Sequential Inhibition of PARP and BET as a Rational Therapeutic Strategy for Glioblastoma.

PARP inhibitors (PARPi) hold substantial promise in treating glioblastoma (GBM). However, the adverse effects have restricted their broad application. Through unbiased transcriptomic and proteomic sequencing, it is discovered that the BET inhibitor (BETi) Birabresib profoundly alters the processes of DNA replication and cell cycle progression in GBM cells, beyond the previously reported impact of BET inhibition on homologous recombination repair. Through in vitro experiments using established GBM cell lines and patient-derived primary GBM cells, as well as in vivo orthotopic transplantation tumor experiments in zebrafish and nude mice, it is demonstrated that the concurrent administration of PARPi and BETi can synergistically inhibit GBM. Intriguingly, it is observed that DNA damage lingers after discontinuation of PARPi monotherapy, implying that sequential administration of PARPi followed by BETi can maintain antitumor efficacy while reducing toxicity. In GBM cells with elevated baseline replication stress, the sequential regimen exhibits comparable efficacy to concurrent treatment, protecting normal glial cells with lower baseline replication stress from DNA toxicity and subsequent death. This study provides compelling preclinical evidence supporting the development of innovative drug administration strategies focusing on PARPi for GBM therapy.

Impact of Duration of Diabetes Mellitus on Long-term Outcome in Type 2 Diabetic Patients With Primary Percutaneous Coronary Intervention After the First Myocardial Infarction.

BACKGROUND: Diabetes mellitus (DM) increases the risk of mortality in patients with acute myocardial infarction (AMI). The impact of the diabetes duration on the long-term outcome of those with percutaneous coronary intervention (PCI) after the first AMI is unclear. In this study, we evaluated the predictive value of diabetes duration in the occurrence of major adverse cardiovascular and cerebrovascular events (MACCEs). METHODS: A total of 394 type 2 DM patients with PCI after the first AMI were enrolled and were divided into two groups by the diabetes duration. A short-DM group with diabetes duration of < 5 years and a long-DM group with a duration of ≥ 5 years. The clinical endpoint was MACCEs. RESULTS: Multivariate Cox regression analysis found that the diabetes duration was independently associated with increased occurrence of MACCEs [HR 1.512, 95% CI: (1.033, 2.215), p = 0.034], along with hypertension, Killip class III or IV, creatinine, multivessel disease, and continuous hypoglycemic therapy. After adjusting for the confounding variables, a nested Cox model showed that diabetes duration was still an independent risk factor of MACCEs [HR 1.963, 95% CI: (1.376, 2.801), p < 0.001]. The Kaplan-Meier survival curve illustrated a significantly high risk of MACCEs (HR 2.045, p < 0.0001) in long-duration DM patients. After propensity score matching, a longer diabetes duration was associated with an increased risk of MACCE occurrence. CONCLUSION: Long-duration diabetes was independently associated with poor clinical outcomes after PCI in patients with their first myocardial infarction, Despite the diabetes duration, continuous hypoglycemic therapy significantly improved long-term clinical outcomes.

[Ginsenoside F1 inhibits cholesterol overload in oxidative-damaged cells through SREBP2/HMGCR pathway].

OBJECTIVE: To investigate the inhibitory mechanisms of ginsenoside F1 on hydrogen peroxide induced cholesterol metabolism disorder and oxidative stress in HepG2 cells. METHODS: 1, 1-diphenyl-2-picrylhydrazyl(DPPH) and oxygen radical absorbance capacity(ORAC) tests were used to detect the scavenging effect of ginsenoside F1 on nitrogen and oxygen free radicals. HepG2 cells were treated with 400 µmol/L hydrogen peroxide and pretreated with 10, 20 and 40 µmol/L ginsenoside F1. Mitochondrial membrane potential(MMP) and total cholesterol levels were detected by JC-1 method and cholesterol kit, respectively. The protein expression levels of sterol-regulatory element binding proteins(SREBP2)and 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR) in cholesterol synthesis pathway were detected by Western blot. RESULTS: The DPPH clearance rate of ginsenoside F1 was much lower than that of 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid(Trolox), but the ORAC capability of ginsenoside F1 was stronger, which was comparable to Trolox. The MMP and protein expression of SREBP2 were significantly decreased in injured group(P<0.05). The cholesterol and protein expression of HMGCR were significantly increased(P<0.05). Whereas, compared with the injured group, the MMP and protein expression of SREBP2 were significantly increased after 10, 20 and 40 µmol/L ginsenoside F1 pretreatment of injured cells(P<0.05). The cholesterol level and protein expression of HMGCR were significantly lower than injured group with concentration-dependent decreases(P<0.05). CONCLUSION: Ginsenoside F1 can protect against hydrogen peroxide induced oxidative stress in HepG2 cells by inhibiting oxygen free radicals and protecting mitochondria. And its mechanism may be related to the intervention of SREBP2/HMGCR pathway in regulating cellular cholesterol anabolism.

Proteomic characterization identifies clinically relevant subgroups of soft tissue sarcoma.

Soft tissue sarcoma is a broad family of mesenchymal malignancies exhibiting remarkable histological diversity. We portray the proteomic landscape of 272 soft tissue sarcomas representing 12 major subtypes. Hierarchical classification finds the similarity of proteomic features between angiosarcoma and epithelial sarcoma, and elevated expression of SHC1 in AS and ES is correlated with poor prognosis. Moreover, proteomic clustering classifies patients of soft tissue sarcoma into 3 proteomic clusters with diverse driven pathways and clinical outcomes. In the proteomic cluster featured with the high cell proliferation rate, APEX1 and NPM1 are found to promote cell proliferation and drive the progression of cancer cells. The classification based on immune signatures defines three immune subtypes with distinctive tumor microenvironments. Further analysis illustrates the potential association between immune evasion markers (PD-L1 and CD80) and tumor metastasis in soft tissue sarcoma. Overall, this analysis uncovers sarcoma-type-specific changes in proteins, providing insights about relationships of soft tissue sarcoma.

Comparison of two pathological processing methods for large endoscopic submucosal dissection (ESD) specimens.

AIMS: Accurate histopathological evaluation of the endoscopic submucosal dissection (ESD) specimens is essential for clinicians to guide further triage and management. This study aimed to report a novel processing technique for large ESD (≥4 cm) specimens. METHODS: 92 patients with colorectal neoplasms who had undergone ESD were included. 46 ESD specimens were treated with conventional handling process, while the rest 46 cases were given the optimised method. Macrobiocassettes and L-shaped embedding moulds were applied in the optimised method. We evaluated the efficacy of this improved procedure in terms of the number of paraffin blocks, storage space and time consumption of pathological assessment. RESULTS: The average diameter of ESD specimens was 4.5±0.4 cm and 4.7±0.5 cm in the control and test group (p=0.023), respectively. In control group, 398 paraffin blocks of 46 cases were obtained. With the same cases number and larger lesion size, only 276 blocks were achieved in test group (p<0.001). As for the storage space, the total volume of paraffin blocks and slides (4554.0 cm3 and 1207.5 cm3) of optimised method was significantly reduced compared with the control group (6208.8 cm3 and 1741.3 cm3) (p=0.001, p<0.001). In addition, the optimised method was superior to the conventional one in shortening time consumption of pathological assessment (164.5 min and 269.0 min, p<0.001). CONCLUSIONS: The optimised technique not only reduced the workload and storage space, but also facilitated accurate pathological assessment.

Impact of ternary NADES prepared from proline, glucose and water on the Maillard reaction: Reaction activity, Amadori compound yield, and taste-enhancing ability.

This study employed proline, glucose, and water to prepare natural deep eutectic solvents (NADES) through heating and stirring. The Maillard reaction was then performed, producing a high yield of Amadori rearrangement product (ARP) and physicochemical properties of NADES were examined for impacts on the reaction. Water had a dual function by promoting the formation of hydrogen bonding networks within the NADES when present at less than 15%, and also working as a diluting agent that could potentially disturb its structure when exceed 15%. These changes further affected the subsequent Maillard reaction, especially the ARP accumulation (reached the highest when water content was 15%). Correlation analysis shows strong positive viscosity-ARP and negative water activity-ARP correlations within a range. Moreover, the product (rich in ARP) remarkably enhanced umami and saltiness. This finding provides insights into modulating the Maillard reaction by adjusting NADES properties, demonstrating feasibility of this approach for flavor enhancer development.

The Correlation Between Health Risk Factors and Diabesity and Lipid Profile Indicators: The Role Mediator of TSH.

Introduction: Obesity in adults is a problem, particularly when paired with other metabolic abnormalities. Previous research have linked various screening approaches to diabetes, but additional evidence points to the relevance of combining diabetes screening methods with obesity and its effects. This research examined the impact of thyroid hormones (TSHs) and health risk factors (HRFs) in screening for obesity and diabetes in Chinese populations, and whether age can modulate this association. Methods: From March to July 2022, the Hefei Community Health Service Center connected with the First Affiliated Hospital of Anhui Medical University was chosen, and the multi-stage cluster sample approach was utilized to test adults aged 21-90 in each community. Latent category analysis (LCA) was performed to investigate the clustering patterns of HRFs. A one-way ANOVA was used to examine waist circumference (WC), biochemical markers, and general data. Furthermore, multivariate logistic regression analysis was utilized to investigate the relationship between health risk variables and WC. Results: A total of 750 individuals without a history of major problems who had a community health physical examination were chosen, with missing data greater than 5% excluded. Finally, 708 samples were included in the study with an effective rate of 94.4%. The average WC was (90.0±10.33) cm, the prevalence in the >P75, P50~P75, P25~P50, and ≤P25 groups were 24.7%, 18.9%, 28.7% and 27.7%, respectively. The average TSH was (2.76±2.0) µIU/mL. Male (ß=1.91), HOMA-IR (ß=0.06), TyG (ß=2.41), SBP (ß=0.08), TG (ß=0.94) and UA (ß=0.03) were more likely to have a higher prevalence of WC level. The analyses revealed significant correlations between HRFs, TSH, age, other metabolic indexes and WC (P < 0.05). Discussion: Our findings suggest that the quality of metabolic-related indicators used to successfully decrease diabetes in Chinese individuals with high HRFs levels should be prioritized. Comprehensive indicators might be a useful and practical way for measuring the metabolic evolution of diabetes level levels.

Effects of Percutaneous Coronary Intervention and Coronary Artery Bypass Grafting on Clinical Outcomes in Patients with Reduced Ejection Fraction Heart Failure and Coronary Heart Disease: A Meta-Analysis.

OBJECTIVE: To clarify the effects of percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) on the clinical outcomes of patients with coronary heart disease (CHD) complicated with reduced ejection fraction heart failure (HFrEF) through meta-analysis. METHODS: Three major literature databases - PubMed, Web of Science, and Cochrane - were searched by search terms and the literature retrieval time was publications dating from January 2007 to December 2021. To search for observational studies and randomized controlled trials (RCT) comparing the efficacy of PCI and CABG in patients with CHD and HFrEF, the abstract or full text of the literature was read and the final included literature was determined, according to inclusion and exclusion criteria. The quality of the included literature was evaluated using the Ottawa scale and data extraction was further completed. Data analysis was made using RevMan5.4 and R4.1 software; relevant forest plots and funnel plots were made, according to the extracted data. Egger's test was used to evaluate whether the data had publication bias. Outcomes were the major adverse cardiovascular events (MACE). RESULTS: A total of 10 studies were included and 11,032 subjects were included, made up of 5,521 cases of PCI and 5,511 cases of CABG. The results showed no significant difference between the two groups in cardiac mortality (CM) (RR=1.13, 95% CI 0.98-1.30, P = 0.10) and in overall all-cause mortality (ACM) (RR=1.12, 95% CI 0.92-1.37, P = 0.25). In the subgroup analysis of ACM, in the subgroups with left ventricular ejection fraction (LVEF) less than 35% and exceeding 35% and less than 50% (RR=1.12, 95% CI 0.92-1.37, P = 0.25) between the two groups, there was no statistical difference. However, among other MACE, compared with the PCI group, the CABG group had a lower risk of MACE (RR=1.58, 95%CI 1.49-1.70, P < 0.00001), myocardial infarction (MI) (RR=1.99, 95% CI 1.02-3.88, P = 0.04), heart failure (HF) (RR=1.29, 95% CI 1.17-1.43, P < 0.00001) and revascularization (RR=2.74, 95% CI 1.93-3.90, P < 0.00001). Finally in the CABG group, the risk of stroke or transient ischemic attack (TIA) was higher (RR=0.71, 95% CI 0.58-0.86, P = 0.0006) than the PCI group. CONCLUSIONS: The mortality rates of PCI and CABG were similar in patients with CHD complicated with HFrEF. Compared with PCI, CABG had a lower incidence of MACE, MI, HF, and revascularization, and a higher incidence of stroke or TIA.

Comprehensive proteogenomic characterization of early duodenal cancer reveals the carcinogenesis tracks of different subtypes.

The subtypes of duodenal cancer (DC) are complicated and the carcinogenesis process is not well characterized. We present comprehensive characterization of 438 samples from 156 DC patients, covering 2 major and 5 rare subtypes. Proteogenomics reveals LYN amplification at the chromosome 8q gain functioned in the transmit from intraepithelial neoplasia phase to infiltration tumor phase via MAPK signaling, and illustrates the DST mutation improves mTOR signaling in the duodenal adenocarcinoma stage. Proteome-based analysis elucidates stage-specific molecular characterizations and carcinogenesis tracks, and defines the cancer-driving waves of the adenocarcinoma and Brunner's gland subtypes. The drug-targetable alanyl-tRNA synthetase (AARS1) in the high tumor mutation burden/immune infiltration is significantly enhanced in DC progression, and catalyzes the lysine-alanylation of poly-ADP-ribose polymerases (PARP1), which decreases the apoptosis of cancer cells, eventually promoting cell proliferation and tumorigenesis. We assess the proteogenomic landscape of early DC, and provide insights into the molecular features corresponding therapeutic targets.

Proteogenomics of diffuse gliomas reveal molecular subtypes associated with specific therapeutic targets and immune-evasion mechanisms.

Diffuse gliomas are devastating brain tumors. Here, we perform a proteogenomic profiling of 213 retrospectively collected glioma tumors. Proteogenomic analysis reveals the downstream biological events leading by EGFR-, IDH1-, TP53-mutations. The comparative analysis illustrates the distinctive features of GBMs and LGGs, indicating CDK2 inhibitor might serve as a promising drug target for GBMs. Further proteogenomic integrative analysis combined with functional experiments highlight the cis-effect of EGFR alterations might lead to glioma tumor cell proliferation through ERK5 medicates nucleotide synthesis process. Proteome-based stratification of gliomas defines 3 proteomic subgroups (S-Ne, S-Pf, S-Im), which could serve as a complement to WHO subtypes, and would provide the essential framework for the utilization of specific targeted therapies for particular glioma subtypes. Immune clustering identifies three immune subtypes with distinctive immune cell types. Further analysis reveals higher EGFR alteration frequencies accounts for elevation of immune check point protein: PD-L1 and CD70 in T-cell infiltrated tumors.

Proteogenomic characterization of cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a highly heterogeneous cancer with limited understanding and few effective therapeutic approaches. We aimed at providing a proteogenomic CCA characterization to inform biological processes and treatment vulnerabilities. APPROACH AND RESULTS: Integrative genomic analysis with functional validation uncovered biological perturbations downstream of driver events including DPCR1 , RBM47 mutations, SH3BGRL2 copy number alterations, and FGFR2 fusions in CCA. Proteomic clustering identified three subtypes with distinct clinical outcomes, molecular features, and potential therapeutics. Phosphoproteomics characterized targetable kinases in CCA, suggesting strategies for effective treatment with CDK and MAPK inhibitors. Patients with CCA with HBV infection showed increased antigen processing and presentation (APC) and T cell infiltration, conferring a favorable prognosis compared with those without HBV infection. The characterization of extrahepatic CCA recommended the feasible application of vascular endothelial-derived growth factor inhibitors. Multiomics profiling presented distinctive molecular characteristics of the large bile duct and the small bile duct of intrahepatic CCA. The immune landscape further revealed diverse tumor immune microenvironments, suggesting immune subtypes C1 and C5 might benefit from immune checkpoint therapy. TCN1 was identified as a potential CCA prognostic biomarker, promoting cell growth by enhancing vitamin B12 metabolism. CONCLUSIONS: We characterized the proteogenomic landscape of 217 CCAs with 197 paired normal adjacent tissues and identified their subtypes and potential therapeutic targets. The multiomics analyses with other databases and some functional validations have indicated strategies regarding the clinical, biological, and therapeutic approaches to the management of CCA.

Stellettin B Sensitizes Glioblastoma to DNA-Damaging Treatments by Suppressing PI3K-Mediated Homologous Recombination Repair.

Glioblastoma (GBM) is the most aggressive type of cancer. Its current first-line postsurgery regimens are radiotherapy and temozolomide (TMZ) chemotherapy, both of which are DNA damage-inducing therapies but show very limited efficacy and a high risk of resistance. There is an urgent need to develop novel agents to sensitize GBM to DNA-damaging treatments. Here it is found that the triterpene compound stellettin B (STELB) greatly enhances the sensitivity of GBM to ionizing radiation and TMZ in vitro and in vivo. Mechanistically, STELB inhibits the expression of homologous recombination repair (HR) factors BRCA1/2 and RAD51 by promoting the degradation of PI3Kα through the ubiquitin-proteasome pathway; and the induced HR deficiency then leads to augmented DNA damage and cell death. It is further demonstrated that STELB has the potential to rapidly penetrate the blood-brain barrier to exert anti-GBM effects in the brain, based on zebrafish and nude mouse orthotopic xenograft tumor models. The study provides strong evidence that STELB represents a promising drug candidate to improve GBM therapy in combination with DNA-damaging treatments.

PMS2 Expression With Combination of PD-L1 and TILs for Predicting Survival of Esophageal Squamous Cell Carcinoma.

Background: DNA mismatch repair (MMR) deficiency (dMMR) has been recognized as an important biomarker for immunotherapy in esophageal squamous cell carcinoma (ESCC), along with programmed death ligand 1 (PD-L1) expression and/or tumor-infiltrated lymphocytes (TILs). However, in ESCC, MMR protein assessment has not been well studied at present. Methods: A total of 484 ESCC tissues treated between 2007 and 2010, in our hospital, were enrolled. Immunohistochemical expression of MLH1, MSH2, MSH6, PMS2, and PD-L1 on tissue microarray specimens and clinicopathological features, including TILs, were analyzed retrospectively. Results: Out of the 484 studied cases, loss of MLH1, MSH2, MSH6, and PMS2 expression were found in 6.8%, 2.1%, 8.7%, and 4.8% patients, respectively. dMMR was found in 65 patients, 37 cases involved in one MMR protein, 17 cases involved in two proteins, 7 cases involved in three proteins, and 4 cases involved in four proteins. There was no significant survival difference between pMMR (MMR-proficient) and dMMR patients (P>0.05). However, 224 patients with low PMS2 expression had better DFS and OS than 260 patients with high PMS2 expression (P=0.006 for DFS and 0.008 for OS), which was identified as an independent prognostic factor in multivariate analyses. Positive PD-L1 expression was detected in 341 (70.5%) samples. In stage I-II disease, patients with PD-L1 expression had better DFS and OS than those without PD-L1 expression(P<0.05), which was not found in stage III-IV disease. With the ITWG system, 40.1% of cases were classified as high TILs. Patients in the high-TILs group tended to have better DFS (P=0.055) and OS (P=0.070) than those in the low-TILs group and the differences were statistically significant in pMMR, high MSH6, or PMS2 expression cases (P<0.05). Also, high PMS2 expression patients with both PD-L1 expression and high TILs, had similar DFS and OS compared with low PMS2 expression patients (P>0.05), which were much better than other high PMS2 expression patients. Conclusion: The expression level of MMR proteins could also be used as a prognostic factor in ESCC and PMS2 expression outperformed other MMR proteins for predicting survival. The combination of PD-L1 expression and TILs may lead to more efficient risk stratification of ESCC.

Proteomic analysis reveals key differences between squamous cell carcinomas and adenocarcinomas across multiple tissues.

Squamous cell carcinoma (SCC) and adenocarcinoma (AC) are two main histological subtypes of solid cancer; however, SCCs are derived from different organs with similar morphologies, and it is challenging to distinguish the origin of metastatic SCCs. Here we report a deep proteomic analysis of 333 SCCs of 17 organs and 69 ACs of 7 organs. Proteomic comparison between SCCs and ACs identifies distinguishable pivotal pathways and molecules in those pathways play consistent adverse or opposite prognostic roles in ACs and SCCs. A comparison between common and rare SCCs highlights lipid metabolism may reinforce the malignancy of rare SCCs. Proteomic clusters reveal anatomical features, and kinase-transcription factor networks indicate differential SCC characteristics, while immune subtyping reveals diverse tumor microenvironments across and within diagnoses and identified potential druggable targets. Furthermore, tumor-specific proteins provide candidates with differentially diagnostic values. This proteomics architecture represents a public resource for researchers seeking a better understanding of SCCs and ACs.

Prognostic value and characterization of NTRK1 variation by fluorescence in situ hybridization in esophageal squamous cell carcinoma.

PURPOSE: Rearrangement of the neurotrophic tyrosine kinase receptor (NTRK) 1 gene is a target of tropomyosin receptor kinase A (TRKA) inhibitors, and its targeted drug (larotrectinib) has been approved by the US Food and Drug Administration. We investigated the existence and prognostic importance of NTRK1 variation in esophageal squamous cell carcinoma (ESCC). METHODS: Fluorescence in situ hybridization of a NTRK1 rearrangement was conducted on 523 ESCC samples through tissue microarrays. Kaplan-Meier curves with log-rank tests were used to evaluate survival. RESULTS: We identified 8 (1.5%), 35(6.7%) and 109 (20.8%) cases with a NTRK1 rearrangement using 15%, 10% and 5% as cut-off values, respectively. We observed copy number (CN) variation of NTRK1 in some cases: 79 (15.1%) cases had a gain in NTRK1 CN ≥ 3, and 24 (4.6%) cases had NTRK1 CN ≥ 4. A NTRK1 rearrangement at the above-mentioned thresholds was not related to disease-free survival (DFS, P = 0.45, 0.47, 0.87) and overall survival (OS, P = 0.80, 0.74, 0.57), respectively. Gain in NTRK1 CN was associated with a poor prognosis irrespective of whether NTRK1 CN ≥ 4 (DFS, P = 0.015; OS, P = 0.035) or NTRK1 CN ≥ 3 (DFS, P = 0.039; OS, P = 0.025). CONCLUSION: A NTRK1 rearrangement occurred rarely in ESCC. The increased CN of NTRK1 might be a prognostic indicator for DFS and OS in patients with ESCC.

[Corncornin-3-O-glucoside mediates the NF-κB pathway to inhibit H_2O_2-induced apoptosis].

OBJECTIVE: To study the anti-apoptotic effect of cyanidin-3-O-glucoside(C3 G) on H_2O_2-induced injury in human embryonic kidney(HEK)-293 cells. METHODS: Hydrogen peroxide induced injury of HEK-293 cell was used as the research object. HEK-293 cells were pretreated with different concentrations of C3 G(1. 25, 5, 20 µmol/L). The anti-apoptotic effects of C3 G on injured cells were examined by the release rates of LDH and mitochondrial membrane potential(MMP). Western blot and qRT-PCR were used to detect the protein expression and mRNA expression of NF-κB P65. RESULTS: The result showed that the release rate of LDH was increased, MMP was decreased, the protein and mRNA of P65 was increased after H_2O_2 inducing. Whereas, the release rates of LDH were significantly lower than that of the injured group after 1. 25, 5, 20 µmol/L C3 G pretreatment of injured cells(P<0. 05). The MMP of C3 G group was significantly higher than injured group with concentration-dependent increases. The proteins and mRNA of P65 were also significantly lower than that of injured group(P<0. 05). CONCLUSION: Cyanidin-3-O-glucoside shows anti-apoptotic effect on H_2O_2-induced injury in HEK-293 cell. The mechanisms of anti-apoptotic effects may be to achieve by protecting cell biofilms, and inhibiting the activity of NF-κB signaling pathway.

Site-Specific and Quantitative N-Glycan Heterogeneity Analysis of the Charge Isomers of an Anti-VEGF Recombinant Fusion Protein by High-Resolution Two-Dimensional Gel Electrophoresis and Mass Spectrometry.

Glycan modification prompts important concerns about the quality control of biopharmaceutical production. Conbercept is a multiglycosylated recombinant fusion protein drug approved for the treatment of age-related macular degeneration (AMD). With 14 N-glycosites in the molecule and 7 N-glycosites in the monomer, the charge isomer separation and characterization of conbercept pose great challenges due to its enormous heterogeneities. The batch-to-batch stability on the charge isomer distribution and the possible causation of the pattern necessitate the development of effective analytical approaches. Here, the immobilized pH gradient (IPG)-based two-dimensional gel electrophoresis (2-DE) approach was first optimized to achieve high-resolution, high-reproducible separation and preparation of charge isomers. Then, combined with the quantitative analysis strategy of site-specific N-glycan heterogeneity based on the diagnostic MS2 ion (peptides+GlcNAc, Y1 ions) of glycopeptides, an integrated approach for the quantitation of site-specific N-glycan heterogeneities among charge isomers was established. Finally, the quantitation of site-specific N-glycoforms in each of the 2-DE resolved spots were performed, and the results showed that the sialylation tends to increase for gel spots located in the acidic regions. This study provides an effective approach to separate the charge isomers of the heavily glycosylated protein drugs, and to quantitatively explore the site-specific N-glycans dynamics along with the different charge isomers.

rAAV9-Mediated MEK1 Gene Expression Restores Post-conditioning Protection Against Ischemia Injury in Hypertrophic Myocardium.

PURPOSE: We investigated whether increased expression of activated mitogen-activated protein kinase (MAPK) kinases 1 (MEK1) restores ischemic post-conditioning (IPostC) protection in hypertrophic myocardium following ischemia/reperfusion (I/R) injury. METHODS: C57Bl/6 mice received recombinant adeno-associated virus type 9 (rAAV9)-mediated activated MEK1 gene delivery systemically, then following the induction of cardiac hypertrophy via transverse aortic constriction for 4 weeks. In a Langendorff model, hypertrophic hearts were subjected to 40 min/60 min I/R or with IPostC intervention consisting of 6 cycles of 10 s reperfusion and 10 s no-flow before a 60-min reperfusion. Hemodynamics, infarct size (IS), myocyte apoptosis and changes in expression of reperfusion injury salvage kinase (RISK) pathway were examined. RESULTS: rAAV9-MEK1 gene delivery led to a 4.3-fold and 2.7-fold increase in MEK1 mRNA and protein expression in the heart versus their control values. I/R resulted in a larger IS in hypertrophic than in non-hypertrophic hearts (52.3 ± 4.7% vs. 40.0 ± 2.5%, P < 0.05). IPostC mediated IS reduction in non-hypertrophic hearts (27.6 ± 2.6%, P < 0.05), while it had no significant effect in hypertrophic hearts (46.5 ± 3.1%, P=NS) compared with the IS in non-hypertrophic or hypertrophic hearts subjected to I/R injury only, respectively. Hemodynamic decline induced by I/R was preserved by IPostC in non-hypertrophic hearts but not in hypertrophic hearts. rAAV9-MEK1 gene delivery restored IPostC protection in hypertrophic hearts evidenced by reduced IS (32.0 ± 2.8% vs. 46.5 ± 3.1%) and cardiac cell apoptosis and largely preserved hemodynamic parameters. These protective effects were associated with significantly increased phosphorylation of ERK1/2 and ribosomal protein S6 kinases (p70S6K), but it had no influence on Akt and glycogen synthase kinase-3ß. CONCLUSION: These results demonstrated that rAAV9-mediated activated MEK1 expression restores IPostC protection in the hypertrophic heart against I/R injury through the activation of ERK pathway.

Integrated Strategy for Large-Scale Investigation on Protein Core Fucosylation Stoichiometry Based on Glycan-Simplification and Paired-Peaks-Extraction.

Core fucosylation (CF) is a special form of N-glycosylation and plays an important role in pathological and biological processes. Increasing efforts in this area are focused on the identification of CF glycosites, whereas evidence showed that the stoichiometry of CF occupancy is functionally important. Here, an integrated strategy based on "Glycan-Simplification and Paired-Peaks-extraction" (GSPPE) for detecting large-scale stoichiometries of CF was developed. After HILIC enrichment of intact glycopeptides, sequential cleavages by endoglycosidases H and endoglycosidases F3 were performed to generate simplified glycopeptide forms (SGFs), i.e., peptide-GlcNAc (pep-HN) and peptide-GlcNAc-Fucose (pep-CF). These paired SGFs were found to be eluted consecutively on a reversed-phase chromatography column, which allowed us to obtain peak areas of SGF pairs, even if only one of the peaks was captured by the mass spectrometer (MS), by introducing the Paired-Peaks-Extraction algorithm. Thus, the missing value dilemma of random data-dependent MS/MS acquisition was reduced and the stoichiometry of site-specific CF could be calculated. We systematically evaluated the feasibility of this strategy using standard glycoproteins and then explored urinary samples from healthy individuals and hepatocellular carcinoma (HCC) patients. In total, 1449 highly reliable core fucose glycosites and their corresponding CF stoichiometries were obtained. Dozens of glycosites that differed significantly in the urine of healthy individuals and HCC patients were disclosed. The developed approach and program presented here may promote studies on core fucosylation and lead to a deeper understanding of their dysregulation in physiological- or pathological processes.

Monocyte to high-density lipoprotein cholesterol ratio as long-term prognostic marker in patients with coronary artery disease undergoing percutaneous coronary intervention.

BACKGROUND: The relation between monocyte to high-density lipoprotein cholesterol ratio (MHR) and coronary artery disease (CAD) undergoing percutaneous coronary intervention (PCI) remains controversial. The present study aims to assess the prognostic value of MHR in patients with CAD who underwent PCI. METHODS: A total of 673 CAD patients were retrospectively enrolled and divided into four groups according to MHR values. Multivariate Cox regression analysis was performed to study the effects of different variables to clinical outcomes reported as major adverse cardiac events (MACE) and all-cause mortality (ACM). RESULTS: In a multivariate Cox analysis, after adjustment of other confounders, MHR was found to be an independent predictor of ACM (HR: 3.655; 95% CI: 1.170-11.419, P = 0.026) and MACE (HR =2.390, 95% CI 1.379-4.143, p < 0.002). Having a MHR in the third and fourth quartile were associated with a 2.83-fold and 3.26 -flod increased risk of MACE. CONCLUSIONS: MHR is an independent predictor of ACM and MACE in CAD patients undergoing PCI.