Carbamoyl Phosphate Research Articles

Comparative structural insight into the unidirectional catalysis of ornithine carbamoyltransferases from Psychrobacter sp. PAMC 21119.

Ornithine carbamoyltransferases (OTCs) are involved in the arginine deiminase (ADI) pathway and in arginine biosynthesis. Two OTCs in a pair are named catalytic OTC (cOTC) and anabolic OTC (aOTC). The cOTC is responsible for catalyzing the third step of the ADI pathway to catabolize citrulline into carbamoyl phosphate (CP), as well as ornithine, and displays CP cooperativity. In contrast, aOTC catalyzes the biosynthesis of citrulline from CP and ornithine in vivo and is thus involved in arginine biosynthesis. Structural and biochemical analyses were employed to investigate the CP cooperativity and unidirectional function of two sequentially similar OTCs (32.4% identity) named Ps_cOTC and Ps_aOTC from Psychrobacter sp. PAMC 21119. Comparison of the trimeric structure of these two OTCs indicated that the 80s loop of Ps_cOTC has a unique conformation that may influence cooperativity by connecting the CP binding site and the center of the trimer. The corresponding 80s loop region of in Ps_aOTC was neither close to the CP binding site nor connected to the trimer center. In addition, results from the thermal shift assay indicate that each OTC prefers the substrate for the unidirectional process. The active site exhibited a blocked binding site for CP in the Ps_cOTC structure, whereas residues at the active site in Ps_aOTC established a binding site to facilitate CP binding. Our data provide novel insights into the unidirectional catalysis of OTCs and cooperativity, which are distinguishable features of two metabolically specialized proteins.

Ornithine aminotransferase and carbamoyl phosphate synthetase 1 involved in ammonia metabolism serve as novel targets for early stages of gastric cancer.

ObjectiveThe sensitivity and specificity of current biomarkers for gastric cancer were insufficient. The aim of the present study was to screen novel biomarkers and determine the diagnostic values of ornithine aminotransferase (OAT) and carbamoyl phosphate synthetase 1 (CPS1) for detecting gastric cancer.MethodsWith stable isotope tags, we labelled an initial discovery group of four paired gastric cancer tissue samples and identified with LC‐ESI‐MS/MS. A validation group of 159 gastric cancer samples and 30 healthy controls were used to validate the candidate targets. GSEA was used to explore the pathways activated in gastric cancer.ResultsFour hundred and thirty one proteins were found differentially expressed in gastric cancer tissues. Of these proteins, OAT and CPS1 were found over‐expressed in gastric cancer patients, with sensitivity of 70.4% (95% CI: 63.3%–77.6%) and specificity of 80.5% (95% CI: 74.3%–86.7%) for ornithine aminotransferase, and with sensitivity of 68.6% (95% CI: 61.3%–75.8%) and specificity of 73% (95% CI: 66%–79.9%) for carbamoyl phosphate synthetase 1. The co‐expression of OAT and CPS1 in gastric cancer tissues has a sensitivity of 81% (95% CI: 73.2%–88.8%) and specificity of 89% (95% CI: 83%–95%). Furthermore, both OAT and CPS1 were overexpressed in patients with local invasion T3 and T4 stages than those in patients with T1 and T2 stages. The co‐expression of OAT and CPS1 was strongly correlated with histological grade I 68% (95% CI: 58.7%–77.3%) and TNM stage I/II 52% (95% CI: 42%–62%). The areas under ROC curves were up to 0.758 for the co‐expression of OAT and CPS1 in gastric cancer. GSEA results showed that two gene sets and 30 gene sets were activated in OAT high‐ and CPS1 high‐expression patients with gastric cancer, respectively.ConclusionsThe present findings indicated a tight correlation between the co‐expression of OAT and CPS1 and the histological grade, local invasion, and TNM stages of gastric cancer. Therefore, OAT and CPS1 might be predictors for gastric cancer invasion and potential targets for anticancer drug design for gastric cancer.

Anesthesia management in living-donor liver transplantation in a patient with carbamoyl phosphate synthetase deficiency: a case report

BackgroundCarbamoyl phosphate synthetase deficiency (CPS1D) is a urea-cycle disorder (UCD). We report successful perioperative management of pediatric living donor liver transplantation (LDLT) in a CPS1D patient.Case presentationA 10-year-old female patient with CPS1D underwent LDLT. Proper administration of dextrose 50% and 60 kcal/kg/day with l-arginine and l-carnitine resulted in the avoidance of intraoperative hyperammonemia induced by hypercatabolism. Serum ammonia level transiently increased to 61 mmol/L in the anhepatic phase and decreased to 44 mmol/L after reperfusion.ConclusionsWe suggest anesthesia management with administration of dextrose to avoid hyperammonemia during LDLT in patients with CPS1D.

O-GlcNAcylation enhances CPS1 catalytic efficiency for ammonia and promotes ureagenesis

Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases.

Microscopic and molecular detection of Babesia bovis and Babesia bigemina in female camel from Al-Diwaniyah province, Iraq

This study identified the etiological pathogens responsible or protozoal-like disease conditions in female camels from Al-Diwaniyah Province, Iraq. For this reason, 125 female camels (one blood sample per animal) that showed signs of weakness and pale mucus membranes were considered for the study. The samples of stained blood smears were explored microscopically and via a polymerase chain reaction (PCR) method that the targeted glutamine-dependent carbamoyl phosphate synthase (CPSII) gene for identifying Babesia bovis and 18S rRNA gene for detecting B. bigemina. The results of the microscopic technique uncovered the occurrence of Babesia spp. in 76 (60.8%) of the examined samples, which encourage the use of PCR to identify the protozoal species. The PCR findings demonstrated that B. bovis and B. bigemina were detected in 8 (8.9%) and 11 (12.22%), respectively, of the positive microscopic samples. The study findings reveal that weakness and paleness of mucus membranes in camel females can be attributed to the presence of infections by blood protozoa, mainly Babesia bovis and Babesia bigemina.

Mechanism of electroacupuncture and herb-partitioned moxibustion on ulcerative colitis animal model: A study based on proteomics.

BACKGROUNDUlcerative colitis (UC) is a chronic, nonspecific intestinal inflammatory disease. Acupuncture and moxibustion is proved effective in treating UC, but the mechanism has not been clarified. Proteomic technology has revealed a variety of biological markers related to immunity and inflammation in UC, which provide new insights and directions for the study of mechanism of acupuncture and moxibustion treatment of UC.AIMTo investigate the mechanism of electroacupuncture (EA) and herb-partitioned moxibustion (HM) on UC rats by using proteomics technology.METHODSMale Sprague-Dawley rats were randomly divided into the normal (N) group, the dextran sulfate sodium (DSS)-induced UC model (M) group, the HM group, and the EA group. UC rat model was prepared with 3% DSS, and HM and EA interventions at the bilateral Tianshu and Qihai acupoints were performed in HM or EA group. Haematoxylin and eosin staining was used for morphological evaluation of colon tissues. Isotope-labeled relative and absolute quantification (iTRAQ) and liquid chromatography-tandem mass spectrometry were performed for proteome analysis of the colon tissues, followed by bioinformatics analysis and protein-protein interaction networks establishment of differentially expressed proteins (DEPs) between groups. Then western blot was used for verification of selected DEPs.RESULTSThe macroscopic colon injury scores and histopathology scores in the HM and EA groups were significantly decreased compared to the rats in the M group (P < 0.01). Compared with the N group, a total of 202 DEPs were identified in the M group, including 111 up-regulated proteins and 91 down-regulated proteins, of which 25 and 15 proteins were reversed after HM and EA interventions, respectively. The DEPs were involved in various biological processes such as biological regulation, immune system progression and in multiple pathways including natural killer cell mediated cytotoxicity, intestinal immune network for immunoglobulin A (IgA) production, and FcγR-mediated phagocytosis. The Kyoto Encyclopedia of Genes and Genomes pathways of DEPs between HM and M groups, EA and M groups both included immune-associated and oxidative phosphorylation. Network analysis revealed that multiple pathways for the DEPs of each group were involved in protein-protein interactions, and the expression of oxidative phosphorylation pathway-related proteins, including ATP synthase subunit g (ATP5L), ATP synthase beta subunit precursor (Atp5f), cytochrome c oxidase subunit 4 isoform 1 (Cox4i1) were down-regulated after HM and EA interventions. Subsequent verification of selected DEPs (Synaptic vesicle glycoprotein 2A; nuclear cap binding protein subunit 1; carbamoyl phosphate synthetase 1; Cox4i1; ATP synthase subunit b, Atp5f1; doublecortin like kinase 3) by western blot confirmed the reliability of the iTRAQ data, HM and EA interventions can significantly down-regulate the expression of oxidative phosphorylation-associated proteins (Cox4i1, Atp5f1) (P < 0.01).CONCLUSIONEA and HM could regulate the expression of ATP5L, Atp5f1, Cox4i1 that associated with oxidative phosphorylation, then might regulate immune-related pathways of intestinal immune network for IgA production, FcγR-mediated phagocytosis, thereby alleviating colonic inflammation of DSS-induced UC rats.

The diagnostic and prognostic value of serum exosome-derived carbamoyl phosphate synthase 1 in HEV-related acute liver failure patients.

Early diagnosis and prognosis evaluation are of great significance to hepatitis E virus(HEV)-related acute liver failure (HEV-ALF) patients. We collected serum samples from 200 health controls (HCs), 200 patients with acute hepatitis E (AHE), and 200 HEV-ALF patients to evaluate serum exosome-derived carbamoyl phosphate synthase 1 (CPS1) levels and determine its diagnostic and prognostic value. The exosome-derived CPS1 levels in the HEV-ALF group were significantly higher than those in the AHE and HCs groups. The AUC of exosome-derived CPS1 to predict the occurrence of HEV-ALF was 0.850 (0.811-0.883). Both logistical regression and orthogonal partial least squares discriminant analysis (OPLS-DA) showed that exosome-derived CPS1 is an independent risk factor for HEV-ALF. The exosome-derived CPS1 levels were positively correlated with organ failureand the outcomes in HEV-ALF patients. The exosome-derived CPS1 levels in the worsening group were significantly higher than those in the fluctuating and the improving groups. The AUC of serum exosome-derived CPS1 to predict 30-day mortality was 0.829 (0.770-0.879), which was significantly greater than that of the Child-Pugh, KCH, and MELD models. The level of serum exosome-derived CPS1 might serve as a promising diagnostic and prognostic biomarker for HEV-ALF patients, which may provide better guidance for the diagnosis, prognosis, and treatment of HEV-ALF patients.

Hyperuricemia induces liver injury by upregulating HIF-1α and inhibiting arginine biosynthesis pathway in mouse liver and human L02 hepatocytes

The molecular mechanisms of uric acid (UA)-induced liver injury has not been clearly elucidated. In this study, we aimed to investigate the effect and action mechanisms of UA in liver injury. We analyzed the damaging effect of UA on mouse liver and L02 cells and subsequently performed metabolomics studies on L02 cells to identify abnormal metabolic pathways. Finally, we verified transcription factors that regulate related metabolic enzymes. UA directly activated the hepatic NLRP3 inflammasome and Bax apoptosis pathway invivo and invitro. Related metabolites in the arginine biosynthesis pathway (or urea cycle), l-arginine and l-argininosuccinate were decreased, and ammonia was increased in UA-stimulated L02 cells, which was mediated by carbamoyl phosphate synthase 1 (CPS1), argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) downregulation. UA upregulated hypoxia inducible factor-1alpha (HIF-1α) invivo and invitro, and HIF-1α inhibition alleviated the UA-induced ASS downregulation and hepatocyte injury. In conclusion, UA upregulates HIF-1α and inhibits urea cycle enzymes (UCEs). This leads to liver injury, with evidence of hepatocyte inflammation, apoptosis and oxidative stress.

214-LB: Activating and Disrupting the Liver–Alpha-Cell Axis Respectively Enhances and Impairs Amino Acid Metabolism and Alpha-Cell Growth

Amino acids control alpha cell secretion and growth, while glucagon stimulates hepatic amino acid uptake and ureagenesis, forming the liver-alpha cell axis. Patients with nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes have increased levels of glucagon and amino acids pointing to a disrupted liver-alpha cell axis. Both glucagon agonism and antagonism are explored for diabetes and NAFLD therapy but their effects on the axis are unclear. Female C57Bl/6JRj mice were treated with a long-acting glucagon analogue (GCGA, NNC9204-0043, Novo Nordisk A/S) or PBS twice daily, or once weekly with a GCGR antibody (GCGR Ab, REGN1193, Regeneron) or control antibody (Ctl. Ab, REGN1945, Regeneron) . After four weeks, total plasma amino acids were decreased by 48% in GCGA mice (P=0.0006) and increased by 285% in GCGR Ab mice (P&amp;lt;0.0001) versus week 0. At week 4, 18 of 19 amino acids were reduced in GCGA mice and increased in GCGR Ab mice. Asparagine changed the most in both groups, with a mean fold change of 0.21 and 7.50 in GCGA and GCGR Ab mice, respectively. Plasma urea index ([urea]/[amino acids]) was increased by 36% in GCGA mice (P=0.055) and decreased by 61% in GCGR Ab mice (P=0.001) . Liver RNA-sequencing revealed opposite effects of GCGA and GCGR Ab on expression of amino acid transporters (e.g. Slc43a) and ureagenesis genes (e.g. Ass1) . Activity of carbamoyl phosphate synthetase 1 was increased by 27% in GCGA versus PBS mice (P=0.007) and decreased by 33% in GCGR Ab mice versus Ctl. Ab (P=0.002) . GCGA lowered pancreas weight and glucagon content by 15% (P=0.004) and 95% (P&amp;lt;0.0001) compared to PBS. Conversely, GCGR Ab mice had a 34% increase in pancreas weight versus Ctl. Ab (P=0.0009) . Thus, chronically increased GCGR signaling leads to hypoaminoacidemia and decreased alpha cell mass, while chronically decreased GCGR signaling leads to hyperaminoacidemia and increased alpha cell mass. These changes must be considered when developing glucagon-based therapeutics. Disclosure E. E. Christensen: None. J. J. Holst: Advisory Panel; Novo Nordisk, Board Member; Antag Therapeutics, Bainan Biotech. N. J. Wewer albrechtsen: Research Support; Mercodia AB, Novo Nordisk, Regeneron Pharmaceuticals Inc., Speaker's Bureau; Merck &amp; Co., Inc., Mercodia AB. K. D. Galsgaard: None. C. D. Johansen: None. S. Trammell: None. A. B. Bomholt: None. J. Hunt: None. T. Kruse: Employee; Novo Nordisk. J. F. Lau: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. T. Grevengoed: None. Funding Nicolai J. Wewer Albrechtsen is supported by NNF Excellence Emerging Investigator Grant ? Endocrinology and Metabolism (Application No. NNF19OC0055001) , EFSD Future Leader Award (NNF21SA0072746) and DFF Sapere Aude (1052-00003B) . Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (grant agreement NNF14CC0001) .

A TRUSTED targeted mass spectrometry assay for pan-herpesvirus protein detection.

SUMMARYThe presence and abundance of viral proteins within host cells are part of the essential signatures of the cellular stages of viral infections. However, methods that can comprehensively detect and quantify these proteins are still limited, particularly for viruses with large protein coding capacity. Here, we design and experimentally validate a mass spectrometry-based Targeted herpesviRUS proTEin Detection (TRUSTED) assay for monitoring human viruses representing the three Herpesviridae subfamilies—herpes simplex virus type 1, human cytomegalovirus (HCMV), and Kaposi sarcoma-associated herpesvirus. We demonstrate assay applicability for (1) capturing the temporal cascades of viral replication, (2) detecting proteins throughout a range of virus concentrations and in in vivo models of infection, (3) assessing the effects of clinical therapeutic agents and sirtuin-modulating compounds, (4) studies using different laboratory and clinical viral strains, and (5) discovering a role for carbamoyl phosphate synthetase 1 in supporting HCMV replication.

Regulation of the urea cycle by CPS1 O-GlcNAcylation in response to dietary restriction and aging.

O-linked N-acetyl-glucosamine glycosylation (O-GlcNAcylation) of intracellular proteins is a dynamic process broadly implicated in age-related disease, yet it remains uncharacterized whether and how O-GlcNAcylation contributes to the natural aging process. O-GlcNAc transferase (OGT) and the opposing enzyme O-GlcNAcase (OGA) control this nutrient-sensing protein modification in cells. Here, we show that global O-GlcNAc levels are increased in multiple tissues of aged mice. In aged liver, carbamoyl phosphate synthetase 1 (CPS1) is among the most heavily O-GlcNAcylated proteins. CPS1 O-GlcNAcylation is reversed by calorie restriction and is sensitive to genetic and pharmacological manipulations of the O-GlcNAc pathway. High glucose stimulates CPS1 O-GlcNAcylation and inhibits CPS1 activity. Liver-specific deletion of OGT potentiates CPS1 activity and renders CPS1 irresponsive to further stimulation by a prolonged fasting. Our results identify CPS1 O-GlcNAcylation as a key nutrient-sensing regulatory step in the urea cycle during aging and dietary restriction, implying a role for mitochondrial O-GlcNAcylation in nutritional regulation of longevity.

Novel Highlight in Malarial Drug Discovery: Aspartate Transcarbamoylase.

Malaria remains one of the most prominent and dangerous tropical diseases. While artemisinin and analogs have been used as first-line drugs for the past decades, due to the high mutational rate and rapid adaptation to the environment of the parasite, it remains urgent to develop new antimalarials. The pyrimidine biosynthesis pathway plays an important role in cell growth and proliferation. Unlike human host cells, the malarial parasite lacks a functional pyrimidine salvage pathway, meaning that RNA and DNA synthesis is highly dependent on the de novo synthesis pathway. Thus, direct or indirect blockage of the pyrimidine biosynthesis pathway can be lethal to the parasite. Aspartate transcarbamoylase (ATCase), catalyzes the second step of the pyrimidine biosynthesis pathway, the condensation of L-aspartate and carbamoyl phosphate to form N-carbamoyl aspartate and inorganic phosphate, and has been demonstrated to be a promising target both for anti-malaria and anti-cancer drug development. This is highlighted by the discovery that at least one of the targets of Torin2 – a potent, yet unselective, antimalarial – is the activity of the parasite transcarbamoylase. Additionally, the recent discovery of an allosteric pocket of the human homology raises the intriguing possibility of species selective ATCase inhibitors. We recently exploited the available crystal structures of the malarial aspartate transcarbamoylase to perform a fragment-based screening to identify hits. In this review, we summarize studies on the structure of Plasmodium falciparum ATCase by focusing on an allosteric pocket that supports the catalytic mechanisms.

Liver injury in non-alcoholic fatty liver disease is associated with urea cycle enzyme dysregulation

The main aim was to evaluate changes in urea cycle enzymes in NAFLD patients and in two preclinical animal models mimicking this entity. Seventeen liver specimens from NAFLD patients were included for immunohistochemistry and gene expression analyses. Three-hundred-and-eighty-two biopsy-proven NAFLD patients were genotyped for rs1047891, a functional variant located in carbamoyl phosphate synthetase-1 (CPS1) gene. Two preclinical models were employed to analyse CPS1 by immunohistochemistry, a choline deficient high-fat diet model (CDA-HFD) and a high fat diet LDLr knockout model (LDLr −/−). A significant downregulation in mRNA was observed in CPS1 and ornithine transcarbamylase (OTC1) in simple steatosis and NASH-fibrosis patients versus controls. Further, age, obesity (BMI > 30 kg/m2), diabetes mellitus and ALT werefound to be risk factors whereas A-allele from CPS1 was a protective factor from liver fibrosis. CPS1 hepatic expression was diminished in parallel with the increase of fibrosis, and its levels reverted up to normality after changing diet in CDA-HFD mice. In conclusion, liver fibrosis and steatosis were associated with a reduction in both gene and protein expression patterns of mitochondrial urea cycle enzymes. A-allele from a variant on CPS1 may protect from fibrosis development. CPS1 expression is restored in a preclinical model when the main trigger of the liver damage disappears.

Influence of Liver Extracellular Matrix in Predicting Drug-Induced Liver Injury: An Alternate Paradigm.

In vitro drug-induced liver injury (DILI) models are promising tools for drug development to predict adverse events during clinical usage. However, the currently available DILI models are not specific or not able to predict the injury accurately. This is believed to be mainly because of failure to conserve the hepatocyte phenotype, lack of longevity, and difficulty in maintaining the tissue-specific microenvironment. In this study, we have assessed the potential of decellularized liver extracellular matrix (DLM) in retaining the hepatic cellular phenotype and functionality in the presence of a tissue-specific microenvironment along with its role in influencing the effect of the drug on hepatic cells. We show that DLM helps maintain the phenotype of the hepatic cell line HepG2, a well-known cell line for secretion of human proteins that is easily available. Also, the DLM enhanced the expression of a metabolic marker carbamoyl phosphate synthetase I (CPS1), a regulator of urea cycle, and bile salt export pump (BSEP), a marker of hepatocyte polarity. We further validated the DLM for its influence on the sensitivity of cells toward different classes of drugs. Interestingly, the coculture model, in the presence of endothelial cells and stellate cells, exhibited a higher sensitivity for both acetaminophen and trovafloxacin, a toxic compound that does not show any toxicity on preclinical screening. Thus, our results demonstrate for the first time that a multicellular combination along with DLM can be a potential and reliable DILI model to screen multiple drugs.

Management of Psychiatric Disorders in Patients with Hepatic and Gastrointestinal Diseases.

Management of Psychiatric Disorders in Patients with Hepatic and Gastrointestinal Diseases.

Unfavorable clinical outcomes in patients with carbamoyl phosphate synthetase 1 deficiency

PurposeCarbamoyl phosphate synthetase 1 (CPS1) deficiency affects the first step of urea cycle and is a severe form of urea cycle disorder (UCD). The severity of hyperammonemic encephalopathy determines the clinical course of UCDs. Here, we describe the genetic and clinical characteristics of CPS1 deficiency in Korea. Patient and methodsThis study included seven patients with CPS1 deficiency genetically confirmed from January 1992 to September 2020. The peak ammonia level during the first crisis, the half time of peak ammonia level, the initial plasma amino acid levels, and neurological outcomes were compared between CPS1 deficiency and two common UCDs (i.e., 17 patients with argininosuccinate synthetase 1 deficiency and 24 patients with ornithine transcarbamylase deficiency). ResultEleven CPS1 mutations were identified, including 10 novel mutations. Eight mutations were missense. Six patients with CPS1 deficiency had neonatal type. The peak ammonia level, initial glutamate level, and accompanying rate of irreversible neurological damages were highest in patients with CPS1 deficiency. The patient with late-onset CPS1 deficiency responded dramatically to N-carbamylglutamate treatment. ConclusionThe clinical manifestations of CPS1 deficiency were the most severe among UCDs. Considering the high proportion of missense mutations, responsiveness to N-carbamylglutamate would be evaluated in a future study.

The first comprehensive, multigene molecular phylogeny for big-headed flies (Diptera: Pipunculidae)

AbstractThe first comprehensive molecular phylogeny of Pipunculidae (Diptera) is inferred from analyses of 6963 bp of DNA sequence data from the following five loci: cytochrome c oxidase subunit I (COI), cytochrome b (Cytb), 12S ribosomal DNA, carbamoyl phosphate synthetase region of CAD (CAD) and alanyl-tRNA synthetase (AATS). The monophyly of Pipunculidae and most known subfamilies, including Chalarinae, Nephrocerinae, Pipunculinae and Protonephrocerinae, are well supported, as are most existing generic concepts. Molecular analysis reveals that Nephrocerinae, either with or without Protonephrocerinae, depending on analytical method, is sister to the rest of the family. Within Pipunculinae, Cephalopsini is synonymized with Pipunculini (synon. nov.) and Eudorylini with Tomosvaryellini (synon. nov.), leaving Pipunculinae with three tribes: Microcephalopsini, Pipunculini and Tomosvaryellini. Jassidophaga is proposed as a synonym of Verrallia (synon. nov.). Beckerias, Cephalops s.s., Cephalosphaera s.s., Neocephalosphaera, Parabeckerias and Semicephalops are all raised to generic status (stat. nov.). Eudorylas fusculus and E. vineti are transferred to Clistoabdominalis (comb. nov.) and Clistoabdominalis ruralis and C. doczkali to Eudorylas (comb. nov.). We provide evidence for four new genera of Pipunculidae, one of which is described herein (Tricosus gen. nov.; Australia: New South Wales, contains two new combinations, Tricosus cyclohirtus and Tricosus anorhaebus).

Ammonia induces changes in carbamoyl phosphate synthetase I and its regulation of glutamine synthesis and urea cycle in yellow catfish Pelteobagrus fulvidraco

Fishes can adapt to certain levels of environmental ammonia in water, but the strategies utilized to defend against ammonia toxicity are not exactly the same. The carbamyl phosphate synthase I (CPS I) plays an important role in the regulation of glutamine synthesis and urea cycle, which are the most common strategies for ammonia detoxification. In this study, CPS I was cloned from the yellow catfish. The full-length cDNAs of the CPS I was 5 034 bp, with open reading frames of 4 461 bp. Primary amino acid sequence alignment of CPS I revealed conserved similarity between the functional domains of the yellow catfish CPS I protein with CPS I proteins of other animals. The mRNA expression of CPS I was significantly up-regulated in liver and kidney tissues after acute ammonia stress. The CPS I RNA interference (RNAi) down-regulated the mRNA expressions of CPS I and ornithine transcarbamylase (OTC), but up-regulated glutamine synthetase (GS) and glutamate dehydrogenase (GDH) expressions in primary culture of liver cell after acute ammonia stress. Similarly, the activity of enzymes related to urea cycle decreased significantly, while the activity of enzymes related to glutamine synthesis increased significantly. The results of RNAi in vitro suggested that when the urea cycle is disturbed, the glutamine synthesis will be activated to cope with ammonia toxicity.

Bactericidal Effect of Pseudomonas oryziphila sp. nov., a Novel Pseudomonas Species Against Xanthomonas oryzae Reduces Disease Severity of Bacterial Leaf Streak of Rice.

Pseudomonas is a diverse genus of Gammaproteobacteria with increasing novel species exhibiting versatile trains including antimicrobial and insecticidal activity, as well as plant growth–promoting, which make them well suited as biocontrol agents of some pathogens. Here we isolated strain 1257 that exhibited strong antagonistic activity against two pathovars of Xanthomonas oryzae, especially X. oryzae pv. oryzicola (Xoc) responsible for the bacterial leaf streak (BLS) in rice. The phylogenetic, genomic, physiological, and biochemical characteristics support that strain 1257 is a representative of a novel Pseudomonas species that is most closely related to the entomopathogenic bacterium Pseudomonas entomophila. We propose to name it Pseudomonas oryziphila sp. nov. Comparative genomics analyses showed that P. oryziphila 1257 possesses most of the central metabolic genes of two closely related strains P. entomophila L48 and Pseudomonas mosselii CFML 90-83, as well as a set of genes encoding the type IV pilus system, suggesting its versatile metabolism and motility properties. Some features, such as insecticidal toxins, phosphate solubilization, indole-3-acetic acid, and phenylacetic acid degradation, were disclosed. Genome-wide random mutagenesis revealed that the non-ribosomal peptide catalyzed by LgrD may be a major active compound of P. oryziphila 1257 against Xoc RS105, as well as the critical role of the carbamoyl phosphate and the pentose phosphate pathway that control the biosynthesis of this target compound. Our findings demonstrate that 1257 could effectively inhibit the growth and migration of Xoc in rice tissue to prevent the BLS disease. To our knowledge, this is the first report of a novel Pseudomonas species that displays a strong antibacterial activity against Xoc. The results suggest that the P. oryziphila strain could be a promising biological control agent for BLS.

Effects of fishmeal replacement with eight protein sources on growth performance, blood biochemistry and stress resistance in Opsariichthys bidens

Nine experimental diets were formulated to study the effects of fishmeal replacement on Opsariichthys bidens. Weight gain rate, protein efficiency rate and apparent digestibility coefficient of fish in fishmeal (FM), maggot meal (MM) and soybean meal (SM) groups were significantly the highest. There was no significant difference in intestinal amylase activity between FM and SM groups. Principal component analysis showed that SM group and wild fish (WF) individual had similar essential amino acid compositions in muscle. The lowest value of serum malondialdehyde and the highest value of total antioxidant capacity were found in SM group, but fish in MM group did the opposite. Fish in MM group had the higher whole-body lipid, serum triglycerides, glucose, complement C3, C4 and immunoglobulin contents and lysozyme activity. After ammonia exposure, cumulative mortality of fish in FM and SM groups was significantly the lowest. Liver carbamyl phosphate synthetase activity of fish in FM group was significantly the highest, but the highest values of glutamine synthetase and glutamate dehydrogenase were found in SM group. This study suggests that fishmeal replacement with soybean meal is acceptable, and muscle amino acid compositions of fish in SM group were more similar to wild individual.