Altered expression of the L-arginine/nitric oxide pathway in ovarian cancer: metabolic biomarkers and biological implications.

MOTIVATION: Ovarian cancer (OC) is a highly lethal gynecological malignancy. Extensive research has shown that OC cells undergo significant metabolic alterations during tumorigenesis. In this study, we aim to leverage these metabolic changes as potential biomarkers for assessing ovarian cancer. METHODS: A functional module-based approach was utilized to identify key gene expression pathways that distinguish different stages of ovarian cancer (OC) within a tissue biopsy cohort. This cohort consisted of control samples (n = 79), stage I/II samples (n = 280), and stage III/IV samples (n = 1016). To further explore these altered molecular pathways, minimal spanning tree (MST) analysis was applied, leading to the formulation of metabolic biomarker hypotheses for OC liquid biopsy. To validate, a multiple reaction monitoring (MRM) based quantitative LCMS/MS method was developed. This method allowed for the precise quantification of targeted metabolite biomarkers using an OC blood cohort comprising control samples (n = 464), benign samples (n = 3), and OC samples (n = 13). RESULTS: Eleven functional modules were identified as significant differentiators (false discovery rate, FDR < 0.05) between normal and early-stage, or early-stage and late-stage ovarian cancer (OC) tumor tissues. MST analysis revealed that the metabolic L-arginine/nitric oxide (L-ARG/NO) pathway was reprogrammed, and the modules related to "DNA replication" and "DNA repair and recombination" served as anchor modules connecting the other nine modules. Based on this analysis, symmetric dimethylarginine (SDMA) and arginine were proposed as potential liquid biopsy biomarkers for OC assessment. Our quantitative LCMS/MS analysis on our OC blood cohort provided direct evidence supporting the use of the SDMA-to-arginine ratio as a liquid biopsy panel to distinguish between normal and OC samples, with an area under the ROC curve (AUC) of 98.3%. CONCLUSION: Our comprehensive analysis of tissue genomics and blood quantitative LC/MSMS metabolic data shed light on the metabolic reprogramming underlying OC pathophysiology. These findings offer new insights into the potential diagnostic utility of the SDMA-to-arginine ratio for OC assessment. Further validation studies using adequately powered OC cohorts are warranted to fully establish the clinical effectiveness of this diagnostic test.

Multi-Omics Signatures Link to Ticagrelor Effects on Vascular Function in Patients With Acute Coronary Syndrome.

BACKGROUND: Long-term antiplatelet agents including the potent P2Y12 antagonist ticagrelor are indicated in patients with a previous history of acute coronary syndrome. We sought to compare the effect of ticagrelor with that of aspirin monotherapy on vascular endothelial function in patients with prior acute coronary syndrome. METHODS: This was a prospective, single center, parallel group, investigator-blinded randomized controlled trial. We randomized 200 patients on long-term aspirin monotherapy with prior acute coronary syndrome in a 1:1 fashion to receive ticagrelor 60 mg BD (n=100) or aspirin 100 mg OD (n=100). The primary end point was change from baseline in brachial artery flow-mediated dilation at 12 weeks. Secondary end points were changes to platelet activation marker (CD41_62p) and endothelial progenitor cell (CD34/133) count measured by flow cytometry, plasma level of adenosine, IL-6 (interleukin-6) and EGF (epidermal growth factor), and multi-omics profiling at 12 weeks. RESULTS: After 12 weeks, brachial flow-mediated dilation was significantly increased in the ticagrelor group compared with the aspirin group (ticagrelor: 3.48±3.48% versus aspirin: -1.26±2.85%, treatment effect 4.73 [95% CI, 3.85-5.62], P<0.001). Nevertheless ticagrelor treatment for 12 weeks had no significant effect on platelet activation markers, circulating endothelial progenitor cell count or plasma level of adenosine, IL-6, and EGF (all P>0.05). Multi-omics pathway assessment revealed that changes in the metabolism and biosynthesis of amino acids (cysteine and methionine metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis) and phospholipids (glycerophosphoethanolamines and glycerophosphoserines) were associated with improved brachial artery flow-mediated dilation in the ticagrelor group. CONCLUSIONS: In patients with prior acute coronary syndrome, ticagrelor 60 mg BD monotherapy significantly improved brachial flow-mediated dilation compared with aspirin monotherapy and was associated with significant changes in metabolomic and lipidomic signatures. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03881943.

An intermolecular disulfide bond is required for thermostability and thermoactivity of ß-glycosidase from Thermococcus kodakarensis KOD1.

Scientists are interested in understanding the molecular origin of protein thermostability and thermoactivity for possible biotechnological applications. The enzymes from extremophilic organisms have been of particular interest in the last two decades. ß-glycosidase, Tkßgly is a hyperthermophilic enzyme from Thermococcus kodakarensis KOD1. Tkßgly contains two conserved cysteine residues, C88 and C376. The protein tertiary structure obtained through homology modeling suggests that the C88 residue is located on the surface whereas C376 is inside the protein. To study the role of these cysteine residues, we substituted C88 and C376 with serine residues through site-directed mutagenesis. The wild-type and C376S protein existed in dimeric form and C88S in monomeric form, in an SDS-PAGE gel under non-reducing conditions. Optimal temperature experiments revealed that the wild-type was active at 100 °C whereas the C88S mutant exhibited optimal activity at 70 °C. The half-life of the enzyme at 70 °C was drastically reduced from 266 h to less than 1 h. Although C88 was not present in the active site region, the kcat/Km of C88S was reduced by 2-fold. Based on the structural model and biochemical properties, we propose that C88 is crucial in maintaining the thermostability and thermoactivity of the Tkßgly enzyme.

Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant.

Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.

Association between fetal nuchal translucency thickness in first trimester and subsequent gestational hypertension and preeclampsia.

Increased fetal nuchal translucency (NT) in the first trimester is associated with adverse pregnancy outcomes. Whether the increased NT is also associated with an increased frequency of pregnancy-associated hypertension (PAH) is not known. Seven hundred and seventy-nine pregnant women who received NT-based Down syndrome screening and delivered their babies at our hospital by September 2000 were enrolled into this study. Among these women, there are 46 cases of preeclampsia, 68 cases of gestational hypertension (GH); 665 women without any adverse pregnancy outcomes served as controls. Correlation analysis demonstrated that NT MoM (multiples of median) level had a positive association with maternal diastolic blood pressure at the time of admission for delivery (r = 0.104; p < 0.01). The severity of PAH was concordant with the stepwise increase of mean NT MoM level, which was 0.88 in control, 1.07 in gestational hypertension, and 1.13 in preeclampsia (p < 0.001). Using the 95th (1.52 MoM) and 90th (1.31 MoM) percentiles of NT thickness as cut-offs, the sensitivities and odds ratios of the women at risk for developing GH after 20 weeks of gestation were 8.8%, 19.1% and 1.98, 2.15 respectively, while for preeclampsia were 10.9%, 28.3% and 2.49, 3.58 respectively. It is concluded that the pathological changes in the placenta responsible for the development of PAH may also influence the physiological decrease of NT thickness in late first trimester. However, the sensitivity of fetal NT measurement in first trimester is not sufficient as a single marker for predicting the pregnant women at risk for subsequent PAH.