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Fibrinogen has an established, essential role in both coagulation and inflammatory pathways, and these processes are deeply intertwined in the development of thrombotic and atherosclerotic diseases. Previous studies aimed to better understand the (patho) physiological actions of fibrinogen by characterizing the genomic contribution to circulating fibrinogen levels. Establish an in vitro approach to define functional roles between genes within these loci and fibrinogen synthesis. Candidate genes were selected on the basis of their proximity to genetic variants associated with fibrinogen levels and expression in hepatocytes and HepG2 cells. HepG2 cells were transfected with small interfering RNAs targeting candidate genes and cultured in the absence or presence of the proinflammatory cytokine interleukin-6. Effects on fibrinogen protein production, gene expression, and cell growth were assessed by immunoblotting, real-time polymerase chain reaction, and cell counts, respectively. HepG2 cells secreted fibrinogen, and stimulation with interleukin-6 increased fibrinogen production by 3.4 ± 1.2 fold. In the absence of interleukin-6, small interfering RNA knockdown of FGA, IL6R, or EEPD1 decreased fibrinogen production, and knockdown of LEPR, PDIA5, PLEC, SHANK3, or CPS1 increased production. In the presence of interleukin-6, knockdown of FGA, IL6R, or ATXN2L decreased fibrinogen production. Knockdown of FGA, IL6R, EEPD1, LEPR, PDIA5, PLEC, or CPS1 altered transcription of one or more fibrinogen genes. Knocking down ATXN2L suppressed inducible but not basal fibrinogen production via a post-transcriptional mechanism. We established an in vitro platform to define the impact of select gene products on fibrinogen production. Genes identified in our screen may reveal cellular mechanisms that drive fibrinogen production as well as fibrin(ogen)-mediated (patho)physiological mechanisms. Show less

The aim of this study was to investigate the effect of low-protein diets supplemented with rumen-protected lysine (RPLys) and methionine (RPMet) on growth performance, rumen fermentation, blood biochemical parameters, nitrogen metabolism, and gene expression related to N metabolism in the liver of Holstein bulls. Thirty-six healthy and disease-free Holstein bulls with a similar body weight (BW) (424 ± 15 kg, 13 months old) were selected. According to their BW, they were randomly divided into three groups with 12 bulls in each group in a completely randomized design. The control group (D1) was fed with a high-protein basal diet (CP13%), while bulls in two low-protein groups were supplied a diet with 11% crude protein and RPLys 34 g/d·head + RPMet 2 g/d·head (low protein with low RPAA, T2) or RPLys 55 g/d·head + RPMet 9 g/d·head (low protein with high RPAA, T3). At the end of the experiment, the feces and urine of dairy bulls were collected for three consecutive days. Blood and rumen fluid were collected before morning feeding, and liver samples were collected after slaughtering. The results showed that the average daily gain (ADG) of bulls in the T3 group was higher than those in D1 ( Show less

Mitochondrial dysfunction is a well-known contributor to aging and age-related diseases. The precise mechanisms through which mitochondria impact human lifespan, however, remain unclear. We hypothesize that humans with exceptional longevity harbor rare variants in nuclear-encoded mitochondrial genes (mitonuclear genes) that confer resistance against age-related mitochondrial dysfunction. Here we report an integrated functional genomics study to identify rare functional variants in ~ 660 mitonuclear candidate genes discovered by target capture sequencing analysis of 496 centenarians and 572 controls of Ashkenazi Jewish descent. We identify and prioritize longevity-associated variants, genes, and mitochondrial pathways that are enriched with rare variants. We provide functional gene variants such as those in MTOR (Y2396Lfs*29), CPS1 (T1406N), and MFN2 (G548*) as well as LRPPRC (S1378G) that is predicted to affect mitochondrial translation. Taken together, our results suggest a functional role for specific mitonuclear genes and pathways in human longevity. Show less

Urea cycle disorders (UCD) are inborn errors of metabolism caused by deficiency of enzymes required to convert nitrogen from ammonia into urea. Current paradigms of treatment focus on dietary manipulations, ammonia scavenger drugs, and liver transplantation. The aim of this study was to describe the characteristics and indication of liver transplantation in UCD in a tertiary hospital. We performed a retrospective study of children with UCD seen in the period 2000-2021. Data was collected on clinical onset, hyperammonemia severity, evolution and liver transplantation. There were 33 patients in the study period, whose diagnosis were: ornithine transcarbamylase (OTC, Show less

The glucocorticoid receptor (GR) is an important anti-cancer target in lymphoid cancers but has been understudied in solid tumors like lung cancer, although glucocorticoids are often given with chemotherapy regimens to mitigate side effects. Here, we identify a dexamethasone-GR mediated anti-cancer response in a subset of aggressive non-small cell lung cancers (NSCLCs) that harbor Serine/Threonine Kinase 11 (STK11/LKB1) mutations. High tumor expression of carbamoyl phosphate synthase 1 (CPS1) was strongly linked to the presence of LKB1 mutations, was the best predictor of NSCLC dexamethasone (DEX) sensitivity ( Show less

Tumor radiation resistance is the main obstacle to effective radiation therapy for patients with hepatocellular carcinoma (HCC). We identified the role of urea cycle key enzyme carbamoyl phosphate synthetase 1 (CPS1) in radioresistance of HCC and explored its mechanism, aiming to provide a novel radiosensitization strategy for the CPS1-deficiency HCC subtype. The expression of CPS1 was measured by western blot and immunohistochemistry. Cell growth assay, EdU assay, cell apoptosis assay, cell cycle assay, clone formation assay, and subcutaneous tumor assay were performed to explore the relationship between CPS1 and radioresistance of HCC cells. Lipid metabonomic analysis was used for investigating the effects of CPS1 on lipid synthesis of HCC cells. RNA sequencing and coimmunoprecipitation assay were carried out to reveal the mechanism of CPS1 participating in the regulation of HCC radiation therapy resistance. Furthermore, 10074-G5, the specific inhibitor of c-Myc, was administered to HCC cells to investigate the role of c-Myc in CPS1-deficiency HCC cells. We found that urea cycle key enzyme CPS1 was frequently lower in human HCC samples and positively associated with the patient's prognosis. Functionally, the present study proved that CPS1 depletion could accelerate the development of HCC and induce radiation resistance of HCC in vitro and in vivo, and deficiency of CPS1 promoted the synthesis of some lipid molecules. Regarding the mechanism, we uncovered that inhibition of CPS1 upregulated CyclinA2 and CyclinD1 by stabilizing oncoprotein c-Myc at the posttranscriptional level and generated radioresistance of HCC cells. Moreover, inactivation of c-Myc using 10074-G5, a specific c-Myc inhibitor, could partially attenuate the proliferation and radioresistance induced by depletion of CPS1. Our results recapitulated that silencing CPS1 could promote HCC progression and radioresistance via c-Myc stability mediated by the ubiquitin-proteasome system, suggesting that targeting c-Myc in CPS1-deficiency HCC subtype may be a valuable radiosensitization strategy in the treatment of HCC. Show less

Urea cycle disorders (UCDs) comprise a group of inborn errors of metabolism with impaired ammonia clearance and an incidence of ~1:35 000 individuals. First described in the 1970s, the diagnosis and management of these disorders has evolved dramatically. We report on a 59-year-old woman with a UCD who contributed to advances in the understanding and treatment of this group of disorders. This individual was diagnosed with carbamoyl phosphate synthetase 1 deficiency based on a biochemical assay under a research context predating genetic sequencing, treated longitudinally as having this metabolic disorder, and was among the first participants to trial UCD pharmaceutical therapies. She ultimately succumbed to a SARS-CoV-2 infection while maintaining unexpectedly normal ammonium levels. Postmortem genetic testing revealed ornithine transcarbamylase deficiency. This individual's contributions to the field of UCDs is discussed herein. Show less

Carbamoyl phosphate synthetase 1 (CPS1) is a highly abundant mitochondrial urea cycle enzyme that is expressed primarily in hepatocytes. CPS1 is constitutively and physiologically secreted into bile but is released into the bloodstream upon acute liver injury (ALI). Given its abundance and known short half-life, we tested the hypothesis that it may serve as a prognostic serum biomarker in the setting of acute liver failure (ALF). CPS1 levels were determined using enzyme-linked immunosorbent assay and immunoblotting of sera collected by the ALF Study Group (ALFSG) from patients with ALI and ALF (103 patients with acetaminophen and 167 non-acetaminophen ALF etiologies). A total of 764 serum samples were examined. The inclusion of CPS1 was compared with the original ALFSG Prognostic Index by area under the receiver operating characteristic curve analysis. CPS1 values for acetaminophen-related patients were significantly higher than for non-acetaminophen patients (P < .0001). Acetaminophen-related patients who received a liver transplant or died within 21 days of hospitalization exhibited higher CPS1 levels than patients who spontaneously survived (P = .01). Logistic regression and area under the receiver operating characteristic analysis of CPS1 enzyme-linked immunosorbent assay values improved the accuracy of the ALFSG Prognostic Index, which performed better than the Model for End-Stage Liver Disease, in predicting 21-day transplant-free survival for acetaminophen- but not non-acetaminophen-related ALF. An increase of CPS1 but not alanine transaminase or aspartate transaminase, when comparing day 3 with day 1 levels was found in a higher percentage of acetaminophen transplanted/dead patients (P < .05). Serum CPS1 determination provides a new potential prognostic biomarker to assess patients with acetaminophen-induced ALF. Show less

Cancer cell heterogeneity is a serious problem in the control of tumor progression because it can cause chemoresistance and metastasis. Heterogeneity can be generated by various mechanisms, including genetic evolution of cancer cells, cancer stem cells (CSCs), and niche heterogeneity. Because the genetic heterogeneity of CSCs has been poorly characterized, the genetic mutation status of CSCs was examined using Exome-Seq and RNA-Seq data of liver cancer. Here we show that different surface markers for liver cancer stem cells (LCSCs) showed a unique propensity for genetic mutations. Cluster of differentiation 133 (CD133)-positive cells showed frequent mutations in the Show less

Prostate cancer (PCa) is usually considered as cold tumor. Malignancy is associated with cell mechanic changes that contribute to extensive cell deformation required for metastatic dissemination. Thus, we established stiff and soft tumor subtypes for PCa patients from perspective of membrane tension. Nonnegative matrix factorization algorithm was used to identify molecular subtypes. We completed analyses using software R 3.6.3 and its suitable packages. We constructed stiff and soft tumor subtypes using eight membrane tension-related genes through lasso regression and nonnegative matrix factorization analyses. We found that patients in stiff subtype were more prone to biochemical recurrence than those in soft subtype (HR 16.18; p < 0.001), which was externally validated in other three cohorts. The top ten mutation genes between stiff and soft subtypes were DNAH, NYNRIN, PTCHD4, WNK1, ARFGEF1, HRAS, ARHGEF2, MYOM1, ITGB6 and CPS1. E2F targets, base excision repair and notch signaling pathway were highly enriched in stiff subtype. Stiff subtype had significantly higher TMB and T cells follicular helper levels than soft subtype, as well as CTLA4, CD276, CD47 and TNFRSF25. From the perspective of cell membrane tension, we found that stiff and soft tumor subtypes were closely associated with BCR-free survival for PCa patients, which might be important for the future research in the field of PCa. Show less

Carbamoyl phosphate synthetase I defect (CPS1D) is a rare disease with clinical case reports mainly in early neonates or adults, with few reports of first onset in late neonatal to childhood. We studied the clinical and genotypic characteristics of children with childhood onset CPS1D caused by two loci mutations (one of these is a rarely reported non-frame shift mutation) in the CPS1. We present a rare case of adolescent-onset CPS1D that had been misdiagnosed due to atypical clinical features, and further investigations revealed severe hyperammonemia (287µmol/L; reference range 11.2 ~ 48.2umol/L). MRI of the brain showed diffuse white matter lesions. Blood genetic metabolic screening showed elevated blood alanine (757.06umol/L; reference range 148.8 ~ 739.74umol/L) and decreased blood citrulline (4.26umol/L; reference range 5.45 ~ 36.77umol/L). Urine metabolic screening showed normal whey acids and uracil. Whole-exome sequencing revealed compound heterozygous mutations in the CPS1, a missense mutation (c.1145 C > T) and an unreported de novo non-frame shift mutation (c.4080_c.4091delAGGCATCCTGAT), respectively, which provided a clinical diagnosis. A comprehensive description of the clinical and genetic features of this patient, who has a rare age of onset and a relatively atypical clinical presentation, will facilitate the early diagnosis and management of this type of late onset CPS1D and reduce misdiagnosis, thus helping to reduce mortality and improve prognosis. It also provides a preliminary understanding of the relationship between genotype and phenotype, based on a summary of previous studies, which reminds us that it may help to explore the pathogenesis of the disease and contribute to genetic counselling and prenatal diagnosis. Show less

The present study aims to investigate the alterations of serum proteomic and metabolomic profiles in Chinese patients with severe and active Graves' Orbitopathy (GO). Thirty patients with GO and 30 healthy volunteers were enrolled. The serum concentrations of FT3, FT4, T3, T4, and thyroid-stimulating hormone (TSH) were analyzed, after which TMT labeling-based proteomics and untargeted metabolomics were performed. Metabo- Analyst and Ingenuity Pathway Analysis (IPA) was used for integrated network analysis. A nomogram was established based on the model to explore the disease prediction ability of the identified feature metabolites. One hundred thirteen proteins (19 up-regulated and 94 down-regulated) and 75 metabolites (20 increased and 55 decreased) were significantly altered in GO compared to the control group. By combining the lasso regression, IPA network, and protein-metabolite-disease sub-networks, we extracted feature proteins (CPS1, GP1BA, and COL6A1) and feature metabolites (glycine, glycerol 3-phosphate, and estrone sulfate). The logistic regression analysis revealed that the full model with the prediction factors and three identified feature metabolites had better prediction performance for GO compared to the baseline model. The ROC curve also indicated better prediction performance (AUC = 0.933 vs. 0.789). A new biomarker cluster combined with three blood metabolites with high statistical power can be used to discriminate patients with GO. These findings provide further insights into the pathogenesis, diagnosis, and potential therapeutic targets for this disease. Show less

Carbamoyl phosphate synthetase 1 (CPS1) deficiency is a severe urea cycle disorder. Patients can present with hyperammonemic coma in the first days of life. Treatment includes nitrogen scavengers, reduced protein intake and supplementation with L-arginine and/or L-citrulline. N-carbamoyl glutamate (NCG) has been hypothesized to stimulate the residual CPS1 function, although only few patients are reported. We report a patient with neonatal-onset CPS1 deficiency who received NCG in association with nitrogen scavenger and L-citrulline. The patient carried the novel variants Our data show that the response to NCG can be indicated based on the protein structure. We hypothesize that variants in the C-terminal domain may be responsive to NCG therapy. Show less

Intrahepatic cholangiocarcinoma (IHCC) is the second most common malignant neoplasm of the liver. In spite of the increasing incidence worldwide, it is relatively rare in Western countries. IHCC is relatively common in Eastern and Southeastern Asia. Patients with IHCC are usually diagnosed at an advanced stage, therefore, the clinical outcome is dismal. Dysregulation of urea cycle metabolic enzyme expression is found in different types of cancers. Nevertheless, a comprehensive evaluation of genes related to the urea cycle (i.e., GO:0000050) has not been conducted in IHCC. By performing a comparative analysis of gene expression profiles, we specifically examined genes associated with the urea cycle (GO:0000050) in a publicly accessible transcriptomic dataset (GSE26566). Interestingly, Show less

The carbon fiber reinforced polyetheretherketone (CFR-PEEK) has been increasingly used in orthopedics dentistry due to its excellent biocompatibility and mechanical properties. However, the biological inertness and poor antibacterial activity limit its clinical applications. This paper focused on the performances of CFR-PEEK with porous morphology that were exposed to different sulfonation periods (1, 3, 5, and 10 min, corresponding to CP-S1, CP-S3, CP-S5, and CP-S10, respectively). Residual sulfuric acid was removed by acetone rinsing, NaOH immersion, and hydrothermal treatment before in vitro and in vivo studies. The results showed some significant difference in the physicochemical properties, including energy dispersive X-ray spectroscopy (EDS) map of sulfur atoms, X-ray photoelectron spectroscopy (XPS) of valences of sulfur ions, Fourier transformation infrared spectroscopy (FTIR), hydrophilicity, hardness, and elastic modulus among CP-S3, CP-S5, and CP-S10. However, CP-S5 and CP-S10 were more effective in promoting the proliferation, adhesion, and osteogenic differentiation of seeded bone mesenchymal stem cells (BMSCs) and growth inhibition of S. aureus and P. gingivalis compared with other groups. Furthermore, the CP-S5 and CP-S10 samples achieved better cranial bone repair than the non-sulfonation group in a rat model. Therefore, it can be inferred that both 5 and 10 min are viable sulfonation durations for 30% CFR-PEEK. These findings provide a theoretical basis for developing CFR-PEEK for clinical applications. Show less

Newborn genome sequencing (NBSeq) can detect infants at risk for treatable disorders currently undetected by conventional newborn screening. Despite broad stakeholder support for NBSeq, the perspectives of rare disease experts regarding which diseases should be screened have not been ascertained. To query rare disease experts about their perspectives on NBSeq and which gene-disease pairs they consider appropriate to evaluate in apparently healthy newborns. This survey study, designed between November 2, 2021, and February 11, 2022, assessed experts' perspectives on 6 statements related to NBSeq. Experts were also asked to indicate whether they would recommend including each of 649 gene-disease pairs associated with potentially treatable conditions in NBSeq. The survey was administered between February 11 and September 23, 2022, to 386 experts, including all 144 directors of accredited medical and laboratory genetics training programs in the US. Expert perspectives on newborn screening using genome sequencing. The proportion of experts indicating agreement or disagreement with each survey statement and those who selected inclusion of each gene-disease pair were tabulated. Exploratory analyses of responses by gender and age were conducted using t and χ2 tests. Of 386 experts invited, 238 (61.7%) responded (mean [SD] age, 52.6 [12.8] years [range 27-93 years]; 126 [52.9%] women and 112 [47.1%] men). Among the experts who responded, 161 (87.9%) agreed that NBSeq for monogenic treatable disorders should be made available to all newborns; 107 (58.5%) agreed that NBSeq should include genes associated with treatable disorders, even if those conditions were low penetrance; 68 (37.2%) agreed that actionable adult-onset conditions should be sequenced in newborns to facilitate cascade testing in parents, and 51 (27.9%) agreed that NBSeq should include screening for conditions with no established therapies or management guidelines. The following 25 genes were recommended by 85% or more of the experts: OTC, G6PC, SLC37A4, CYP11B1, ARSB, F8, F9, SLC2A1, CYP17A1, RB1, IDS, GUSB, DMD, GLUD1, CYP11A1, GALNS, CPS1, PLPBP, ALDH7A1, SLC26A3, SLC25A15, SMPD1, GATM, SLC7A7, and NAGS. Including these, 42 gene-disease pairs were endorsed by at least 80% of experts, and 432 genes were endorsed by at least 50% of experts. In this survey study, rare disease experts broadly supported NBSeq for treatable conditions and demonstrated substantial concordance regarding the inclusion of a specific subset of genes in NBSeq. Show less

Carbamoyl phosphate synthetase 1 (CPS1) is the most abundant hepatocyte mitochondrial matrix protein. Hypoosmotic stress increases CPS1 release in isolated mouse hepatocytes without cell death. We hypothesized that increased CPS1 release during hypoosmosis is selective and associates with altered mitochondrial morphology. Both ex vivo and in vivo models were assessed. Mouse hepatocytes and livers were challenged with isotonic or hypoosmotic (35 mosM) buffer. Mice were injected intraperitoneally with water (10% body weight) with or without an antidiuretic. Mitochondrial and cytosolic fractions were isolated using differential centrifugation, then analyzed by immunoblotting to assess subcellular redistribution of four mitochondrial proteins: CPS1, ornithine transcarbamylase (OTC), pyrroline-5-carboxylate reductase 1 (PYCR1), and cytochrome c. Mitochondrial morphology alterations were examined using electron microscopy. Hypoosmotic treatment of whole livers or hepatocytes led to preferential or increased mitochondrial release, respectively, of CPS1 as compared with two mitochondrial matrix proteins (OTC/PYCR1) and with the intermembrane space protein, cytochrome c. Mitochondrial apoptosis-induced channel opening using staurosporine in hepatocytes led to preferential CPS1 and cytochrome c release. The CPS1-selective changes were accompanied by dramatic alterations in ultrastructural mitochondrial morphology. In mice, hypoosmosis/hyponatremia led to increased liver vascular congestion and increased CPS1 in bile but not blood, coupled with mitochondrial structural alterations. In contrast, isotonic increase of intravascular volume led to a decrease in mitochondrial size with limited change in bile CPS1 compared with hypoosmotic conditions and absence of the hypoosmosis-associated histological alterations. Taken together, hepatocyte CPS1 is selectively released in response to hypoosmosis/hyponatremia and provides a unique biomarker of mitochondrial injury. Show less

To determine whether hyperammonemia has a direct impact on steatohepatitis in mice fed with a high-fat diet (HFD). Male C57BL/6 mice were divided into two groups receiving either chow diet or HFD. After 12-week NASH modeling, hyperammonemia was induced by intragastric administration of ammonium chloride solution (NH NH Our study revealed a limited role of ammonia in aggravating the progression of NASH. Further studies are needed to clarify the role and mechanism of ammonia in NASH development. Show less

Toxoplasma gondii is an obligate intracellular protozoan parasite that commonly infects mammals and birds throughout the world. This protocol describes murine models of acute T. gondii infection, toxoplasmic encephalitis and toxoplasma retinochoroiditis. T. gondii infection in severe combined immunodeficient (SCID) mice, deficient in T and B cells, has allowed for the study of T cell-independent mechanisms of defense against intracellular organisms, as described here. The uracil auxotroph strain cps1-1 and temperature-sensitive mutant strains of T. gondii induce protection against challenge with virulent strains of the parasite. They have allowed studies of immunization and adoptive-transfer experiments. A protocol is provided for infection with these mutant strains. The EGS strain of T. gondii has the unique feature of spontaneously forming tissue cysts in cell culture. Dual fluorescent reporter stains of this strain have allowed the study of tachyzoite to bradyzoite transitions in vitro and in vivo. A protocol for in vitro and in vivo growth of this strain and tissue cyst isolation is provided. Genetic manipulation of T. gondii and mice has led to the development of parasites that express fluorescent proteins as well as mice with fluorescently labeled leukocytes. This together with the use of T. gondii that express model antigens and transgenic mice that express the appropriate T cell receptor have facilitated the in vivo study of parasite host-interaction. In addition, parasites that express bioluminescent markers have made it possible to study the dynamics of infection in real time using bioluminescence imaging. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite that includes these newer methodologies. In addition, support protocols address the maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of acute T. gondii infection in mice Basic Protocol 2: Model of toxoplasmic encephalitis and toxoplasma retinochoroiditis in chronically infected mice Basic Protocol 3: Assessment of T. gondii invasion into neural tissue Basic Protocol 4: T. gondii infection in scid/scid (SCID) mice Basic Protocol 5: Infection with the uracil auxotroph strain CPS1-1 or the temperature-sensitive TS-4 strain of T. gondii Basic Protocol 6: In vivo and in vitro maintenance of the EGS strain of T. gondii Support Protocol 1: Assessment of progression of infection and immune response to T. gondii Support Protocol 2: Maintenance of a bank of T. gondii cysts of the ME49 strain Support Protocol 3: Maintenance of T. gondii tachyzoites using human foreskin fibroblasts Support Protocol 4: Maintenance of T. gondii tachyzoites in mice Support Protocol 5: Preparation of T. gondii lysate antigens Support Protocol 6: Isolation of T. gondii tissue cysts from brain. Show less

Two polysaccharides (CPS1 and CPW2) from Corydalis decumbens were obtained to develop insights into natural medical resources. Optimal extraction conditions of total sugars were researched using the method of response surface methodology, polysaccharides were purified using a combination of ethanol precipitation and anion-exchange chromatography, and structure features were analyzed by scanning electron microscopy, transmission electron microscopy, and Congo-red assay. The bioactivities were estimated in terms of antioxidant and anti-inflammatory effects. Total sugars were extracted with an experimental yield of 32.74% under optimum conditions. CPS1 and CPW2 were purified with yields of 12.01% and 8.23%, respectively. CPS1 was a unique polysaccharide with a molecular weight (Mw) of 360 kDa and consisted of glucose, galactose, mannose, and arabinose in a ratio of 4.9:2.0:1:1.9, and CPW2 was composed of glucose with the Mw of 550 kDa. CPS1 possessed a four-helix conformation, and CPW2 was identified as a linear molecule without branched and entangled chains. The mRNA expressions of TNF-α (71.80%), IL-1β (56.55%), IL-6 (43.98%), and COX-2 (91.88%) in LPS-stimulated RAW 264.7 cells were significantly inhibited by 75 μg/mL CPS1 (P < 0.0001), while CPW2 showed lower inhibitory effects than CPS1. Compared with CPW2, CPS1 showed stronger scavenging abilities for hydroxyl (EC50 = 520.46 μg/mL), ABTS (EC50 = 533.99 μg/mL), and superoxide (EC50 = 1512.06 μg/mL) radicals. CPS1 with four-helix conformation exhibited more outstanding bioactivities than CPW2 without entangled chains. Show less

Lung adenocarcinoma (LUAD) is a highly heterogeneous disease that ranks first in morbidity and mortality. Abnormal arginine metabolism is associated with inflammatory lung disease and may influence alterations in the tumor immune microenvironment. However, the potential role of arginine and proline metabolic patterns and immune molecular markers in LUAD is unclear. Gene expression, somatic mutations, and clinicopathological information of LUAD were downloaded from The Cancer Genome Atlas (TCGA) database. Univariate Cox regression analysis was performed to identify metabolic genes associated with overall survival (OS). Unsupervised clustering divided the sample into two subtypes with different metabolic and immunological profiles. Gene set enrichment analysis (GESA) and gene set variation analysis (GSVA) were used to analyze the underlying biological processes of the two subtypes. Drug sensitivity between subtypes was also predicted; then prognostic features were developed by multivariate Cox regression analysis. In addition, validation was obtained in the GSE68465, and GSE50081 dataset. Then, gene expression, and clinical characterization of hub genes CPS1 and SMS were performed; finally, in vitro validation experiments for knockdown of SMS were performed in LUAD cell lines. In this study, we first identified 12 arginine and proline-related genes (APRGs) significantly associated with OS and characterized the clinicopathological features and tumor microenvironmental landscape of two different subtypes. Then, we established an arginine and proline metabolism-related scoring system and identified two hub genes highly associated with prognosis, namely CPS1, and SMS. In addition, we performed CCK8, transwell, and other functional experiments on SMS to obtain consistent results. Our comprehensive analysis revealed the potential molecular features and clinical applications of APRGs in LUAD. A model based on 2 APRGs can accurately predict survival outcomes in LUAD, improve our understanding of APRGs in LUAD, and pave a new pathway to guide risk stratification and treatment strategy development for LUAD patients. Show less

Carbamoyl-phosphate synthetase 1 (CPS1) deficiency is an autosomal recessive congenital urea cycle disorder (UCD) characterized by hyperammonemia. The recipients of liver transplantation (LT) for UCD are often children, and the potential donors are often the parents. Hereditary congenital diseases involving UCD entail the possibility of both parents being genetically heterozygous. Herein, we describe the case of a 12-year-old girl with CPS1 deficiency receiving a liver transplant (soon after birth) from her father, who had a heterozygous Show less

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. Show less

To investigate the effect of canagliflozin (20 mg/kg) on hepatic steatosis and atherosclerosis, and further to explore its possible mechanism. Blood glucose, blood lipid, oxidative stress response and inflammatory cytokines were examined by intraperitoneal glucose tolerance test and ELISA assay. HE and Oil Red O staining were used to estimate the extent of hepatic steatosis and atherosclerosis. RNA-seq and qRT-PCR were used to further investigate the potential mechanism. The effects of canagliflozin on autophagy were detected using transmission electron microscopy and Western blotting. The endothelial function-related markers were determined by qRT-PCR. Canagliflozin notably alleviated the elevation in blood glucose and insulin resistance in western diet-fed ApoE-/- mice. In ApoE-/-+Cana group, ApoE-/- mice had lower levels of TG, TC, LDL-C, TNF-α, IL-6, IL-1β, and MCP-1. HE and Oil Red O staining presented that canagliflozin restrained the atherosclerotic plaque development and lipid accumulation. RNA-seq showed that 87 DEGs were relevant to improvement of hepatic steatosis and atherosclerosis by canagliflozin. Among them, CPS1, ASS1, ASL, ARG1, MATLA, GLS2, GOT1, SREBP1, Plin5, Retreg1, and C/EBPβ were verified. KEGG enrichment analysis indicated that DEGs were mainly involved in amino acid metabolism. Besides, we observed that canagliflozin reduced the contents of aspartic acid and citrulline in liver. Western blotting showed that ASS1 and p-AMPK/AMPK was remarkably elevated after administration of canagliflozin. Correspondingly, canagliflozin down-regulated SREBP1, FAS, ACC1, HMGCR, p-mTOR/m-TOR, p-ULK1/ULK1 and p62, but up-regulated CPT1, Beclin 1 and LC3 II/LC3I. TEM showed that canagliflozin reduced the number of lipid droplets and increased the autophagosomes. Moreover, we found that canagliflozin elevated the aortic endothelial function-associated markers including ASS1, ASL and eNOS. Canagliflozin may attenuate hepatic steatosis by improving lipid metabolism, enhancing autophagy, and reducing inflammatory response through ASS1/AMPK pathway. Besides, canagliflozin further effectively improves the aortic endothelial function, thereby suppressing atherosclerosis development. Show less

Hypertension is a multifactorial condition in which several genetic and environmental elements contribute. Recent investigations have revealed contribution of non-coding region of the transcriptome in this trait. CDKN2B-AS1, AK098656, MEG3, H19, PAXIP1-AS1, TUG1, GAS5, CASC2 and CPS1-IT are among long non-coding RNAs participating in the pathophysiology of hypertension. Several miRNAs have also been found to be implicated in this disorder. miR-296, miR-637, miR-296, miR-637, hsa-miR-361-5p, miR-122-5p, miR-199a-3p, miR-208a-3p, miR-423-5p, miR-223-5p and miR-140-5p are among dysregulated miRNAs in this condition whose application as diagnostic biomarkers for hypertension has been evaluated. Finally, hsa-circ-0005870, hsa_circ₀₀₃₇₉₁₁ and hsa_circ₀₀₁₄₂₄₃ are examples of dysregulated circular RNAs in hypertensive patients. In the current review, we describe the role of these non-coding RNAs in the pathophysiology of hypertension. Show less

Carbamoyl phosphate synthetase 1 (CPS1) deficiency is an autosomal recessive urea cycle disorder with varying presentations. Patients with a neonatal-onset phenotype are initially healthy but develop severe hyperammonemia days after birth and often have poor or lethal outcomes, while patients who present later in life may exhibit less severe clinical manifestations. CPS1 deficiency is rarely found on newborn screening because most states do not screen for this disease due to the technical difficulties. We report a case of an 11-year-old, previously healthy girl who presented with hyperammonemia and acute psychosis after eating large amounts of meat at summer camp. A diagnosis of carbamoyl phosphate synthetase type 1 deficiency was suspected by biochemical profiles and confirmed by molecular analysis. Subsequent follow up lab results revealed ammonia to be only 25-39 μmol/L shortly after glutamine reached levels as high as 770-1432 μmol/L with concurrent alanine elevations, highlighting the compensating mechanisms of the human body. Her initial hospital course also demonstrated the importance of continuous renal replacement therapy (CRRT) in avoiding rebound hyperammonemia and high glutamine and the benefits of intracranial pressure (ICP) monitoring, providing 3% hypertonic saline and temperature control to avoid fever in treating cerebral edema. Carglumic acid was not considered helpful in this case, with BUN levels ranging between 2 and 4 mg/dL after administration. Show less