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The urea cycle is the main pathway for the disposal of excess nitrogen. Carbamoylphosphate synthetase 1 (CPS1), the first and rate-limiting enzyme of urea cycle, is activated by N-acetylglutamate (NAG), and thus N-acetylglutamate synthase (NAGS) is an essential part of the urea cycle. Although NAGS deficiency is the rarest urea cycle disorder, it is the only one that can be specifically and effectively treated by a drug, N-carbamylglutamate, a stable structural analogous of NAG that activates CPS1. Here we report an infant with NAGS deficiency who presented with neonatal hyperammonemia. She was found to have a novel homozygous splice-site mutation, c.1097-2A>T, in the NAGS gene. We describe the clinical course of this infant, who had rapid response to N-carbamylglutamate treatment. In addition, we reviewed the clinical and molecular spectra of previously reported individuals with NAGS deficiency, which presents in most cases with neonatal hyperammonemia, and in some cases the presentation is later, with a broad spectrum of ages and manifestations. With this broad later-onset phenotypic spectrum, maintaining a high index of suspicion is needed for the early diagnosis of this treatable disease. Show less

Urea cycle disorders (UCDs) are rare inherited metabolic defects of ammonia detoxification. In about half of patients presenting with a UCD, the first symptoms appear within a few days after birth. These neonatal onset patients generally have a severe defect of urea cycle function and their survival and outcome prognoses are often limited. To understand better the current situation of neonatal onset in UCDs, we have performed a multicentre, retrospective, non-interventional case series study focussing on the most severe UCDs, namely defects of carbamoyl phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), and argininosuccinate synthetase (ASS). Data of 63 patients were collected (27 patients with ASS deficiency, 23 patients with OTC deficiency, and 12 patients with CPS1 deficiency, one patient definite diagnosis not documented). The majority of patients (43/63, 68 %) had an initial ammonia concentration exceeding 500 μmol/L (normal < 100), of which most (26/43, 60.5 %) were also encephalopathic and were treated with hemodialysis. In patients surviving the initial crisis, recurrence of hyperammonemic events within the first 1.5 years of life occurred frequently (mean 3.6 events, range 0-20). Of all patients, 16 (25.4 %) died during or immediately after the neonatal period. We observed in this cohort of neonatal onset UCD patients a high rate of initial life-threatening hyperammonemia and a high risk of recurrence of severe hyperammonemic crises. These corresponded to a high mortality rate during the entire study period (30.2 %) despite the fact that patients were treated in leading European metabolic centers. This underlines the need to critically re-evaluate the current treatment strategies in these patients. Show less

The rat pancreatic progenitor cell line B-13 is of interest for research on drug metabolism and toxicity since the cells trans-differentiate into functional hepatocyte-like cells (B-13/H) when treated with glucocorticoids. In this study we investigated the trans-differentiation and liver-specific functions of B-13/H cells in a three-dimensional (3D) multi-compartment bioreactor, which has already been successfully used for primary liver cell culture. Undifferentiated B-13 cells were inoculated into the bioreactor system and exposed to dexamethasone to promote hepatic trans-differentiation (B-13/HT). In a second approach, pre-differentiated B-13 cells were cultured in bioreactors for 15 days to evaluate the maintenance of liver-typical functions (B-13/HP). During trans-differentiation of B-13 cells into hepatocyte-like cells in the 3D bioreactor system (approach B-13/HT), an increase in glucose metabolism and in liver-specific functions (urea and albumin synthesis; cytochrome P450 [CYP] enzyme activity) was observed, whereas amylase - characteristic for exocrine pancreas and undifferentiated B-13 cells - decreased over time. In bioreactors with pre-differentiated cells (approach B-13/HP), the above liver-specific functions were maintained over the whole culture period. Results were confirmed by gene expression and protein analysis showing increased expression of carbamoyl-phosphate synthase 1 (CPS-1), albumin, CYP2E1, CYP2C11 and CYP3A1 with simultaneous loss of amylase. Immunohistochemical studies showed the formation of 3D structures with expression of liver-specific markers, including albumin, cytokeratin (CK) 18, CCAAT/enhancer-binding protein beta (CEBP-β), CYP2E1 and multidrug resistance protein 2 (MRP2). In conclusion, successful culture and trans-differentiation of B-13 cells in the 3D bioreactor was demonstrated. The requirement for only one hormone and simple culture conditions to generate liver-like cells makes this cell type useful for Show less

Perfluorododecanoic acid (PFDoA) is a ubiquitous environmental pollutant known to cause hepatocellular hypertrophy; however, the mechanisms of hepatotoxicity remain poorly understood. In this study, male rats were exposed to 0, 0.05, 0.2 and 0.5 mg/kg/day of PFDoA for 110 days. After two-dimensional differential gel electrophoresis and MALDI-TOF/TOF analysis, 73 differentially expressed proteins involved in lipid metabolism, inflammation, stress response and other functions were successfully identified. Among them, six significantly changed proteins (CTE1, MTE1, HADHA, ECH1, ALDH2 and CPS1) were found to be regulated by peroxisome proliferator-activated receptor alpha (PPARα). The anti-oxidant enzyme activity assays of superoxide dismutase and glutathione peroxidase and the content of thiobarbituric acid-reactive substances in the liver implied that PFDoA caused oxidative stress. The mRNA levels of PPARα in rat primary hepatocytes were knocked down by lentivirus-mediated RNAi. Furthermore, targeted protein levels of CTE1 and MTE1 were down-regulated, while those of HADHA, ALDH2 and CPS1 were up-regulated. After PFDoA exposure, however, the targeted protein levels of CTE1 and ALDH2 increased compared with those of the knockdown untreated group. The reactive oxygen species (ROS) content in rat hepatocytes assayed by flow cytometry significantly increased in the PPARα knockdown groups, consistent with the PPARα antagonist GW6471- and agonist WY14643-treated groups. These results strongly suggested that PPARα played an important role in suppressing ROS content in hepatocytes following PFDoA exposure. Show less

Circulating blood cell counts and indices are important indicators of hematopoietic function and a number of clinical parameters, such as blood oxygen-carrying capacity, inflammation, and hemostasis. By performing whole-exome sequence association analyses of hematologic quantitative traits in 15,459 community-dwelling individuals, followed by in silico replication in up to 52,024 independent samples, we identified two previously undescribed coding variants associated with lower platelet count: a common missense variant in CPS1 (rs1047891, MAF = 0.33, discovery + replication p = 6.38 × 10(-10)) and a rare synonymous variant in GFI1B (rs150813342, MAF = 0.009, discovery + replication p = 1.79 × 10(-27)). By performing CRISPR/Cas9 genome editing in hematopoietic cell lines and follow-up targeted knockdown experiments in primary human hematopoietic stem and progenitor cells, we demonstrate an alternative splicing mechanism by which the GFI1B rs150813342 variant suppresses formation of a GFI1B isoform that preferentially promotes megakaryocyte differentiation and platelet production. These results demonstrate how unbiased studies of natural variation in blood cell traits can provide insight into the regulation of human hematopoiesis. Show less

The CPS1 gene was identified as a virulence factor in the maize pathogen Cochliobolus heterostrophus Hypothesizing that the homologous gene in Coccidioides posadasii could be important for virulence, we created a Δcps1 deletion mutant which was unable to cause disease in three strains of mice (C57BL/6, BALB/c, or the severely immunodeficient NOD-scid,γc(null) [NSG]). Only a single colony was recovered from 1 of 60 C57BL/6 mice following intranasal infections of up to 4,400 spores. Following administration of very high doses (10,000 to 2.5 × 10(7) spores) to NSG and BALB/c mice, spherules were observed in lung sections at time points from day 3 to day 10 postinfection, but nearly all appeared degraded with infrequent endosporulation. Although the role of CPS1 in virulence is not understood, phenotypic alterations and transcription differences of at least 33 genes in the Δcps1 strain versus C. posadasii is consistent with both metabolic and regulatory functions for the gene. The in vitro phenotype of the Δcps1 strain showed slower growth of mycelia with delayed and lower spore production than C. posadasii, and in vitro spherules were smaller. Vaccination of C57BL/6 or BALB/c mice with live Δcps1 spores either intranasally, intraperitoneally, or subcutaneously resulted in over 95% survival with mean residual lung fungal burdens of <1,000 CFU from an otherwise lethal C. posadasii intranasal infection. Considering its apparently complete attenuation of virulence and the high degree of resistance to C. posadasii infection when used as a vaccine, the Δcps1 strain is a promising vaccine candidate for preventing coccidioidomycosis in humans or other animals. Show less

The origin of carbapenem-hydrolyzing metallo-β-lactamases (MBLs) acquired by clinical bacteria is largely unknown. We investigated the frequency, host range, diversity, and functionality of MBLs in the soil microbiota. Twenty-five soil samples of different types and geographical origins were analyzed by antimicrobial selective culture, followed by phenotypic testing and expression of MBL-encoding genes in Escherichia coli, and whole-genome sequencing of MBL-producing strains was performed. Carbapenemase activity was detected in 29 bacterial isolates from 13 soil samples, leading to identification of seven new MBLs in presumptive Pedobacter roseus (PEDO-1), Pedobacter borealis (PEDO-2), Pedobacter kyungheensis (PEDO-3), Chryseobacterium piscium (CPS-1), Epilithonimonas tenax (ESP-1), Massilia oculi (MSI-1), and Sphingomonas sp. (SPG-1). Carbapenemase production was likely an intrinsic feature in Chryseobacterium and Epilithonimonas, as it occurred in reference strains of different species within these genera. The amino acid identity to MBLs described in clinical bacteria ranged between 40 and 69%. Remarkable features of the new MBLs included prophage integration of the encoding gene (PEDO-1), an unusual amino acid residue at a key position for MBL structure and catalysis (CPS-1), and overlap with a putative OXA β-lactamase (MSI-1). Heterologous expression of PEDO-1, CPS-1, and ESP-1in E. coli significantly increased the MICs of ampicillin, ceftazidime, cefpodoxime, cefoxitin, and meropenem. Our study shows that MBL producers are widespread in soil and include four genera that were previously not known to produce MBLs. The MBLs produced by these bacteria are distantly related to MBLs identified in clinical samples but constitute resistance determinants of clinical relevance if acquired by pathogenic bacteria. Show less

Previous studies suggest that elevated total homocysteine levels and the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, which correlates with plasma total homocysteine levels, are risk factors for schizophrenia (SCZ). Recently, a large genome-wide association study (GWAS) of plasma total homocysteine levels in individuals of European ancestry identified many single-nucleotide polymorphisms (SNPs) (n=13,974). The primary purpose of this study was to examine the association between these plasma total homocysteine-related SNPs and SCZ in the Japanese population. First, we investigated associations between six SNPs and plasma total homocysteine levels in non-psychiatric subjects in the Japanese population (n=1030). Then, we evaluated the cumulative effects of three SNPs on SCZ risk by calculating the Genotype Risk Score (GRS) (1120 cases, 2643 controls). Of the six SNPs examined, we replicated similar associations with the European GWAS at four loci (CENPQ, CPS1, MTHFR, and MUT). GRS based on three SNPs (CENPQ, CPS1, and MTHFR) was significantly associated with SCZ. Our findings suggest that common polygenic variations, which are associated with the plasma total homocysteine levels, may contribute to the risk of SCZ. Show less

Carbamoyl phosphate synthetase 1 (CPS1) deficiency is a rare autosomal recessive disorder of ureagenesis presenting as life-threatening hyperammonemia. In this study, we present the main clinical features and biochemical and molecular data of six Malaysian patients with CPS1 deficiency. All the patients have neonatal-onset symptoms, initially diagnosed as infections before hyperammonemia was recognized. They have typical biochemical findings of hyperglutaminemia, hypocitrullinemia, and low to normal urinary excretion of orotate. One neonate succumbed to the first hyperammonemic decompensation. Five neonatal survivors received long-term treatment consisting of dietary protein restriction and ammonia-scavenging drugs. They have delayed neurocognitive development of varying severity. Genetic analysis revealed eight mutations in CPS1 gene, five of which were not previously reported. Five mutations were missense changes while another three were predicted to create premature stop codons. In silico analyses showed that these new mutations affected different CPS1 enzyme domains and were predicted to interrupt interactions at enzyme active sites, disturb local enzyme conformation, and destabilize assembly of intact enzyme complex. All mutations are private except one mutation; p.Ile1254Phe was found in three unrelated families. Identification of a recurrent p.Ile1254Phe mutation suggests the presence of a common and unique mutation in our population. Our study also expands the mutational spectrum of the CPS1 gene. Show less

Carbamyl phosphate synthetase 1 (CPS1) was down-regulated in hepatocellular carcinoma (HCC), as treated by aflatoxin B1 (AFB1), a potent hepatocarcinogenesis mycotoxin. In this study, we firstly confirmed that AFB1 down-regulated the expression of CPS1 in a dose-dependent manner. At the meantime, both siRNA knock down of CPS1 and AFB1 treatment inhibited cell proliferation, and induced cell apoptosis. To further analysis the function of CPS1, the interacting proteins of CPS1 were searched by Co-IP, and three interacting proteins including type II cytoskeletal 1 (KRT1), albumin (ALB), and ubiquitin C (UBC) were found. Both KRT1 and ALB were new interacting proteins for CPS1. Our further study showed that CPS1 was regulating interacted and colocalized with KRT1 and ALB, and the intensity correlation was changed by AFB1. KRT1, ALB and CPS1 were all reported to play an important role in differentiation and tissue specialization. These results may offer an increasing understand that CPS1 might have a function in differentiation. Show less

Recently, increasing numbers of long noncoding RNAs (lncRNAs), with both oncogenic and tumor-suppressive potential, have been found to be aberrantly expressed in various human cancers. However, the function of lncRNAs in hepatocellular carcinoma (HCC) progression remains largely unknown. In this study, we performed a comprehensive microarray analysis of lncRNA expression using human HCC specimens. After validation in 119 human HCC tissues, we identified a novel tumor suppressor lncRNA, CPS1 intronic transcript 1 (CPS1-IT1). To elucidate the clinical significance of CPS1-IT1 in HCC, correlations between CPS1-IT1 levels, clinical parameters, and survival outcomes were analyzed. In vitro and in vivo functional assays were also performed to dissect the potential underlying mechanisms. Expression of CPS1-IT1 was significantly decreased in 73% of HCC tissues, and patients with low CPS1-IT1 expression had poor survival outcomes. Furthermore, in vitro functional assays indicated that CPS1-IT1 significantly reduced cell proliferation, migration and invasion capacities through reduced Hsp90 binding to and activation of HIF-1α, thereby suppressing the epithelial-mesenchymal transition (EMT). An in vivo animal model also demonstrated the tumor suppressor role of CPS1- IT1 via decreased tumor growth and metastasis. In conclusion, lncRNA CPS1-IT1 acts as a tumor suppressor in HCC by reducing HIF-1α activation and suppressing EMT. The findings of this study establish a function for CPS1-IT1 in HCC progression and suggest its potential as a new prognostic biomarker and target for HCC therapy. Show less

It is well-known that the conversion of normal colon epithelium to adenoma and then to carcinoma stems from acquired molecular changes in the genome. The genetic basis of colorectal cancer has been elucidated to a certain extent, and much remains to be known about the identity of specific cancer genes that are associated with the advancement of colorectal cancer from one stage to the next. Here in this study we attempted to identify novel cancer genes that could underlie the stage-specific progression and metastasis of colorectal cancer. We conducted a stage-based meta-analysis of the voluminous tumor genome-sequencing data and mined using multiple approaches for novel genes driving the progression to stage-II, stage-III and stage-IV colorectal cancer. The consensus of these driver genes seeded the construction of stage-specific networks, which were then analyzed for the centrality of genes, clustering of subnetworks, and enrichment of gene-ontology processes. Our study identified three novel driver genes as hubs for stage-II progression: DYNC1H1, GRIN2A, GRM1. Four novel driver genes were identified as hubs for stage-III progression: IGF1R, CPS1, SPTA1, DSP. Three novel driver genes were identified as hubs for stage-IV progression: GSK3B, GGT1, EIF2B5. We also identified several non-driver genes that appeared to underscore the progression of colorectal cancer. Our study yielded potential diagnostic biomarkers for colorectal cancer as well as novel stage-specific drug targets for rational intervention. Our methodology is extendable to the analysis of other types of cancer to fill the gaps in our knowledge. Show less

The hepatocyte paraffin 1 (Hep Par 1) antibody is widely used as a hepatocyte marker, recognizing carbamoyl phosphate synthetase 1 (CPS1), an essential component of the urea cycle. Various missense, nonsense, and frameshift mutations occur in the CPS1 gene. In neonatal patients with homozygous CPS1 deficiency (CPS1D), urea cycle defects with resulting severe hyperammonemia can be fatal, though liver transplantation provides a complete cure for CPS1D. We performed Hep Par 1 immunostaining in the explanted livers of 10 liver transplant patients with CPS1D. Seven were negative for Hep Par 1 in the hepatocytes and the other three showed normal diffuse granular cytoplasmic staining. As expected, all three Hep Par 1-positive patients had at least one missense mutation, and all four patients who had only nonsense or frameshift mutations were Hep Par 1-negative. The other three patients were unexpectedly negative for Hep Par 1, even though each had one missense mutation. These results suggest that CPS1D can be related to the loss of Hep Par 1 reactivity due to the loss of protein production, a one amino acid substitution resulting in an abortive protein product, or both. Hep Par 1 immunohistochemistry can be used as a simple method to confirm CPS1D. Show less

Cisplatin (Cis) is one of the most commonly used antineoplastic drugs. It is used as chemotherapy for many solid organ malignancies such as brain, neck, male and female urogenital, vesical and pulmonary cancers. Infliximab blocks tumor necrosis factor alpha (TNF-α). Several studies have reported that infliximab ameliorates cell damage by reducing cytokine levels. We aimed to investigate whether infliximab has a preventive effect against cisplatin-induced hepatotoxicity and whether it has a synergistic effect when combined with Cis. Animal experimentation. Male Wistar albino rats were divided in three groups as follows: Cis group, infliximab + Cis (CIN) group and the control group. Each group comprised 10 animals. Animals in the Cis group received an intraperitoneal single-dose injection of Cis (7 mg/kg). In the CIN group, a single dose of infliximab (7 mg/kg) was administered 72 h prior to the Cis injection. After 72 h, a single dose of Cis (7 mg/kg) was administered. All rats were sacrificed five days after Cis injection. TNF-α levels in the Cis group were significantly higher (345.5±40.0 pg/mg protein) than those of the control (278.7±62.1 pg/mg protein, p=0.003) and CIN groups (239.0±64.2 pg/mg protein, p=0.013). The Cis group was found to have high carbonic anhydrase (CA)-II and low carbamoyl phosphate synthetase-1 (CPS-1) levels. Aspartate transaminase (AST) and alanine transaminase (ALT) levels were lower in the CIN group as compared to the Cis group. Total histological damage was greater in the Cis group as compared to the control and CIN groups. Cis may lead to liver damage by increasing cytokine levels. It may increase oxidative stress-induced tissue damage by increasing carbonic anhydrase II (CA-II) enzyme levels and decreasing CPS-1 enzyme levels. Infliximab decreases Cis-induced hepatic damage by blocking TNF-α and it may also protect against liver damage by regulating CPS-1 and CA-II enzyme levels. Show less

Excess ammonia is produced during fasting when amino acids are used for glucogenesis. Together with ureagenesis, glucogenesis occurs in periportal hepatocytes mediated mainly through the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). In vivo experiments showed that fasting strongly stimulated mice glucagon secretion, hepatic PGC-1α, sirtuin 3 (SIRT3) and sirtuin 5 (SIRT5) expression and ureagenesis enzymatic activity such as carbamoyl phosphate synthetase 1 (CPS1) and ornithine transcarbamoylase (OTC). Interestingly, (15)N-labeled urea and (13)C-labeled glucose production in wild-type mice were significantly increased compared with PGC-1α null mice by [(15)N,(13)C]alanine perfused liver. Glucagon significantly stimulated ureagenesis, expression of SIRT3, SIRT5 and the activities of CPS1 and OCT but did not stimulate PGC-1α silencing hepatocytes in mice periportal hepatocytes. Contrarily, PGC-1α overexpression significantly increased the expression of SIRT3, SIRT5 and the activities of CPS1 and OTC, but induced no significant changes in CPS1 and OTC expression. Morever, SIRT3 directly deacetylates and upregulates the activity of OTC, while SIRT5 deacetylates and stimulates the activity of CPS1. During fasting, PGC-1α facilitates ureagenesis in mouse periportal hepatocytes by deacetylating CPS1 and OTC modulated by mitochondrial deacetylase, SIRT3 and SIRT5. This mechanism may be relevant to ammonia detoxification and metabolic homeostasis in liver during fasting. Show less

MicroRNAs, whose transcription is regulated by members of the tumor protein p53 family, modulate the expression of numerous metabolic enzymes, significantly altering tumor cell response to chemotherapeutic treatments. The role for ΔNp63α-regulated microRNAs in regulation of cell cycle arrest, apoptosis and autophagy in squamous cell carcinoma (SCC) cells upon cisplatin exposure has been reported. The current study indicated that the selected microRNA targets differentially regulated by ΔNp63α in cisplatin-sensitive and cisplatin-resistant SCC cells could alter the expression of a few metabolic enzymes, thereby potentially contributing to the metabolic changes in SCC cells upon cisplatin exposure. Finally, the modulation of specific targets (e.g., SREBF2, AKT2, G6PD, CPS1, FADS1, and ETNK1) using a combination of microRNA mimics and siRNA silencing has shown that a suppression of these metabolic factors/ enzymes could confer a sensitivity of SCC cells to cisplatin. Thus, the Δ Np63α-regulated microRNAs were found to regulate the levels of several metabolic factors and enzymes, thereby potentially contributing to the response of larynx and tongue-derived SCC cells to platinum chemotherapy. Show less

Genome-wide association studies with metabolic traits (mGWAS) uncovered many genetic variants that influence human metabolism. These genetically influenced metabotypes (GIMs) contribute to our metabolic individuality, our capacity to respond to environmental challenges, and our susceptibility to specific diseases. While metabolic homeostasis in blood is a well investigated topic in large mGWAS with over 150 known loci, metabolic detoxification through urinary excretion has only been addressed by few small mGWAS with only 11 associated loci so far. Here we report the largest mGWAS to date, combining targeted and non-targeted 1H NMR analysis of urine samples from 3,861 participants of the SHIP-0 cohort and 1,691 subjects of the KORA F4 cohort. We identified and replicated 22 loci with significant associations with urinary traits, 15 of which are new (HIBCH, CPS1, AGXT, XYLB, TKT, ETNPPL, SLC6A19, DMGDH, SLC36A2, GLDC, SLC6A13, ACSM3, SLC5A11, PNMT, SLC13A3). Two-thirds of the urinary loci also have a metabolite association in blood. For all but one of the 6 loci where significant associations target the same metabolite in blood and urine, the genetic effects have the same direction in both fluids. In contrast, for the SLC5A11 locus, we found increased levels of myo-inositol in urine whereas mGWAS in blood reported decreased levels for the same genetic variant. This might indicate less effective re-absorption of myo-inositol in the kidneys of carriers. In summary, our study more than doubles the number of known loci that influence urinary phenotypes. It thus allows novel insights into the relationship between blood homeostasis and its regulation through excretion. The newly discovered loci also include variants previously linked to chronic kidney disease (CPS1, SLC6A13), pulmonary hypertension (CPS1), and ischemic stroke (XYLB). By establishing connections from gene to disease via metabolic traits our results provide novel hypotheses about molecular mechanisms involved in the etiology of diseases. Show less

Metabolite quantitative traits carry great promise for epidemiological studies, and their genetic background has been addressed using Genome-Wide Association Studies (GWAS). Thus far, the role of less common variants has not been exhaustively studied. Here, we set out a GWAS for metabolite quantitative traits in serum, followed by exome sequence analysis to zoom in on putative causal variants in the associated genes. 1H Nuclear Magnetic Resonance (1H-NMR) spectroscopy experiments yielded successful quantification of 42 unique metabolites in 2,482 individuals from The Erasmus Rucphen Family (ERF) study. Heritability of metabolites were estimated by SOLAR. GWAS was performed by linear mixed models, using HapMap imputations. Based on physical vicinity and pathway analyses, candidate genes were screened for coding region variation using exome sequence data. Heritability estimates for metabolites ranged between 10% and 52%. GWAS replicated three known loci in the metabolome wide significance: CPS1 with glycine (P-value  = 1.27×10-32), PRODH with proline (P-value  = 1.11×10-19), SLC16A9 with carnitine level (P-value  = 4.81×10-14) and uncovered a novel association between DMGDH and dimethyl-glycine (P-value  = 1.65×10-19) level. In addition, we found three novel, suggestively significant loci: TNP1 with pyruvate (P-value  = 1.26×10-8), KCNJ16 with 3-hydroxybutyrate (P-value  = 1.65×10-8) and 2p12 locus with valine (P-value  = 3.49×10-8). Exome sequence analysis identified potentially causal coding and regulatory variants located in the genes CPS1, KCNJ2 and PRODH, and revealed allelic heterogeneity for CPS1 and PRODH. Combined GWAS and exome analyses of metabolites detected by high-resolution 1H-NMR is a robust approach to uncover metabolite quantitative trait loci (mQTL), and the likely causative variants in these loci. It is anticipated that insight in the genetics of intermediate phenotypes will provide additional insight into the genetics of complex traits. Show less

To investigate the transdifferentiation relationship between eight types of liver cell during rat liver regeneration (LR). 114 healthy Sprague-Dawley (SD) rats were used in this experimental study. Eight types of liver cell were isolated and purified with percoll density gradient centrifugation and immunomagentic bead methods. Marker genes for eight types of cell were obtained by retrieving the relevant references and databases. Expression changes of markers for each cell of the eight cell types were measured using microarray. The relationships between the expression profiles of marker genes and transdifferentiation among liver cells were analyzed using bioinformatics. Liver cell transdifferentiation was predicted by comparing expression profiles of marker genes in different liver cells. During LR hepatocytes (HCs) not only express hepatic oval cells (HOC) markers (including PROM1, KRT14 and LY6E), but also express biliary epithelial cell (BEC) markers (including KRT7 and KRT19); BECs express both HOC markers (including GABRP, PCNA and THY1) and HC markers such as CPS1, TAT, KRT8 and KRT18; both HC markers (KRT18, KRT8 and WT1) and BEC markers (KRT7 and KRT19) were detected in HOCs. Additionally, some HC markers were also significantly upregulated in hepatic stellate cells ( HSCs), sinusoidal endothelial cells (SECs) , Kupffer cells (KCs) and dendritic cells (DCs), mainly at 6-72 hours post partial hepatectomy (PH). Our findings indicate that there is a mutual transdifferentiation relationship between HC, BEC and HOC during LR, and a tendency for HSCs, SECs, KCs and DCs to transdifferentiate into HCs. Show less

Ammonia detoxification is essential for physiological well-being, and the urea cycle in liver plays a predominant role in ammonia disposal. Nobiletin (NOB), a natural dietary flavonoid, is known to exhibit various physiological efficacies. In the current study, we investigated a potential role of NOB in ammonia control and the underlying cellular mechanism. C57BL/6 mice were fed with regular chow (RC), high-fat (HFD) or high-protein diet (HPD) and treated with either vehicle or NOB. Serum and/or urine levels of ammonia and urea were measured. Liver expression of genes encoding urea cycle enzymes and C/EBP transcription factors was determined over the circadian cycle. Luciferase reporter assays were carried out to investigate function of CCAAT consensus elements on the carbamoyl phosphate synthetase (Cps1) gene promoter. A circadian clock-deficient mouse mutant, Clock (Δ19/Δ19) , was utilized to examine a requisite role of the circadian clock in mediating NOB induction of Cps1. NOB was able to lower serum ammonia levels in mice fed with RC, HFD or HPD. Compared with RC, HFD repressed the mRNA and protein expression of Cps1, encoding the rate-limiting enzyme of the urea cycle. Interestingly, NOB rescued CPS1 protein levels under the HFD condition via induction of the transcription factors C/EBPα and C/EBPβ. Expression of other urea cycle genes was also decreased by HFD relative to RC and again restored by NOB to varying degrees, which, in conjunction with Cps1 promoter reporter analysis, suggested a C/EBP-dependent mechanism for the co-induction of urea cycle genes by NOB. In comparison, HPD markedly increased CPS1 levels relative to RC, yet NOB did not further enrich CPS1 to a significant extent. Using the circadian mouse mutant Clock (Δ19/Δ19) , we also showed that a functional circadian clock, known to modulate C/EBP and CPS1 expression, was required for NOB induction of CPS1 under the HFD condition. NOB, a dietary flavonoid, exhibits a broad activity in ammonia control across varying diets, and regulates urea cycle function via C/EBP-and clock-dependent regulatory mechanisms. Show less

Human carbamoyl phosphate synthetase (CPS1), a 1500-residue multidomain enzyme, catalyzes the first step of ammonia detoxification to urea requiring N-acetyl-L-glutamate (NAG) as essential activator to prevent ammonia/amino acids depletion. Here we present the crystal structures of CPS1 in the absence and in the presence of NAG, clarifying the on/off-switching of the urea cycle by NAG. By binding at the C-terminal domain of CPS1, NAG triggers long-range conformational changes affecting the two distant phosphorylation domains. These changes, concerted with the binding of nucleotides, result in a dramatic remodeling that stabilizes the catalytically competent conformation and the building of the ~35 Å-long tunnel that allows migration of the carbamate intermediate from its site of formation to the second phosphorylation site, where carbamoyl phosphate is produced. These structures allow rationalizing the effects of mutations found in patients with CPS1 deficiency (presenting hyperammonemia, mental retardation and even death), as exemplified here for some mutations. Show less

To improve an asialoglycoprotein receptor (ASGPR)-based enrichment method for detection of circulating tumor cells (CTCs) of hepatocellular carcinoma (HCC). Peripheral blood samples were collected from healthy subjects, patients with HCC or various other cancers, and patients with hepatic lesions or hepatitis. CTCs were enriched from whole blood by extracting CD45-expressing leukocytes with monoclonal antibody coated-beads following density gradient centrifugation. The remaining cells were cytocentrifuged on polylysine-coated slides. Isolated cells were treated by triple immunofluorescence staining with CD45 antibody and a combination of antibodies against ASGPR and carbamoyl phosphate synthetase 1 (CPS1), used as liver-specific markers, and costained with DAPI. The cell slide was imaged and stained tumor cells that met preset criteria were counted. Recovery, sensitivity and specificity of the detection methods were determined and compared by spiking experiments with various types of cultured human tumor cell lines. Expression of ASGPR and CPS1 in cultured tumor cells and tumor tissue specimens was analyzed by flow cytometry and triple immunofluorescence staining, respectively. CD45 depletion of leukocytes resulted in a significantly greater recovery of multiple amounts of spiked HCC cells than the ASGPR(+) selection (Ps < 0.05). The expression rates of either ASGPR or CPS1 were different in various liver cancer cell lines, ranging between 18% and 99% for ASGPR and between 9% and 98% for CPS1. In both human HCC tissues and liver cancer cell lines, there were a few HCC cells that did not stain positive for ASGPR or CPS1. The mixture of monoclonal antibodies against ASGPR and CPS1 identified more HCC cells than either antibody alone. However, these antibodies did not detect any tumor cells in blood samples spiked with the human breast cancer cell line MCF-7 and the human renal cancer cell line A498. ASGPR(+) or/and CPS1(+) CTCs were detected in 29/32 (91%) patients with HCC, but not in patients with any other kind of cancer or any of the other test subjects. Furthermore, the improved method detected a higher CTC count in all patients examined than did the previous method (P = 0.001), and consistently achieved 12%-21% higher sensitivity of CTC detection in all seven HCC patients with more than 40 CTCs. Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells. Show less

N-acetylglutamate synthase (NAGS) catalyzes the production of N-acetylglutamate (NAG) from acetyl-CoA and L-glutamate. In microorganisms and plants, the enzyme functions in the arginine biosynthetic pathway, while in mammals, its major role is to produce the essential co-factor of carbamoyl phosphate synthetase 1 (CPS1) in the urea cycle. Recent work has shown that several different genes encode enzymes that can catalyze NAG formation. A bifunctional enzyme was identified in certain bacteria, which catalyzes both NAGS and N-acetylglutamate kinase (NAGK) activities, the first two steps of the arginine biosynthetic pathway. Interestingly, these bifunctional enzymes have higher sequence similarity to vertebrate NAGS than those of the classical (mono-functional) bacterial NAGS. Solving the structures for both classical bacterial NAGS and bifunctional vertebrate-like NAGS/K has advanced our insight into the regulation and catalytic mechanisms of NAGS, and the evolutionary relationship between the two NAGS groups. Show less

Food consumption with different bioactive compounds could reduce the risk of diabetic complications. This study was designed to evaluate the effect of cooked common beans on differentially expressed genes in whole kidney homogenates of streptozotocin-induced diabetic rats. After 4weeks of treatment with a cooked bean supplemented (10%) diet, animals fed with Flor de Mayo bean (FMB) exerted the greatest protective effect, since they presented the lowest blood glucose levels, consistent with an increase in blood insulin levels, a decrease in urine albumin and urea levels and an increase in creatinine clearance (P≤.05). Regarding the gene expression of kidneys evaluated using expressed sequence tag, consumption of cooked beans improved the expression of Glu1, Cps1, Ipmk, Cacna1c, Camk1, Pdhb, Ptbp3 and Pim1, which are related to the elimination of ammonium groups, the regulation of inflammatory and oxidative response, as well as cell signaling and apoptosis. In addition, the beneficial effects observed were not related to their polyphenolic and saponin profile, suggesting the activity of other bioactive compounds or the synergistic interaction of these compounds. These results suggest that the consumption of cooked common beans (FMB) might be used as an alternative for the regulation of genes related to renal alterations. Show less

Carbamoyl phosphate synthetase 1 (CPS1) deficiency (CPS1D) is an inborn error of the urea cycle having autosomal (2q34) recessive inheritance that can cause hyperammonemia and neonatal death or mental retardation. We analyzed the effects on CPS1 activity, kinetic parameters and enzyme stability of missense mutations reported in patients with CPS1 deficiency that map in the 20-kDa C-terminal domain of the enzyme. This domain turns on or off the enzyme depending on whether the essential allosteric activator of CPS1, N-acetyl-L-glutamate (NAG), is bound or is not bound to it. To carry out the present studies, we exploited a novel system that allows the expression in vitro and the purification of human CPS1, thus permitting site-directed mutagenesis. These studies have clarified disease causation by individual mutations, identifying functionally important residues, and revealing that a number of mutations decrease the affinity of the enzyme for NAG. Patients with NAG affinity-decreasing mutations might benefit from NAG site saturation therapy with N-carbamyl-L-glutamate (a registered drug, the analog of NAG). Our results, together with additional present and prior site-directed mutagenesis data for other residues mapping in this domain, suggest an NAG-triggered conformational change in the β4-α4 loop of the C-terminal domain of this enzyme. This change might be an early event in the NAG activation process. Molecular dynamics simulations that were restrained according to the observed effects of the mutations are consistent with this hypothesis, providing further backing for this structurally plausible signaling mechanism by which NAG could trigger urea cycle activation via CPS1. Show less

Although there are seven mammalian sirtuins (SIRT1-7), little is known about their expression in the aging brain. To characterize the change(s) in mRNA and protein expression of SIRT1-7 and their associated proteins in the brain of "physiologically" aged Wistar rats. We tested mRNA and protein expression levels of rat SIRT1-7, and the levels of associated proteins in the brain using RT-PCR and western blotting. Our data shows that SIRT1 expression increases with age, concurrently with increased acetylated p53 levels in all brain regions investigated. SIRT2 and FOXO3a protein levels increased only in the occipital lobe. SIRT3-5 expression declined significantly in the hippocampus and frontal lobe, associated with increases in superoxide and fatty acid oxidation levels, and acetylated CPS-1 protein expression, and a reduction in MnSOD level. While SIRT6 expression declines significantly with age acetylated H3K9 protein expression is increased throughout the brain. SIRT7 and Pol I protein expression increased in the frontal lobe. This study identifies previously unknown roles for sirtuins in regulating cellular homeostasis and healthy aging. Show less

Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs) using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs) were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG), conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5), transporters (SULT2A1, SLC13A5, and SLCO2B1), and urea cycle-related enzymes (ARG1 and CPS1). In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density) in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the necessity of optimizing culture conditions to generate other specific lineage-oriented hiPSCs, allowing for a very simple differentiation. Show less

To explore the role of CD44 in monocyte adhesion to human brain microvascular endothelial cells (HBMECs) and monocyte migration across an in vitro model of blood-brain barrier (BBB) infected by Cryptococcus neoformans (Cn). An in vitro blood-brain barrier model was constructed using a transwell chamber covered with a HBMEC monolayer. The wild-type strain of Cn B4500FO2, TYCC645#32 strain with CPS1 gene deletion and PCIP strain with CPS1 complementation were chosen to infect the monolayer HBMECs. THP-1 cells were added to the upper chamber of transwell, and the relative migration rate was determined by counting the number of the cells entering the lower chambers. The inhibitory effects of anti-CD44 monoclonal antibody and the CD44 inhibitor bikunin were examined on THP-1 binding to and migration across HBMECs. Cn infection of the HBMECs caused markedly enhanced THP-1 cell adhesion and migration across the monolyers (P<0.01) dependent on Cn concentration and exposure time. Addition of anti-CD44 monoclonal antibody and bikunin significantly lowered THP-1 adhesion and migration rates in the BBB model with Cn-infected HBMECs (P<0.01) with a dose dependence of the antibody (within 0-1 µg) and inhibitor (within 0-20 nmol/L). Both THP-1 adhesion rate and migration rate were lowered in the BBB model infected with CPS1 gene-deleted Cn but increased in the model infected with the complemented strain compared with those in the wild-type strain-infected model. In the in vitro BBB model, CD44 expressed on HBMECs may play an essential role in monocyte adhesion to and migration across the BBB. The capsular hyaluronic acid may mediate Cn-induced monocyte adhesion and migration. Show less

Gestational alloimmune liver disease (GALD) produces severe neonatal liver disease that is notable for paucity of hepatocytes, large numbers of parenchymal tubules, and extensive fibrosis. Liver specimens from 19 GALD cases were studied in comparison with 14 infants without liver disease (normal newborn liver; NNL) to better understand the pathophysiology that would produce this characteristic histopathology. GALD liver parenchyma contained large numbers of tubules comprising epithelium expressing KRT7/19, EPCAM, and SOX9, suggesting biliary progenitor status. Quantitative morphometry demonstrated that in GALD, the area density of KRT19+ tubules was 16.4 ± 6.2 versus 2.0 ± 2.6 area% in NNL (P < .0001). Functional hepatocyte mass was markedly reduced in GALD, 16.3 ± 6.2 versus 61.9 ± 11.0 area% of CPS1+ cells in NNL (P < .0001). A strong inverse correlation was established between CPS1+ area density and KRT19+ area density (r(2) = 0.66, P < .0001). Tubules showed active hedgehog signaling as determined by SHH and nuclear GLI2 expression and expressed the profibrogenic cytokine SPP1. SPP1 protein content and SPP1 expression were greater in GALD than NNL (15- and 13-fold respectively; P = .002). GALD liver contained large numbers of activated myofibroblasts and showed greater than 10-fold more fibrosis than NNL. The extent of fibrosis correlated with the area density of KRT19+ tubules (r(2) = 0.387, P = .001). The data support a pathogenic model in which immune injury to fetal hepatocytes provides a stimulus for expansion of parenchymal tubules, which, by way of Hh activation, produce fibrogenic signals leading to vibrant fibrosis. Show less

Pyrrolizidine alkaloids (PAs) are a group of phytotoxins that can induce human liver injury, particularly hepatic sinusoidal obstruction syndrome (HSOS). To date, the molecular targets of PA-induced HSOS are largely unknown. In this study, retrorsine (RTS), a known hepatotoxic PA, was used as a representative PA for proteomic studies. Toxicological assessment demonstrated that 35 mg/kg RTS (designated as RTS-L) caused early lesions of HSOS at 24 h after dosing. A proteomic approach revealed 17 up-regulated and 31 down-regulated proteins in RTS-L-treated rats. Subsequently, bioinformatic analysis suggested that two proteins, carbamoyl-phosphate synthase (CPS1) (p < 0.05) and ATP synthase subunit beta (ATP5B) (p < 0.01) were associated with RTS-L intoxication. Using immunohistochemical staining, we further verified the down-regulation of CPS1 and ATP5B in RTS-L-treated rats. These findings indicated that CPS1 and ATP5B were altered in the RTS-induced early lesions of HSOS in rats, and therefore, these two proteins and their involved pathways might play important roles in the initiation of HSOS. To the best of our knowledge, our study using a proteomic approach combined with conventional toxicological assessment is the first systems toxicology study on PA-induced HSOS. The results of this study provide novel findings on protein profiles in response to PA exposure, which can serve as a starting point to further investigate potential protein targets and their interactions with PAs to induce HSOS. Show less