NONO promotes gallbladder cancer cell proliferation by enhancing oncogenic RNA splicing of DLG1 through interaction with IGF2BP3/RBM14.

Gallbladder cancer (GBC) is a highly malignant and rapidly progressing tumor of the human biliary system, and there is an urgent need to develop new therapeutic targets and modalities. Non-POU domain-containing octamer-binding protein (NONO) is an RNA-binding protein involved in the regulation of transcription, mRNA splicing, and DNA repair. NONO expression is elevated in multiple tumors and can act as an oncogene to promote tumor progression. Here, we found that NONO was highly expressed in GBC and promoted tumor cells growth. The dysregulation of RNA splicing is a molecular feature of almost all tumor types. Accordingly, mRNA-seq and RIP-seq analysis showed that NONO promoted exon6 skipping in DLG1, forming two isomers (DLG1-FL and DLG1-S). Furthermore, lower Percent-Spliced-In (PSI) values of DLG1 were detected in tumor tissue relative to the paraneoplastic tissue, and were associated with poor patient prognosis. Moreover, DLG1-S and DLG1-FL act as tumor promoters and tumor suppressors, respectively, by regulating the YAP1/JUN pathway. N6-methyladenosine (m6A) is the most common and abundant RNA modification involved in alternative splicing processes. We identified an m6A reader, IGF2BP3, which synergizes with NONO to promote exon6 skipping in DLG1 in an m6A-dependent manner. Furthermore, IP/MS results showed that RBM14 was bound to NONO and interfered with NONO-mediated exon6 skipping of DLG1. In addition, IGF2BP3 disrupted the binding of RBM14 to NONO. Overall, our data elucidate the molecular mechanism by which NONO promotes DLG1 exon skipping, providing a basis for new therapeutic targets in GBC treatment.

Neoadjuvant Chemotherapy or Adjuvant Chemotherapy for Esophageal Squamous Cell Carcinoma.

BACKGROUND: Chemotherapy and chemoradiation have become essential adjuncts to improve the survival of patients with resectable esophageal squamous cell carcinoma (ESCC) in the perioperative period. Although preoperative treatment plus surgery is commonly used, controversy remains regarding the optimal treatment strategy for patients with locally advanced ESCC. METHODS: A retrospective review of clinical stage II and III ESCC patients who underwent esophagectomy at Henan Cancer Hospital between October 2014 and October 2017 was performed. The patients were divided into a neoadjuvant chemotherapy (NAC) group and an adjuvant chemotherapy (AC) group. Propensity score matching (PSM) was used to exclude confounders. Survival was estimated using Kaplan‒Meier analysis and compared by the log-rank test. The Cox proportional hazards regression model was used for both the univariate and multivariate analyses. RESULTS: A total of 684 patients were enrolled, including 365 (53.4%) patients in the NAC group. After PSM, 294 pairs of patients were left. NAC prolonged the OS (not reached versus 57.3 months, P = 0.002) and DFS (57.2 vs. 36.4 months, P = 0.010) and decreased the total rate of recurrence (50.1% vs. 59.2%, P = 0.025) and local recurrence (27.9% vs. 36.7%, P = 0.022) compared with AC. The multivariable analyses showed that NAC plus surgery modality was an independent predictor for improved OS (HR: 0.582, 95% CI: 0.467-0.786, P = 0.001). CONCLUSION: NAC plus surgery prolonged OS and DFS, and significantly decreased the total rate of recurrence compared with surgery plus AC in patients with clinical stage II and III ESCC.

Simultaneous Quantification of Biomarkers for Bis-(2-chloroethyl) Sulfide and 1,2-Bis(2-chloroethylthio) Ethane Exposure in Human Urine at Trace Exposure Levels by Gas Chromatography Tandem Mass Spectrometry via Simultaneous Incubation and Extraction.

Sulfur mustard [HD; bis-(2-chloroethyl) sulfide] and other analogues are a kind of highly toxic vesicant and have been prohibited by the Organization for the Prohibition of Chemical Weapons (OPCW) since 1997. Exposures to HD could generate several adducts in the plasma and hydrolysis products in the urine, which are widely applied as biomarkers to identify HD exposure in forensic analysis. Several methods have been developed for the detection of related biomarkers. However, most methods are based on complex derivatization, and not enough attention is paid to HD analogues. A modified and convenient analytical method reported herein includes simultaneous incubation and organic solvent extraction. The biomarkers such as thiodiglycol and 1,2-bis (2-hydroxyethylthio) are transferred to HD and 1,2-bis(2-chloroethylthio) ethane via hydrochloric acid at the appropriate temperature. The analytes are analyzed by gas chromatography tandem mass spectrometry (GC-MS/MS) with 2-chloroethyl ethyl sulfide (2-CEES) applied as the internal standard. The interday and intraday study according to FDA rules has been achieved to evaluate the accuracy and precision of the method. The two targets are detected with a good linearity (R2 > 0.99) in the concentration ranges from 5 to 1000 ng/mL and 10 to 1000 ng/mL, with small relative standard deviations (RSD ≤6.62% and RSD ≤6.93%) and favorable recoveries between 90.3 and 107.3% and between 89.4 and 108.7%, respectively. The established method can be used for retrospective detection of sulfur mustards in biological samples and successfully applied in the biomedical proficiency testing organized by the OPCW.

A protein standard absolute quantification strategy for enhanced absolute quantification of ricin in complex matrices using in vitro synthesized mutant holoprotein as internal standard by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Ricin is a highly toxic protein toxin that poses a potential bioterrorism threat due to its potency and widespread availability. However, the accurate quantification of ricin through absolute mass spectrometry (MS) using a protein standard absolute quantification (PSAQ) strategy is not widely practiced. This limitation primarily arises from the presence of interchain disulfide bonds, which hinder the production of full-length isotope-labeled ricin as an internal standard (IS) in vitro. In this study, we have developed a novel approach for the absolute quantification of ricin in complex matrices using recombinant single-chain and full-length mutant ricin as the protein IS, instead of isotope-labeled ricin, in conjunction with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The amino acid sequence of the ricin mutant internal standard (RMIS) was designed by introducing site mutations in specific amino acids of trypsin/Glu-C enzymatic digestion marker peptides of ricin. To simplify protein expression, the A-chain and B-chain of RMIS were directly linked to replace the original interchain disulfide bonds. The RMISs were synthesized using an Escherichia coli expression system. An appropriate RMIS was selected as the protein IS based on consistent digestion efficiency, UHPLC-MS/MS behavior, antibody recognition function, lectin activity, and proper depurination activity with intact ricin. The RMIS was utilized to simultaneously quantify A- and B-chain marker peptides of ricin through UHPLC-MS/MS. This method was thoroughly validated using a milk matrix. By employing internal protein standards, this quantitative strategy overcomes the challenges posed by variations in extraction recoveries, matrix effects, and digestion efficiency encountered when working with different matrices. Consequently, calibration curves generated from milk matrix-spiked samples were utilized to accurately and precisely quantify ricin in river water and plasma samples. Moreover, the established method successfully detected intact ricin in samples obtained from the sixth Organization for the Prohibition of Chemical Weapons (OPCW) exercise on biotoxin analysis. This study presents a novel PSAQ strategy that enables the accurate quantification of ricin in complex matrices.

A multicenter randomized, controlled clinical trial of adjuvant sintilimab for esophageal squamous cell carcinoma.

No adjuvant treatment has been established for patients who remain at high risk of recurrence and incidental pathologic lymph node metastasis for esophageal squamous cell carcinoma (ESCC). In this open-label, multicenter, phase III, randomized controlled trial, ESCC patients who did not achieve pathologic complete response after neoadjuvant chemotherapy plus surgery and clinical T1-2 N0 patients with incidental pathologic lymph node metastasis following initial surgery were randomized at a 2:1 ratio to receive either a sintilimab regimen or observational management (NCT05495152). The primary end point was disease-free survival for all randomized patients. The results of this randomized controlled trial addressed controversy regarding the survival benefits of adjuvant sintilimab treatment for patients with resected locally advanced ESCC. Clinical Trial Registration: NCT05495152 (ClinicalTrials.gov).

Ponicidin inhibited gallbladder cancer proliferation and metastasis by decreasing MAGEB2 expression through FOXO4.

BACKGROUND: Gallbladder cancer (GBC) is the most aggressively malignant tumor in the bile duct system. The prognosis for patients with GBC is extremely poor. Ponicidin is a diterpenoid compound extracted and purified from the traditional Chinese herb Rabdosia rubescens, and showed promising anti-cancer effects in a variety of tumors. However, Ponicidin has not been investigated in GBC. METHODS: CCK-8, colony formation assay and EdU-488 DNA synthesis assay were performed to investigate the effect of Ponicidin on GBC cells proliferation. Cell invasion and migration assays and wound-healing assay were used to explore the effect of Ponicidin on invasion and migration ability of GBC cells. mRNA-seq was adopted to explore the underlying mechanisms. Western blot and immunohistochemical staining were conducted to detect the protein level. CHIP assay and dual-luciferase assay were used to validate binding motif. Nude mouse model of GBC was used to assess the anti-tumor effect and safety of Ponicidin. RESULTS: Ponicidin inhibited the proliferation and cell invasion and migration of GBC cells in vitro. Moreover, Ponicidin exerted anti-tumor effects by down-regulating the expression of MAGEB2. Mechanically, Ponicidin upregulated the FOXO4 expression and promoted it to accumulate in nucleus to inhibit the transcript of MAGEB2. Furthermore, Ponicidin suppressed tumor growth in the nude mouse model of GBC with excellent safety. CONCLUSION: Ponicidin may be a promising agent for the treatment of GBC effectively and safely.

Generic detection of organophosphorus nerve agent adducts to butyrylcholinesterase in plasma using liquid chromatography-tandem mass spectrometry combined with an improved procainamide-gel separation and pepsin digestion method.

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be applied to confirm exposure in humans. A sensitive method for generic detection of G- and V-series OPNA adducts to BChE in plasma was developed by combining an improved procainamide-gel separation (PGS) and pepsin digestion protocol with ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Residual matrix interferences from prior PGS purification of OPNA-BChE adducts from plasma were found to be a critical cause of significantly reduced UHPLC-MS/MS detection sensitivity. In our developed on-column PGS approach, the matrix interference was successfully removed by adding an appropriate concentration of NaCl to the washing buffer, and it could capture ≥92.5% of the BChE in plasma. The lower pH value and the longer digestion time in all previous pepsin digestion methods were found to be a key accelerated aging factor of several adducts such as tabun (GA)-, cyclohexylsarin (GF)-, and soman (GD)-BChE nonapeptide adducts, making them difficult to detect. The aging event of several OPNA-BChE nonapeptide adducts was so successfully addressed that the formic acid level in enzymatic buffer and digestion time were lowered to 0.05% (pH 2.67) and 0.5 h, respectively, and the post-digestion reaction was immediately terminated. The improved condition parameters were optimal for pepsin digestion of all types of OPNA-BChE adducts into their individual unaged nonapeptide adducts with the highest yields, expanding the applicability of the method. The method had a nearly one-fold decrease in sample preparation time through the reduction of digestion time and removal of ultrafiltration procedure after digestion. The limit of identification (LOI) were determined respectively as 0.13 ng mL-1, 0.28 ng mL-1, 0.50 ng mL-1, 0.41 ng mL-1 and 0.91 ng mL-1 for VX-, sarin (GB)-, GA-, GF-, and GD-exposed human plasma, being low exposure value compared to previously documented approaches. The approach was utilized to fully characterize the adducted (aged and unaged) BChE levels of five OPNAs in a series of their individual exposed concentration (1.00-400 nM) of plasma sample, and successfully detect OPNA exposure from all unknown plasma samples from OPCW's second and third biomedical proficiency tests. The OPNA-BChE adducts, their aged adducts, and unadducted BChE from OPNA-exposed plasma can simultaneously be measured using the method. The study provides a recommended diagnostic tool for generic verification of any OPNA exposure with high confidence by detecting its corresponding BChE adduct.

Initial experience with modified en bloc robot-assisted minimally invasive oesophagectomy for thoracic oesophageal squamous cell carcinoma.

BACKGROUND: The feasibility and safety of en bloc robot-assisted minimally invasive oesophagectomy (RAMIE) need to be verified. METHODS: Forty-seven patients who received conventional RAMIE and 31 who received modified en bloc RAMIE at Henan Cancer Hospital were included in the study cohort. We compared the perioperative outcomes of conventional RAMIE and modified en bloc RAMIE. RESULTS: Compared with the conventional RAMIE group, the en bloc RAMIE group yielded a higher total number of lymph nodes (p = 0.001), especially thoracic lymph nodes (p = 0.025) and left recurrent laryngeal nerve (RLN) lymph nodes (p = 0.005). No notable differences were found in the rate of total complications (p = 0.663) or RLN injury (p = 0.891) between the two groups. The preoperative and postoperative serological indicators were comparable between the two groups. CONCLUSIONS: Modified en bloc RAMIE was safe and feasible for patients with oesophageal squamous cell carcinoma and improved lymph node dissection, especially thoracic and left RLN lymph node dissection.

Inhibition of XPO1 impairs cholangiocarcinoma cell proliferation by triggering p53 intranuclear accumulation.

BACKGROUND: XPO1 mediates the nuclear export of several proteins, mainly tumor suppressors. KPT-330 (Selinexor) is a selective inhibitor of XPO1 that has demonstrated good therapeutic effects in hematologic cancers. METHODS: We used TCGA and GTEx pan-cancer database to evaluate XPO1 mRNA expression in various tumors. Cell proliferation assay and colony formation assay were used to analyze the in vitro antitumor effects of XPO1 inhibitor KPT-330. Western blot was performed to explore the specific mechanisms. RESULTS: We found that XPO1 was highly expressed across a range of cancers and associated with poor prognosis in hepatobiliary and pancreatic tumors. We revealed that the XPO1 inhibitor KPT-330 triggered the nuclear accumulation of the p53 protein and significantly disrupted the proliferation of cholangiocarcinoma cells. Mechanistically, the XPO1 inhibitor, KPT-330, reduced BIRC6 expression by inhibiting the PI3K/AKT pathway to decrease p53 degradation and improve its stability. CONCLUSION: Therefore, XPO1 may be a potential therapeutic target in cholangiocarcinoma, mediated by its effects on KPT-330.

Perioperative Outcomes and Learning Curve of Robot-Assisted McKeown Esophagectomy.

BACKGROUND: This study aimed to evaluate the perioperative outcomes of patients undergoing robot-assisted McKeown esophagectomy (RAME) and the learning curves of surgeons performing RAME at a single center. METHODS: Perioperative outcomes of RAME and video-assisted McKeown esophagectomy (VAME) were compared after eliminating confounding factors by propensity score matching (PSM). The cumulative sum (CUSUM) method was used to evaluate the learning curves of RAME for a single surgical team. RESULTS: In general, a total of 198 patients with esophageal cancer (RAME: 45 patients, VAME: 153 patients) were included in this study, and 43 pairs of patients receiving RAME or VAME were matched using 1:1 PSM analysis. Those in the RAME group had more lymph nodes dissected in the total lymph nodes (median 29.0 vs. 26.0, P = 0.011) and the upper mediastinum (median 8.0 vs. 6.0, P < 0.001), especially the left recurrent laryngeal nerve (RLN) lymph node (median 4.0 vs. 2.0, P = 0.001). According to the trend of the CUSUM plot, the learning curve was divided into two stages at the 20th RAME procedure. After mastering the learning curve, RAME harvested a significantly higher number of upper mediastinal lymph nodes (median 9.0 vs. 6.0, P = 0.001), left RLN lymph nodes (median 5.0 vs. 3.5, P = 0.003), and right RLN lymph nodes (median 4.0 vs. 2.0, P = 0.002). Meanwhile, the incidence of postoperative pneumonia in the proficiency phase was significantly lower than that in the learning phase (4.0% vs. 25.0%, P = 0.04). CONCLUSIONS: RAME improved left RLN lymph node dissection. Surgeons with extensive VAME experience need at least 20 cases to achieve early proficiency in RAME.

Inhibition of XPO1 with KPT-330 induces autophagy-dependent apoptosis in gallbladder cancer by activating the p53/mTOR pathway.

BACKGROUND: Gallbladder cancer (GBC) is a highly aggressive malignant cancer in the biliary system with poor prognosis. XPO1 (chromosome region maintenance 1 or CRM1) mediates the nuclear export of several proteins, mainly tumor suppressors. Thus, XPO1 functions as a pro-oncogenic factor. KPT-330 (Selinexor) is a United States Food and Drug Administration approved selective inhibitor of XPO1 that demonstrates good therapeutic effects in hematologic cancers. However, the function of XPO1 and the effect of KPT-330 have not been reported in GBC. METHODS: We analyzed the correlation between XPO1 expression levels by q-PCR and clinical features of GBC patients. Cell proliferation assays were used to analyze the in vitro antitumor effects of XPO1 inhibitor KPT-330. mRNA sequencing was used to explore the underlying mechanisms. Western blot was performed to explore the relationship between apoptosis and autophagy. The in vivo antitumor effect of KPT-330 was investigated in a nude mouse model of gallbladder cancer. RESULTS: We found that high expression of XPO1 was related to poor prognosis of GBC patients. We observed that XPO1 inhibitor KPT-330 inhibited the proliferation of GBC cells in vitro. Furthermore, XPO1 inhibitor KPT-330 induced apoptosis by reducing the mitochondrial membrane potential and triggering autophagy in NOZ and GBC-SD cells. Indeed, XPO1 inhibitor KPT-330 led to nuclear accumulation of p53 and activated the p53/mTOR pathway to regulate autophagy-dependent apoptosis. Importantly, KPT-330 suppressed tumor growth with no obvious toxic effects in vivo. CONCLUSION: XPO1 may be a promising prognostic indicator for GBC, and KPT-330 appears to be a potential drug for treating GBC effectively and safely.

Simultaneous solvent extraction and quantification of eleven amine compounds related to Chemical Weapon Convention in soils via hydrophilic interaction liquid chromatography-tandem mass spectrometry.

The detection of Chemical Weapon Convention (CWC)-related amine compounds including the precursors or degradation products of V-type organophosphorus nerve agent, nitrogen mustard and 3-quinuclidinyl benzilate is an important aspect for verifying their intact chemical warfare agents. This work focuses on the development of a novel formulation for the simultaneous solvent extraction of eleven CWC-related amine compounds, from the four-type soil matrices including environmental standard soil, sand, clay, and loam. Extracts were well separated on the hydrophilic interaction liquid chromatography (HILIC) and then detected by MS/MS multiple reaction monitoring mode. The type and component of solvent mixtures were optimized to cover a wide range of polarity over all eleven amine compounds with high extraction efficiencies. Extraction parameters, such as the proportion of methanol, water and NH4OH, the times and the period of extraction, and volumes of extraction solution were optimized. The results indicated that a mixed solvent of methanol/water (44:53, v/v) in 3.0% NH4OH was the optimal formulation for extraction of all 11 analytes with high mean extraction recoveries (64.4-96.1%). Specificity and sensitivity were well improved by the good separation of 11 analytes from four-type soil matrices using these optimized HILIC parameters. This method was fully validated for each analyte in four soil matrices. The linear range of 11 analytes was 0.50/0.75-500 ng·g-1 with correlation coefficient (R2) ≥0.990, and intra/inter-day accuracies were 70.3-125% with relative standard deviation (RSD) ≤19.3%. Limit of detection (LOD) of 11 analytes ranged from 0.01 to 0.5 ng·g-1, which was far lower than those reported in previous studies. The built method accomplishes simultaneously quantitative and trace measurement of all eleven CWC-related amine compounds within a single solvent extraction and detection. It only takes a small amount of soil samples and possesses the highest sensitivity over all previous methods. This study provides an optional recommended operating procedure for determination of CWC-related amine compounds in four typical types of complex soils during chemical weapons verification.

UBAP2L promotes gastric cancer metastasis by activating NF-κB through PI3K/AKT pathway.

Ubiquitin-associated protein 2-like (UBAP2L) is highly expressed in various types of tumors and has been shown to participate in tumor growth and metastasis; however, its role in gastric cancer (GC) remains unknown. In this study, we observed that UBAP2L expression was markedly elevated in GC tissues and five GC cell lines. Higher expression of UBAP2L was associated with poor prognosis as revealed by bioinformatics analysis on online websites and laboratory experiments. Knockdown of UBAP2L impeded the migration and invasion abilities of GC cell lines. In contrast, its overexpression enhanced the migration and invasion abilities of GC cell lines. Overexpression of UBAP2L also increased the number and size of lung metastatic nodules in vivo. According to the results of mass spectrometry and pathway annotation of the identified proteins, the PI3K/AKT pathway was found to be related to UBAP2L regulation. Further exploration and rescue experiments revealed that UBAP2L stimulates the expression and nuclear aggregation of p65 and promotes the expression of SP1 by activating the PI3K/AKT pathway. In summary, our findings indicate that UBAP2L regulates GC metastasis through the PI3K/AKT/SP1/NF-κB axis. Thus, targeting UBAP2L may be a potential therapeutic strategy for GC.

Gemcitabine and XCT790, an ERRα inverse agonist, display a synergistic anticancer effect in pancreatic cancer.

Pancreatic cancer (PC) is one of the most fatal and chemoresistant malignancies with a poor prognosis. The current therapeutic options for PC have not achieved satisfactory results due to drug resistance. Therefore, it is urgent to develop novel treatment strategies with enhanced efficacy. This study sought to investigate the anticancer effect of gemcitabine and XCT790, an estrogen-related receptor alpha (ERRα) inverse agonist, as monotherapies or in combination for the treatment of PC. Here we demonstrated that the drug combination synergistically suppressed PC cell viability, its proliferative, migratory, invasive, apoptotic activities, and epithelial-to-mesenchymal transition (EMT), and it triggered G0/G1 cell cycle arrest and programmed cell death in vitro. In addition, in vivo assays using xenograft and mini-PDX (patient-derived xenograft) models further confirmed the synergistic antitumor effect between gemcitabine and XCT790 on PC. Mechanistically, gemcitabine and XCT790 suppressed PC by inhibiting ERRα and MEK/ERK signaling pathway. In conclusion, our current study demonstrated for the first time that gemcitabine combined with XCT790 displayed synergistic anticancer activities against PC, suggesting that their combination might be a promising treatment strategy for the therapy of PC.

Preoperative lymphocyte to C-reactive protein ratio as a new prognostic indicator in patients with resectable gallbladder cancer.

BACKGROUND: Inflammation is often related to cancer, and several inflammatory scores have been established to predict the prognosis of various types of cancer. Our study aimed to determine the prognostic value of the preoperative lymphocyte to C-reactive protein ratio (LCR) for predicting postoperative outcomes in patients with resectable gallbladder cancer (GBC). METHODS: A retrospective analysis of 104 GBC patients who received curative surgery at Xinhua Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine from January 2000 to December 2016 was performed. A time-dependent receiver operating characteristic curve was constructed to evaluate the accuracy of different markers. Univariate and multivariate Cox proportional hazard models were used to define factors associated with overall survival. RESULTS: Among the assessed variables, the preoperative LCR showed the highest accuracy in predicting the overall survival of GBC patients (AUC: 0.736). Decreased preoperative LCR was significantly associated with advanced tumor stage, including tumor invasion (P = 0.018), lymph node metastasis (P = 0.011) and TNM stage (P = 0.022). A low preoperative LCR (cutoff threshold = 145.5) was an independent risk factor for overall survival in patients with resectable GBC (P < 0.001). CONCLUSIONS: The preoperative LCR is a novel and valuable prognostic indicator of postoperative survival in patients with resectable GBC.

ESRRA promotes gastric cancer development by regulating the CDC25C/CDK1/CyclinB1 pathway via DSN1.

Background: Estrogen-related receptor-α (ESRRA) is an orphan nuclear receptor, expressing at high level in exuberant metabolism organs and acting as transcription factor. High expression was found in many malignances but no research was done in gastric cancer (GC), where lipid metabolism disorder is common. Methods: Kaplan-Meier plot was utilized to find the relationship between ESRRA expression and patients' prognoses. The expression level of ESRRA was measured by real-time PCR. The protein expression levels were tested with western-blot and immunohistochemistry. Cell cycle and apoptosis was identified with flow cytometry. RNA-seq, bioinformatics analysis, dual-luciferase assay and ChIP assay were used to predict and validate ESRRA's target gene and binding motif. Animal models were also introduced in our study. Results: ESRRA expression is notably higher in GC cell lines and high ESRRA levels are correlated to poor prognoses. ESRRA silencing decreased GC cell viability, migration, and invasion capacities. Its downstream gene DSN1 was spotted by RNA-seq and confirmed by later bioinformatics analyses, dual-luciferase, and ChIP assays. Western-blot showed G2M arrest caused by ESRRA silencing was via CDC25C-CDK1-Cyclin B1 pathway. Conclusion: ESRRA/DSN1/CDC25C-CDK1-Cyclin B1 is of great importance in GC development. ESRRA could be a potential target as well as prognostic marker in GC.

Rapid Differential Detection of Abrin Isoforms by an Acetonitrile- and Ultrasound-Assisted On-Bead Trypsin Digestion Coupled with LC-MS/MS Analysis.

The high toxic abrin from the plant Abrus precatorius is a type II ribosome-inactivating protein toxin with a human lethal dose of 0.1-1.0 µg/kg body weight. Due to its high toxicity and the potential misuse as a biothreat agent, it is of great importance to developing fast and reliable methods for the identification and quantification of abrin in complex matrices. Here, we report rapid and efficient acetonitrile (ACN)- and ultrasound-assisted on-bead trypsin digestion method combined with HPLC-MS/MS for the quantification of abrin isoforms in complex matrices. Specific peptides of abrin isoforms were generated by direct ACN-assisted trypsin digestion and analyzed by HPLC-HRMS. Combined with in silico digestion and BLASTp database search, fifteen marker peptides were selected for differential detection of abrin isoforms. The abrin in milk and plasma was enriched by immunomagnetic beads prepared by biotinylated anti-abrin polyclonal antibodies conjugated to streptavidin magnetic beads. The ultrasound-assisted on-bead trypsin digestion method was carried out under the condition of 10% ACN as denaturant solvent, the entire digestion time was further shortened from 90 min to 30 min. The four peptides of T3Aa,b,c,d, T12Aa, T15Ab, and T9Ac,d were chosen as quantification for total abrin, abrin-a, abrin-b, and abrin-c/d, respectively. The absolute quantification of abrin and its isoforms was accomplished by isotope dilution with labeled AQUA peptides and analyzed by HPLC-MS/MS (MRM). The developed method was fully validated in milk and plasma matrices with quantification limits in the range of 1.0-9.4 ng/mL for the isoforms of abrin. Furthermore, the developed approach was applied for the characterization of abrin isoforms from various fractions from gel filtration separation of the seeds, and measurement of abrin in the samples of biotoxin exercises organized by the Organization for the Prohibition of Chemical Weapons (OPCW). This study provided a recommended method for the differential identification of abrin isoforms, which are easily applied in international laboratories to improve the capabilities for the analysis of biotoxin samples.

Metal-Free Intramolecular [3+2] Cycloaddition of γ-Hydroxy Acetylenic Ketones with Alkynes for the Synthesis of Naphtho[1,2-c]furan-5-ones and Its Derivatization via a Selective C(sp2)-H Deuteration Reaction.

A metal-free intramolecular [3+2] cycloaddtion has been achieved by treating benzene-linked propynol-ynes with AcOH/H2O in a one-pot manner. The reaction provides greener, 100% atom-economic, highly regioselective, and more practical access to functionalized naphtho[1,2-c]furan-5-ones with valuable and versatile applications. The regioselective α-deuteration of naphtho[1,2-c]furan-5-ones has been also presented with excellent deuterium incorporation and chemical yields. Moreover, the fluorescent properties of naphtho[1,2-c]furan-5-one products have been investigated in solution.