Carbamoyl Phosphate Synthetase Research Articles

Use of pure recombinant human enzymes to assess the disease-causing potential of missense mutations in urea cycle disorders, applied to N-acetylglutamate synthase deficiency.

N-acetylglutamate synthase (NAGS) makes acetylglutamate, the essential activator of the first, regulatory enzyme of the urea cycle, carbamoyl phosphate synthetase 1 (CPS1). NAGS deficiency (NAGSD) and CPS1 deficiency (CPS1D) present identical phenotypes. However, they must be distinguished, because NAGSD is cured by substitutive therapy with the N-acetyl-L-glutamate analogue N-carbamyl-L-glutamate, while curative therapy of CPS1D requires liver transplantation. Since their differentiation is done genetically, it is important to ascertain the disease-causing potential of CPS1 and NAGS genetic variants. With this goal, we previously carried out site-directed mutagenesis studies with pure recombinant human CPS1. We could not do the same with human NAGS (HuNAGS) because of enzyme instability, leading to our prior utilization of a bacterial NAGS as an imperfect surrogate of HuNAGS. We now use genuine HuNAGS, stabilized as a chimera of its conserved domain (cHuNAGS) with the maltose binding protein (MBP), and produced in Escherichia coli. MBP-cHuNAGS linker cleavage allowed assessment of the enzymatic properties and thermal stability of cHuNAGS, either wild-type or hosting each one of 23 nonsynonymous single-base changes found in NAGSD patients. For all but one change, disease causation was accounted by the enzymatic alterations identified, including, depending on the variant, loss of arginine activation, increased Km Glutamate, active site inactivation, decreased thermal stability, and protein misfolding. Our present approach outperforms experimental in vitro use of bacterial NAGS or in silico utilization of prediction servers (including AlphaMissense), illustrating with HuNAGS the value for UCDs of using recombinant enzymes for assessing disease-causation and molecular pathogenesis, and for therapeutic guidance.

Urea cycle promotion via ammonia-upregulated CPS1 is involved in arsenite-induced pulmonary fibrosis through enhancing collagen synthesis

Arsenic exposure is connected with lung toxicity and is related to lung fibrotic changes. Idiopathic pulmonary fibrosis (IPF) is characterized by extracellular matrix (ECM) deposition. Various genetic mechanisms and environmental factors induce or exacerbate pulmonary fibrosis. Collagen synthesis induced by sodium arsenite (NaAsO2) is closely associated with IPF. Fibroblasts tend to fine-tune their metabolic networks to support their synthetic requirements in response to environmental stimuli. Alterations in metabolism have an influential role in the pathogenesis of IPF. However, it is unclear how arsenic affects the metabolism in IPF. The urea cycle (UC) is needed for collagen formation, which provides adequate levels of proline (Pro) for biosynthesis of collagen. Carbamoyl phosphate synthetase 1 (CPS1) converts the ammonia to carbamoyl phosphate, which controls the first reaction of the UC. We show that, in arsenite-exposed mice, high amounts of ammonia in the lung microenvironment promotes the expression levels of CPS1 and the Pro metabolism. Reduction of ammonia and CPS1 ablation inhibit collagen synthesis and ameliorate IPF phenotypes induced by arsenite. This work takes advantage of multi-omics data to enhance understanding of the underlying pathogenic mechanisms, the key molecules and the complicated cellular responses to this pollutant, which provide a target for the prevention of pulmonary fibrosis caused by arsenic.

Prognostic and Immunotherapeutic Predictive Value of CAD Gene in Hepatocellular Carcinoma: Integrated Bioinformatics and Experimental Analysis.

Hepatocellular carcinoma (HCC) is a common type of cancer that ranks first in cancer-associated death worldwide. Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) are the key components of the pyrimidine pathway, which promotes cancer development. However, the function of CAD in HCC needs to be clarified. In this study, the clinical and transcriptome data of 424 TCGA-derived HCC cases were analyzed. The results demonstrated that high CAD expression was associated with poor prognosis in HCC patients. The effect of CAD on HCC was then investigated comprehensively using GO annotation analysis, KEGG enrichment analysis, Gene Set Enrichment Analysis (GSEA), and CIBERSORT algorithm. The results showed that CAD expression was correlated with immune checkpoint inhibitors and immune cell infiltration. In addition, low CAD levels in HCC patients predicted increased sensitivity to anti-CTLA4 and PD1, while HCC patients with high CAD expression exhibited high sensitivity to chemotherapeutic and molecular-targeted agents, including gemcitabine, paclitaxel, and sorafenib. Finally, the results from clinical sample suggested that CAD expression increased remarkably in HCC compared with non-cancerous tissues. Loss of function experiments demonstrated that CAD knockdown could significantly inhibit HCC cell growth and migration both in vitro and in vivo. Collectively, the results indicated that CAD is a potential oncogene during HCC metastasis and progression. Therefore, CAD is recommended as a candidate marker and target for HCC prediction and treatment.

Cytotoxicity of Quantum Dots in Receptor-Mediated Endocytic and Pinocytic Pathways in Yeast.

Despite the promising applications of the use of quantum dots (QDs) in the biomedical field, the long-lasting effects of QDs on the cell remain poorly understood. To comprehend the mechanisms underlying the toxic effects of QDs in yeast, we characterized defects associated with receptor-mediated endocytosis (RME) as well as pinocytosis using Saccharomyces cerevisiae as a model in the presence of cadmium selenide/zinc sulfide (CdSe/ZnS) QDs. Our findings revealed that QDs led to an inefficient RME at the early, intermediate, and late stages of endocytic patch maturation at the endocytic site, with the prolonged lifespan of GFP fused yeast fimbrin (Sac6-GFP), a late marker of endocytosis. The transit of FM1-43, a lipophilic dye from the plasma membrane to the vacuole, was severely retarded in the presence of QDs. Finally, QDs caused an accumulation of monomeric red fluorescent protein fused carbamoyl phosphate synthetase 1 (mRFP-Cps1), a vacuolar lumen marker in the vacuole. In summary, the present study provides novel insights into the possible impact of CdSe/ZnS QDs on the endocytic machinery, enabling a deeper comprehension of QD toxicity.

The histone demethylase JMJD1C regulates CPS1 expression and promotes the proliferation of paroxysmal nocturnal haemoglobinuria clones through cell metabolic reprogramming.

Paroxysmal nocturnal haemoglobinuria (PNH) is a disorder resulting from erythrocyte membrane deficiencies caused by PIG-A gene mutations. While current treatments alleviate symptoms, they fail to address the underlying cause of the disease-the pathogenic PNH clones. In this study, we found that the expression of carbamoyl phosphate synthetase 1 (CPS1) was downregulated in PNH clones, and the level of CPS1 was negatively correlated with the proportion of PNH clones. Using PIG-A knockout K562 (K562 KO) cells, we demonstrated that CPS1 knockdown increased cell proliferation and altered cell metabolism, suggesting that CPS1 participates in PNH clonal proliferation through metabolic reprogramming. Furthermore, we observed an increase in the expression levels of the histone demethylase JMJD1C in PNH clones, and JMJD1C expression was negatively correlated with CPS1 expression. Knocking down JMJD1C in K562 KO cells upregulated CPS1 and H3K36me3 expression, decreased cell proliferation and increased cell apoptosis. Chromatin immunoprecipitation analysis further demonstrated that H3K36me3 regulated CPS1 expression. Finally, we demonstrated that histone demethylase inhibitor JIB-04 can suppressed K562 KO cell proliferation and reduced the proportion of PNH clones in PNH mice. In conclusion, aberrant regulation of the JMJD1C-H3K36me3-CPS1 axis contributes to PNH clonal proliferation. Targeting JMJD1C with a specific inhibitor unveils a potential strategy for treating PNH patients.

L-Ornithine-l-Aspartate (LOLA) Normalizes Metabolic Parameters in Models of Steatosis, Insulin Resistance and Metabolic Syndrome.

l-Ornithine- l-aspartate (LOLA) reduces toxic ammonium (NH3) plasma levels in hepatic encephalopathy. NH3 detoxification/excretion is achieved by its incorporation into urea and glutamine via activation of carbamoyl phosphate synthetase 1 (CSP1) by l-ornithine and stimulation of arginase by l-aspartate. We aimed at identifying additional molecular targets of LOLA as a potential treatment option for non-alcoholic fatty liver disease (NAFLD). In primary hepatocytes from NAFLD patients, urea cycle enzymes CSP1 and ornithine transcarbamylase (OTC) increase, while the catabolism of branched-chain amino acids (BCAAs) decreases with disease severity. In contrast, LOLA increased the expression rates of the BCAA enzyme transcripts bcat2, bckdha, and bckdk. In untreated HepG2 hepatoblastoma cells and HepG2-based models of steatosis, insulin resistance, and metabolic syndrome (the latter for the first time established herein), LOLA reduced the release of NH3; beneficially modulated the expression of genes related to fatty acid import/transport (cd36, cpt1), synthesis (fasn, scd1, ACC1), and regulation (srbf1); reduced cellular ATP and acetyl-CoA; and favorably modulated the expression of master regulators/genes of energy balance/mitochondrial biogenesis (AMPK-α, pgc1α). Moreover, LOLA reconstituted the depolarized mitochondrial membrane potential, while retaining mitochondrial integrity and avoiding induction of superoxide production. Most effects were concentration-dependent at ≤40 mM LOLA. We demonstrate for l-ornithine-l-aspartate a broad range of reconstituting effects on metabolic carriers and targets of catabolism/energy metabolism impaired in NAFLD. These findings strongly advocate further investigations to establish LOLA as a safe, efficacious, and cost-effective basic medication for preventing and/or alleviating NAFLD.

Abstract 7206: Inhibition of CAD, an enzyme of de novo pyrimidine synthesis, to overcome EGFR-TKI resistance in NSCLC

Abstract Given the fast-growing cancer cells, there is an increased demand for large amounts of energy and nucleotides for DNA and RNA synthesis. Thus, targeting nucleotide metabolism, specifically de novo nucleotide biosynthesis, is a promising strategy for cancer treatment. Hyperactivation of Carbamoyl-phosphate synthetase, aspartate transcarbamylase (CAD), a key enzyme catalyzing the first three steps of de novo pyrimidine synthesis, is a common event in tumors and has been associated with chemoresistance in tumors. Our analysis, using TCGA data, revealed that increased expression of CAD is associated with poor prognosis in non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated significant benefits to patients with NSCLC harboring EGFR-activating mutations; however, acquired resistance limits their long-term efficacy. Therefore, it remains urgent to discover the underlying mechanisms for discovering novel therapeutic strategies for overcoming TKI resistance. Our previous findings demonstrated that blockade of S6K1 overcomes acquired resistance to EGFR-TKIs in NSCLC. It was reported that S6K1 is involved in de novo pyrimidine biosynthesis. In this study, we aimed to investigate whether CAD mediates S6K1-induced EGFR-TKI resistance in NSCLC. Immunohistochemistry analysis on a tissue microarray of lung adenocarcinoma tissues indicated that increased co-expression of p-CAD and S6K1 proteins is associated with clinicopathological parameters and poor survival outcomes. Our in vitro results showed an increased expression level of CAD and p-CAD proteins in TKI-resistant cells compared with parental cells upon TKI treatment. Knockdown of CAD by siRNA led to sensitizing TKI-resistant cells to osimertinib, as indicated by BrdU cell proliferation assay. Inhibition of S6K1 by a siRNA or specific inhibitors PF-470867 and LY2584702 effectively reduced the phosphorylation levels of CAD protein in TKI-resistant cells, suggesting that S6K1 may serve as an upstream regulator of CAD for TKI resistance. The String database predicted a protein-protein interaction between S6K1 and CAD. Indeed, our co-immunoprecipitation (co-IP) results revealed a direct association between S6K1 and CAD proteins. Importantly, TKI treatment enhanced the binding between S6K1 and CAD. Taken together, these findings provide evidence that CAD may mediate S6K1-mediated EGFR-TKI resistance in NSCLC. Our ongoing and future work will assess whether inhibition of CAD sensitizes resistant cells to TKI in vivo and elucidate the associated mechanism. Citation Format: Leili Saeednejad Zanjani, Lei Zhao, Shaomin Wang, Margot Federoff, Xin Sun, Jun He. Inhibition of CAD, an enzyme of de novo pyrimidine synthesis, to overcome EGFR-TKI resistance in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7206.

Generation of Highly Functional Hepatocyte-like Organoids from Human Adipose-Derived Mesenchymal Stem Cells Cultured with Endothelial Cells.

Previously, we successfully established a highly functional, three-dimensional hepatocyte-like cell (3D-HLC) model from adipose-derived mesenchymal stem cells (ADSCs) via a three-step differentiation protocol. The aim of the present study was to investigate whether generating hepatocyte-like organoids (H-organoids) by adding endothelial cells further improved the liver-like functionality of 3D-HLCs and to assess H-organoids' immunogenicity properties. Genes representing liver maturation and function were detected by quantitative reverse transcription-PCR analysis. The expression of hepatic maturation proteins was measured using immunofluorescence staining. Cytochrome P (CYP)450 metabolism activity and ammonia metabolism tests were used to assess liver function. H-organoids were successfully established by adding human umbilical vein endothelial cells at the beginning of the definitive endoderm stage in our 3D differentiation protocol. The gene expression of alpha-1 antitrypsin, carbamoyl-phosphate synthase 1, and apolipoprotein E, which represent liver maturation state and function, was higher in H-organoids than non-organoid 3D-HLCs. H-organoids possessed higher CYP3A4 metabolism activity and comparable ammonia metabolism capacity than 3D-HLCs. Moreover, although H-organoids expressed human leukocyte antigen class I, they expressed little human leukocyte antigen class II, cluster of differentiation (CD)40, CD80, CD86, and programmed cell death ligand 1, suggesting their immunogenicity properties were not significantly upregulated during differentiation from ADSCs. In conclusion, we successfully established an H-organoid model with higher liver-like functionality than previously established 3D-HLCs and comparable immunogenicity to ADSCs.

Gasdermin-E-Dependent Non-Canonical Pyroptosis Promotes Drug-Induced Liver Failure by Promoting CPS1 deISGylation and Degradation.

Drug-induced liver injury (DILI) is a significant global health issue that poses high mortality and morbidity risks. One commonly observed cause of DILI is acetaminophen (APAP) overdose. GSDME is an effector protein that induces non-canonical pyroptosis. In this study, the activation of GSDME, but not GSDMD, in the liver tissue of mice and patients with APAP-DILI is reported. Knockout of GSDME, rather than GSDMD, in mice protected them from APAP-DILI. Mice with hepatocyte-specific rescue of GSDME reproduced APAP-induced liver injury. Furthermore, alterations in the immune cell pools observed in APAP-induced DILI, such as the replacement of TIM4+ resident Kupffer cells (KCs) by monocyte-derived KCs, Ly6C+ monocyte infiltration, MerTk+ macrophages depletion, and neutrophil increase, reappeared in mice with hepatocyte-specific rescue of GSDME. Mechanistically, APAP exposure led to a substantial loss of interferon-stimulated gene 15 (ISG15), resulting in deISGylation of carbamoyl phosphate synthetase-1 (CPS1), promoted its degradation via K48-linked ubiquitination, causing ammonia clearance dysfunction. GSDME deletion prevented these effects. Delayed administration of dimethyl-fumarate inhibited GSDME cleavage and alleviated ammonia accumulation, mitigating liver injury. This findings demonstrated a previously uncharacterized role of GSDME in APAP-DILI by promoting pyroptosis and CPS1 deISGylation, suggesting that inhibiting GSDME can be a promising therapeutic option for APAP-DILI.

Establishment of iPS cell line (SDQLCHi061-A) from a patient with carbamoylphosphate synthetase I deficiency due to CPS1 mutation

The induced pluripotent stem cells (iPSCs) line was generated using peripheral blood mononuclear cells (PBMCs) from a patient with compound heterozygous mutation of c.2374A > G/p.M792V and c.3949C > T/p.R1317W in the CPS1 gene by non-integrating vectors. The expression of pluripotency markers, potential for in vitro trilineage differentiation and exhibiting normal karyotype were demonstrated in the SDQLCHi061-A cell line. This cell line could provide a useful CPS1D model in vitro for further study.

Hijacking of nucleotide biosynthesis and deamidation-mediated glycolysis by an oncogenic herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS) and multiple types of B cell malignancies. Emerging evidence demonstrates that KSHV reprograms host-cell central carbon metabolic pathways, which contributes to viral persistence and tumorigenesis. However, the mechanisms underlying KSHV-mediated metabolic reprogramming remain poorly understood. Carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) is a key enzyme of the de novo pyrimidine synthesis, and was recently identified to deamidate the NF-κB subunit RelA to promote aerobic glycolysis and cell proliferation. Here we report that KSHV infection exploits CAD for nucleotide synthesis and glycolysis. Mechanistically, KSHV vCyclin binds to and hijacks cyclin-dependent kinase CDK6 to phosphorylate Ser-1900 on CAD, thereby activating CAD-mediated pyrimidine synthesis and RelA-deamidation-mediated glycolytic reprogramming. Correspondingly, genetic depletion or pharmacological inhibition of CDK6 and CAD potently impeded KSHV lytic replication and thwarted tumorigenesis of primary effusion lymphoma (PEL) cells in vitro and in vivo. Altogether, our work defines a viral metabolic reprogramming mechanism underpinning KSHV oncogenesis, which may spur the development of new strategies to treat KSHV-associated malignancies and other diseases.

Hyperammonemic encephalopathy after tyrosine kinase inhibitors: A literature review and a case example.

To review the evidence of uncommon but fatal adverse event of hyperammonemic encephalopathy by tyrosine kinase inhibitors (TKI) and the possible mechanisms underlying this condition and to describe the case of a patient that developed drug-induced hyperammonemic encephalopathy related to TKI. Literature search of different databases was performed for studies published from 1 January 1992 to 7 May 2023. The search terms utilized were hyperammonemic encephalopathy, TKI, apatinib, pazopanib, sunitinib, imatinib, sorafenib, regorafenib, trametinib, urea cycle regulation, sorafenib, carbamoyl-phosphate synthetase 1, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase 1, Mitogen activated protein kinases (MAPK) pathway and mTOR pathway, were used individually search or combined. Thirty-seven articles were included. The articles primarily focused in hyperammonemic encephalopathy case reports, management of hyperammonemic encephalopathy, urea cycle regulation, autophagy, mTOR and MAPK pathways, and TKI. Eighteen cases of hyperammonemic encephalopathy were reported in the literature from various multitargeted TKI. The mechanism of this event is not well-understood but some authors have hypothesized vascular causes since some of TKI are antiangiogenic, however our literature review shows a possible relationship between the urea cycle and the molecular inhibition exerted by TKI. More preclinical evidence is required to unveil the biochemical mechanisms responsible involved in this process and clinical studies are necessary to shed light on the prevalence, risk factors, management and prevention of this adverse event. It is important to monitor neurological symptoms and to measure ammonia levels when manifestations are detected.

Integrated transcriptomic and metabolomic investigation of the genes and metabolites involved in swine follicular cyst formation.

Follicular cysts are a common reproductive disorder in mammals that is usually caused by stress. However, the pathogenesis of follicular cysts in sows remains unclear. To provide new insights into the mechanisms of follicular cyst formation in pigs, we conducted a combined transcriptomic and metabolomic analysis on theca interna and mural granulosa cells of follicular cysts and mature follicles. We identified 2,533 up-regulated and 1,355 down-regulated genes in follicular cysts, compared with mature follicles. These differentially expressed genes were mainly found in signaling pathways related to tumor formation and cortisol synthesis and secretion as shown by Ingenuity Pathway Analysis, which predicted 4,362 upstream regulatory factors. The combined gene expression and pathway analysis identified the following genes as potential biomarkers for porcine follicular cysts: cytochrome P450 family 2 subfamily C polypeptide 18, L-lactate dehydrogenase, carbamoyl-phosphate synthase, fibroblast growth factor 7, integrin binding sialoprotein, interleukin 23 receptor, prolactin receptor, epiregulin, interleukin 1 receptor type II, arginine vasopressin receptor 1A, fibroblast growth factor 10, claudin 7, G Protein Subunit Gamma 3, cholecystokinin B receptor and cytosolic phospholipase A2. Metabolomics analysis found significant differences in 87 metabolites, which were enriched in unsaturated fatty acid biosynthesis, and sphingolipid signaling pathways. These results provide valuable information on the molecular mechanisms of follicular cyst formation, which may facilitate the development of new therapeutics to prevent and treat follicular cysts.

Beyond protein synthesis: the emerging role of arginine in poultry nutrition and host-microbe interactions.

Arginine is a functional amino acid essential for various physiological processes in poultry. The dietary essentiality of arginine in poultry stems from the absence of the enzyme carbamoyl phosphate synthase-I. The specific requirement for arginine in poultry varies based on several factors, such as age, dietary factors, and physiological status. Additionally, arginine absorption and utilization are also influenced by the presence of antagonists. However, dietary interventions can mitigate the effect of these factors affecting arginine utilization. In poultry, arginine is utilized by four enzymes, namely, inducible nitric oxide synthase arginase, arginine decarboxylase and arginine: glycine amidinotransferase (AGAT). The intermediates and products of arginine metabolism by these enzymes mediate the different physiological functions of arginine in poultry. The most studied function of arginine in humans, as well as poultry, is its role in immune response. Arginine exerts immunomodulatory functions primarily through the metabolites nitric oxide (NO), ornithine, citrulline, and polyamines, which take part in inflammation or the resolution of inflammation. These properties of arginine and arginine metabolites potentiate its use as a nutraceutical to prevent the incidence of enteric diseases in poultry. Furthermore, arginine is utilized by the poultry gut microbiota, the metabolites of which might have important implications for gut microbial composition, immune regulation, metabolism, and overall host health. This comprehensive review provides insights into the multifaceted roles of arginine and arginine metabolites in poultry nutrition and wellbeing, with particular emphasis on the potential of arginine in immune regulation and microbial homeostasis in poultry.

Long-term follow-up of children with carbamoyl phosphate synthase 1 deficiency detected in newborn screening.

To investigate genotype-phenotype characteristics and long-term prognosis of neonatal carbamoyl phosphate synthetase 1 (CPS1) deficiency among children through newborn screening in Zhejiang province. The clinical and follow-up data of children with CPS1 deficiency detected through neonatal screening and confirmed by tandem mass spectrometry and genetic testing in Zhejiang Province Newborn Disease Screening Center from September 2013 to August 2023 were retrospectively analyzed. A total of 4 056 755 newborns were screened and 6 cases of CPS1 deficiency were diagnosed through phenotypic and genetic testing. Ten different variations of CPS1 genewere identified in genetic testing, including 2 known pathogenic variations (c.2359C>T and c.1549+1G>T) and 8 unreported variations (c.3405-1G>T, c.2372C>T, c.1436C>T, c.2228T>C, c.2441G>A, c.3031G>A, c.3075T>C and c.390-403del). All patients had decreased citrulline levels (2.72-6.21 μmol/L), and varying degrees of elevated blood ammonia. The patients received restricted natural protein intake (special formula), arginine and supportive therapy after diagnosis, and were followed-up for a period ranging from 9 months to 10 years. Three patients experienced hyperammonemia, and one patient each had attention deficit hyperactivity disorder, transient facial twitching and increased muscle tone. One patient died, while the other five surviving patients had normal scores of the Ages & Stages Questionnaires (ASQ) and Griffiths Development Scales up to the present time; 4 cases had combined height or weight lag and one case was normal in height and weight. Low citrulline levels and hyperammonemia are common in CPS1 deficiency patients in Zhejiang. Most gene variants identified were specific to individual families, and no hotspot mutations were found. Early diagnosis through newborn screening and following standardized treatment can significantly improve the prognosis of the patients.

First specific detection and validation of tomato wilt caused by Fusarium brachygibbosum using a PCR assay.

Tomato wilt is a widespread soilborne disease of tomato that has caused significant yield losses in many tomato growing regions of the world. Previously, it was reported that tomato wilt can be caused by many pathogens, such as Fusarium oxysporum, Ralstonia solanacearum, Ralstonia pseudosolanacearum, Fusarium acuminatum, and Plectosphaerella cucumerina. In addition, we have already reported that Fusarium brachygibbosum caused symptomatic disease of tomato wilt for the first time in China. The symptoms of tomato wilt caused by these pathogens are similar, making it difficult to distinguish them in the field. However, F. brachygibbosum specific identification method has not been reported. Therefore, it is of great importance to develop a rapid and reliable diagnostic method for Fusarium brachygibbosum to establish a more effective plan to control the disease. In this study, we designed F. brachygibbosum-specific forward primers and reverse primers with a fragment size of 283bp located in the gene encoding carbamoyl phosphate synthase arginine-specific large chain by whole genome sequence comparison analysis of the genomes of eight Fusarium spp.. We then tested different dNTP, Mg2+ concentrations, and annealing temperatures to determine the optimal parameters for the PCR system. We evaluated the specificity, sensitivity and stability of the PCR system based on the optimized reaction system and conditions. The PCR system can specifically identify the target pathogens from different fungal pathogens, and the lower detection limit of the target pathogens is at concentrations of 10 pg/uL. In addition, we can accurately identify F. brachygibbosum in tomato samples using the optimized PCR method. These results prove that the PCR method developed in this study can accurately identify and diagnose F. brachygibbosum.

Evolutionary history of a cold-adapted limnephilid caddisfly: Effects of climate change and topography on genetic structure

The distribution of organisms is influenced by complex factors such as the phylogenetic evolutionary histories of species, the physiological and ecological characteristics of organisms, climate, and geographical and geohistorical features. In this study, we focused on a caddisfly, Asynarchus sachalinensis (Trichoptera: Limnephilidae), which has adapted to cold habitats. From phylogeographic analyses based on the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) and 16S rRNA regions and the nuclear DNA (nDNA) 18S rRNA, 28S rRNA, carbamoyl-phosphate synthetase (CAD), elongation factor-1 alpha (EF1-α), and RNA polymerase II (POLII) regions, two distinct genetic clades were detected. Clade I was shown to be widely distributed from Sakhalin to Honshu, whereas Clade II was only distributed within Honshu. The distributions of these clades overlapped in Honshu. The habitats were located at relatively lower altitudes for Clade I and higher altitudes for Clade II. The divergence time of these clades was estimated to be during the Pleistocene, indicating that repeated climatic changes facilitated distributional shifts. Haplotype network and demographic analyses based on the mtDNA COI region showed contrasting genetic structures in the two clades. It was indicated that the population sizes of Clade I had expanded rapidly in a recent period, whereas Clade II had maintained stable population sizes. The habitats of Clade II were typically isolated and scattered at high altitudes, resulting in restricted migration and dispersal because of their discontinuous “Sky Island” habitats. The habitats of Clade I were located at relatively low altitudes, and it was assumed that the populations were continuous, which resulted in a higher frequency of migration and dispersal between populations. Thus, differences in the spatial scale of the adapted habitats of each clade may have resulted in different patterns of population connectivity and fragmentation associated with repeated climatic changes during the Pleistocene. Our study provided new insight into the distributional patterns of cold-adapted aquatic insects in the Japanese Archipelago. Furthermore, the distributional shifts predicted by ecological niche modeling under future climatic change conditions were different for each clade. Therefore, different principles are required in the assessment of each clade to predict temporal changes in their distributions.

TMET-17. LEVERAGING METABOLIC VULNERABILITIES INDUCED BY TERT EXPRESSION FOR GLIOBLASTOMA THERAPY IN VIVO

Abstract Telomerase reverse transcriptase (TERT) is essential for telomere maintenance and tumor immortality. Although TERT is an attractive therapeutic target, targeting TERT directly is hampered by toxicity to stem cells that also rely on TERT for proliferation and the lag period before telomere attrition is observed. Targeting metabolic reprogramming associated with TERT is an alternate strategy. Our studies indicate that TERT is essential for redox homeostasis in glioblastoma cells (GBM; GBM6 and U251). Specifically, TERT increases levels of glutathione (GSH) via upregulation of the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in de novo GSH synthesis. Inhibiting GCLC using buthionine sulfoximine (BSO) or siRNA reduces synthesis of 13C-GSH from [U-13C]-glutamine and inhibits viability of GBM6 and U251 cells. However, GCLC inhibition causes a compensatory increase in synthesis of glutamate, aspartate, and pyrimidine nucleotides from [U-13C]-glutamine via upregulation of two key glutamine-utilizing enzymes i.e., glutaminase (GLS), the enzyme that generates glutamate from glutamine, and carbamoyl phosphate synthetase II (CPSII), the enzyme that converts glutamine to carbamoyl phosphate during pyrimidine biosynthesis. We then examined the therapeutic potential of inhibiting GLS and CPSII in combination with GCLC in GBMs. 6-diazo-5-oxy-L-norleucin (DON), a potent inhibitor of glutamine-utilizing enzymes including GLS and CPSII, synergizes with BSO in inhibiting viability of GBM6 and U251 cells. This effect is specific to TERT-expressing GBM cells because the combination of DON and BSO does not affect viability of normal astrocytes or astrocytomas that use the ALT pathway for telomere maintenance. Importantly, in vivo stable isotope tracing confirmed that combined treatment with JHU-083 (a brain-penetrant prodrug of DON) and BSO abrogates synthesis of GSH, aspartate, and pyrimidine nucleotides from [U-13C]-glutamine and induces apoptotic death in mice bearing intracranial GBM6 tumors.Collectively, our studies highlight the therapeutic potential of targeting metabolic vulnerabilities induced by TERT expression in GBMs in vivo.

Abstract 14779: Whole Exome Sequencing Study in Pediatric Postural Orthostatic Tachycardia Syndrome Unveils a Path Forward From Genes to Drugs

Introduction: Postural orthostatic tachycardia syndrome (POTS) is more common in Caucasian individuals with a positive family history, while the risk in females is ~4-times of that in males. To date, the etiology of POTS is poorly understood. Hypothesis: Integrative sequencing and translational approaches to identify its genetic underpinning may better understand its pathogenesis and molecular mechanisms for the development of novel precision-based therapies. Methods: We adopted exome sequencing to evaluate the etiology and molecular mechanisms of POTS. We examined a unique collection of 87 unrelated pediatric POTS cases (61 females and 26 males, 6~21 years old), using exome sequencing. Non-relationship between individuals were validated by genome wide genotyping and identity by descent (IBD) analysis. Results: All the individuals were classified as European by principal component analysis (Figure 1). We identified 184 pathogenic (P) or likely pathogenic (LP) variants (in 162 genes). Sixty-eight (78%) cases have at least one P/LP variant. Each P/LP mutation is supported by a minimum of two databases, including ClinVar, InterVar or HGMD_Pro_2023.1. Over-representation analysis (ORA) by the DisGeNET approach showed the enriched gene sets of muscle hypotonia and epilepsy/seizures with statistical significance (FDR<E-10). Clinically, among the 87 WES patients, 41 (47%) suffered from chronic fatigue, and 13 (15%) patients have the symptoms of epilepsy, seizure or convulsion. A number of drug targets in these gene sets are highlighted, including vitamin D receptor, carbamoyl-phosphate synthase 1, methylmalonyl-CoA mutase, androgen receptor, and phenylalanine hydroxylase. Conclusions: Our results suggests that chronic fatigue and epilepsy/seizure symptoms are comorbid symptoms in POTS patients, resulting from common genetic defects. Future precision-based therapies targeting these specific genetic defects may lead to improved outcome in POTS.

Sericin enhances ammonia detoxification by promotes urea cycle enzyme genes and activates hepatic autophagy in relation to CARD-9/MAPK pathway

Urea cycle is an important metabolic process that initiates in liver mitochondria and converts ammonia to urea. The impairment of ammonia detoxification, both primary and secondary causes, lead to hyperammonemia, a life-threatening condition affecting to the brain. Current treatments are not enough effective. In addition, our recent proteomics study in hypercholesterolemic rat model demonstrated that sericin enhances hepatic nitrogenous waste removal through carbamoyl-phosphate synthase 1 (CPS-1), aldehyde dehydrogenase-2 (ALDH-2), and uricase proteins. However, the underlining mechanisms regard to this property is not clarified yet. Therefore, the present study aims to examine the effect of sericin on urea cycle enzyme genes (CPS-1 and ornithine transcarbamylase; OTC) and proteins (mitogen-activated protein kinase; MAPK, caspase recruitment domain-containing protein 9; CARD-9, Microtubule-associated protein light chain 3; LC-3), which relate to urea production and liver homeostasis in hepatic cell line (HepG2) and hypercholesterolemic rat treated with or without sericin. qRT-PCR, immunohistochemistry, and electron microscopy techniques were performed. In vitro study determined that high dose of sericin at 1 mg/ml increased liver detoxification enzyme (Cytochrome P450 1A2; CYP1A2 and ALDH-2) and urea cycle enzyme (CPS-1 and OTC) genes. Both in HepG2 cell and rat liver mitochondria, sericin significantly downregulated CARD-9 (apoptotic protein) expression while upregulated MAPK (hepatic homeostasis protein) and LC-3 (autophagic protein) expressions. Hence, it might be concluded that sericin promotes ammonia detoxification by both increases urea cycle enzyme genes and enhances hepatic autophagy in associated with CARD-9/MAPK pathway (as shown by their own negative relationship). This study presents another beneficial property of sericin to develop an upcoming candidate for ammonia toxicity alleviation and liver function improvement.