile, and 200 mL methanol as Mobile Phase B in gradient mode with flow rate of 1.2 mL/min. The citrulline concentration was 22 µmol/L in healthy infants and 220 µmol/L in infants suffering from the disorder.

Molecular networks of insulin signaling and amino acid metabolism in subcutaneous adipose tissue are altered by body condition in periparturient Holstein cows

Peripartal cows mobilize not only body fat but also body protein to satisfy their energy requirements. The objective of this study was to determine the effect of prepartum BCS on blood biomarkers related to energy and nitrogen metabolism, and mRNA and protein abundance associated with AA metabolism and insulin signaling in subcutaneous adipose tissue (SAT) in peripartal cows. Twenty-two multiparous Holstein cows were retrospectively classified into a high BCS (HBCS; n = 11, BCS ≥ 3.5) or normal BCS (NBCS; n = 11, BCS ≤ 3.17) group at d 28 before expected parturition. Cows were fed the same diet as a total mixed ration before parturition and were fed the same lactation diet postpartum. Blood samples collected at -10, 7, 15, and 30 d relative to parturition were used for analyses of biomarkers associated with energy and nitrogen metabolism. Biopsies of SAT harvested at -15, 7, and 30 d relative to parturition were used for mRNA (real time-PCR) and protein abundance (Western blotting) assays. Data were subjected to ANOVA using the MIXED procedure of SAS (v. 9.4; SAS Institute Inc., Cary, NC), with P ≤ 0.05 being the threshold for significance. Cows in HBCS had greater overall plasma nonesterified fatty acid concentrations, due to marked increases at 7 and 15 d postpartum. This response was similar (BCS × Day effect) to protein abundance of phosphorylated (p) protein kinase B (p-AKT), the insulin-induced glucose transporter (SLC2A4), and the sodium-coupled neutral AA transporter (SLC38A1). Abundance of these proteins was lower at -15 d compared with NBCS cows, and either increased (SLC2A4, SLC38A1) or did not change (p-AKT) at 7 d postpartum in HBCS. Unlike protein abundance, however, overall mRNA abundances of the high-affinity cationic (SLC7A1), proton-coupled (SLC36A1), and sodium-coupled amino acid transporters (SLC38A2) were greater in HBCS than NBCS cows, due to upregulation in the postpartum phase. Those responses were similar to protein abundance of p-mTOR, which increased (BCS × Day effect) at 7 d in HBCS compared with NBCS cows. mRNA abundance of argininosuccinate lyase (ASL) and arginase 1 (ARG1) also was greater overall in HBCS cows. Together, these responses suggested impaired insulin signaling, coupled with greater postpartum AA transport rate and urea cycle activity in SAT of HBCS cows. An in vitro study using adipocyte and macrophage cocultures stimulated with various concentrations of fatty acids could provide some insights into the role of immune cells in modulating adipose tissue immunometabolic status, including insulin resistance and AA metabolism.

Clinical and Biochemical Profiles of Filipino Patients with Distal Urea Cycle Disorders Detected by Abnormal Expanded Newborn Screening

Objective. The study is a retrospective review which provides preliminary data on the correlation between biochemical profiles and initial clinical manifestation of patients diagnosed to have argininosuccinate synthetase deficiency (ASSD) and argininosuccinate lyase deficiency (ASLD) detected by expanded newborn screening (ENBS).Methods. This is a study of five distal UCD patients initially detected by elevated citrulline on ENBS. Medical charts of the patients were reviewed. The initial clinical manifestations of the patients were correlated with results of biochemical tests.Results. There were four cases of ASLD and one case of ASSD reviewed in this study. All cases of ASLD were confirmed by the presence of argininosuccinic acid (ASA) in the urine metabolic screen (UMS). The plasma citrulline level of the ASSD patient is significantly elevated as compared to the ASLD patients (2,690 µmol/L; NV: 10-45 µmol/L). The ASSD patient and one ASLD patient were symptomatic within the first six days of life. Both presented with significantly elevated plasma ammonia, citrulline and glutamine levels. Three ASLD patients were asymptomatic on initial screening.Conclusion. ENBS has shown importance in the early detection and management of ASSD and ASLD. Early initiation of management may prevent hyperammonemic crises. Long term outcome studies are needed to look into the correlation of neurodevelopmental outcome with lifelong accumulation of citrulline and glutamine in ASSD and ASA in ASLD.

Molecular docking studies of natural compounds of naringin on enzymes involved in the urea cycle pathway in hyperammonemia

Purpose: To investigate the anti-hyperammonemic activity of naringin by molecular docking via in silico studies.Methods: Urea cycle proteins were docked to the natural compound naringin as well as a standard drug, sodium benzoate. Hydrogen bonds and binding energy were obtained using Catalytic Site Atlas and Cast P Finder Software Tool.Results: There were six urea cycle enzymes, including N-acetyl glutamate synthase, carbamoyl phosphate synthase I, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase I. On evaluating protein interactions with naringin, which is dynamically connected to the urea cycle pathway with hyperammonemia, naringin showed more hydrogen bonds and also produced higher binding energy when compared to the standard drug, sodium benzoate.Conclusion: The results of the molecular docking study show that naringin interacts with urea cycle enzymes with more hydrogen bonds and higher bonding energy than the standard drug, sodium benzoate. This supports the hypothesis that naringin can prevent experimental hyperammonemia.
 Keywords: Naringin, Sodium benzoate, Hyperammonemia, Urea cycle enzymes, In silico studies

Lactic acid biosynthesis pathways and important genes of Lactobacillus panis L7 isolated from the Chinese liquor brewing microbiome

Lactic acid is a major organic acid and precursor of ethyl lactate, which confers the important flavor of Chinese Maotai-flavor liquor. Moreover, lactic acid helps modulate the microbiome pH, which coordinates the microbial community. Lactobacillus panis L7 is the major lactic acid producer, however, the whole genome sequence is not available and the important genes for lactic acid biosynthesis have not been unidentified, which limits understanding of lactic acid biosynthesis. Whole genome sequencing of L. panis L7 was undertaken, obtaining 127 genome sequence scaffolds with an N50 of 33,743 bp. The genome total length was 2,026,099 bp encoding 1932 genes with a GC content of 47.0%. Heterolactic acid fermentation pathways and the involved genes were identified, including multiple copies of lactate dehydrogenase encoding genes. To identify important genes for lactic acid production, L. panis L7 was genetically perturbed using atmospheric and room temperature plasma followed by high-throughput screening of mutants with improved or reduced lactic acid production. Comparative genomic analysis of L. panis L7 and its mutants identified genes encoding for glutamate racemase, methylated-DNA-protein-cysteine methyltransferase, fumarate reductase flavoprotein subunit, and amidophosphoribosyltransferase as potentially important factors to improve lactic acid biosynthesis; genes encoding for ribokinase, fructokinase, argininosuccinate lyase, and large subunit ribosomal protein L30 as potentially important factors for reduced lactic acid biosynthesis. The results improved the understanding of the mechanism of lactic acid metabolism by L. panis for the benefit of the Chinese liquor brewing industry, and will, hopefully, help develop effective measures to modulate lactic acid content during brewing.

The kidney urea cycle is perturbed by gadolinium‐based contrast agent treatment

IntroductionGadolinium‐based contrast agents (GBCAs) are used extensively in magnetic resonance imaging. GBCA administration causes systemic fibrosis and long‐term retention of gadolinium in every vital (and non‐vital) organ, including the brain. Gadolinium‐based contrast agents are metabolic disruptors. GBCA administration leads to hyperlipidemia, insulin resistance, and renal disease on par of that induced by high fat diet‐induced experimental obesity (Do C et al, Toxicol Appl Pharmacol 2019 Jul 15;375: 32–45). Gadolinium‐based contrast agents induce glycolytic switching in renal cortex similar to that induced by a high fat diet. The urea cycle is requisite for urinary concentration in the mammal and for elimination of nitrogenous waste. Because high fat diet can also alter the urea cycle, we investigated the impact of gadolinium‐based contrast agent treatment on the urea cycle using metabolomic data.MethodsWild‐type C57/BL6 mice were randomized by age and weight into either a 60% saturated fat diet (20% protein, 20% carbohydrate) ad libitum (n = 20) or control chow (n = 20) for 20 weeks. At Week 17 groups were then sub‐randomized into untreated (n = 10) or GBCA‐treated (n = 10) (Omniscan, 2.5mmol/kg, intraperitoneally) subgroups for the remaining 4 weeks. Kidneys were isolated from mice and then snap frozen in liquid nitrogen. Metabolites were profiled by liquid chromatography‐mass spectrometry (Metabolon, Morrisville, NC). Comparisons were performed by two‐way ANOVA with post‐hoc Tukey HSD.ResultsGBCA treatment (P ≤ 0.05) reduced citrulline relative to the non‐treated group. GBCA treatment decreased aspartate relative to no treatment (P ≤ 0.001). Although high fat diet increased renal aspartate, the effect was reduced by the combination with gadolinium‐based contrast agent. Arginosuccinate was reduced by high fat diet with respect to the control (with similar trends for gadolinium treatment and the combination). Fumarate (also a tricarboxylic acid cycle metabolite) was greatly reduced by high fat diet and the combination with gadolinium‐based contrast agents (P < 0.0015 and P < 0.05, respectively). Although there was a trend for gadolinium to reduce urea levels relative to control, this effect of gadolinium was reversed in the setting of high fat diet (P < 0.005).ConclusionThese studies have been instrumental in defining the metabolic impact of GBCAs. Our results imply that high fat diet activates argininosuccinate lyase (thereby increasing L‐arginine). Gadolinium‐induced suppression of citrulline is either suppression of arginosuccinate synthetase or an increase of ornithine transcarbamoylase. The fact that gadolinium‐based contrast agents impact the same metabolic pathway as an experimental high fat diet suggest that there are physiologic disruptions (potentially pathologic) that will be aggravated by the combination, and that metabolic syndrome is a risk factor for gadolinium‐induced disease.Support or Funding InformationThe research was funded by the Veterans Administration Merit Award (I01 BX001958, BW); the National Institutes of Health Grant R01 DK‐102085 (BW); and the DCI/Kidney Institute of New Mexico.

Neonatal factors related to survival and intellectual and developmental outcome of patients with early-onset urea cycle disorders

PurposeWe aimed to identify prognostic factors for survival and long-term intellectual and developmental outcome in neonatal patients with early-onset urea cycle disorders (UCD) experiencing hyperammonaemic coma. MethodsWe retrospectively analysed ammonia (NH3) and glutamine levels, electroencephalogram and brain images obtained during neonatal coma of UCD patients born between 1995 and 2011 and managed at a single centre and correlated them to survival and intellectual and developmental outcome. ResultsWe included 38 neonates suffering from deficiencies of argininosuccinate synthetase (ASSD, N = 12), ornithine transcarbamylase (OTCD, N = 10), carbamoylphosphate synthetase 1 (CPSD, N = 7), argininosuccinate lyase (ASLD, N = 7), N-acetylglutamate synthase (NAGS, N = 1) or arginase (ARGD, N = 1). Symptoms occurred earlier in mitochondrial than in cytosolic UCD. Sixty-eight percent of patients survived, with a mean (standard deviation-SD) follow-up of 10.4 (5.3) years. Mortality was mostly observed in OTCD (N = 7/10) and CPSD (N = 4/7) patients. Plasma NH3 level during the neonatal period, expressed as area under the curve, but not glutamine level was associated with mortality (p = .044 and p = .610). 62.1% of the patients had normal intellectual and developmental outcome. Intellectual and developmental outcome tended to correlate with UCD subtype (p = .052). No difference in plasma NH3 or glutamine level during the neonatal period among developmental outcomes was identified. EEG severity was linked to UCD subtypes (p = .004), ammonia levels (p = .037), duration of coma (p = .043), and mortality during the neonatal period (p = .020). Status epilepticus was recorded in 6 patients, 3 of whom died neonatally, 1 developed a severe intellectual disability while the 2 last patients had a normal development. ConclusionUCD subtypes differed by survival rate, intellectual and developmental outcome and EEG features in the neonatal period. Hyperammonaemia expressed as area under the curve was associated with survival but not with intellectual and developmental outcome whereas glutamine was not associated with one of these outcomes. Prognostic value of video-EEG monitoring and the association between status epilepticus and mortality should be assessed in neonatal hyperammonaemic coma in further studies.