A Recombinant Thermophilic and Glucose-Tolerant GH1 ß-Glucosidase Derived from Hehua Hot Spring.

As a crucial enzyme for cellulose degradation, ß-glucosidase finds extensive applications in food, feed, and bioethanol production; however, its potential is often limited by inadequate thermal stability and glucose tolerance. In this study, a functional gene (lq-bg5) for a GH1 family ß-glucosidase was obtained from the metagenomic DNA of a hot spring sediment sample and heterologously expressed in E. coli and the recombinant enzyme was purified and characterized. The optimal temperature and pH of LQ-BG5 were 55 °C and 4.6, respectively. The relative residual activity of LQ-BG5 exceeded 90% at 55 °C for 9 h and 60 °C for 6 h and remained above 100% after incubation at pH 5.0-10.0 for 12 h. More importantly, LQ-BG5 demonstrated exceptional glucose tolerance with more than 40% activity remaining even at high glucose concentrations of 3000 mM. Thus, LQ-BG5 represents a thermophilic ß-glucosidase exhibiting excellent thermal stability and remarkable glucose tolerance, making it highly promising for lignocellulose development and utilization.

Ultrahigh-performance liquid chromatography-high-resolution mass spectrometry-based plasma metabolomics study of thymoma and thymic hyperplasia.

RATIONALE: Thymoma is a rare malignant tumor but it is the most common primary tumor of the anterior mediastinum. The current imaging methods for thymoma screening suffer from false positive rate problems, and thymoma pathogenesis remains elusive. Study of thymoma metabolic characteristics could provide clues for improving the diagnosis and understanding the pathogenesis of thymoma. METHODS: Metabolic profiling of plasma from thymoma and thymic hyperplasia patients was performed using ultrahigh-performance liquid chromatography combined with high-resolution mass spectrometry in both positive and negative ionization modes. After pre- and post-processing, the dataset was divided into three age groups and statistical analysis was performed to select differential metabolites of thymoma. For feature identification, experimental tandem mass spectra were matched to those of databases and available chemical standards, and also manually annotated with plausible chemical structures to ensure high identification confidence. RESULTS: A total of 47 differential metabolites were identified in thymoma. Significantly higher levels of histidine, sphinganine 1-phosphate, lactic acid dimer, phenylacetylglutamine, LPC (18:3) and LPC (16:1), and significantly lower levels of phenylalanine, indole-3-propionic acid (IPA), hippuric acid and mesobilirubinogen were associated with thymoma. Tryptophan level in thymoma-associated myasthenia gravis (TAMG) was significantly lower than that of the MG(-) group. IPA and hippuric acid abundances exhibited increasing trends from indolent to aggressive thymoma. CONCLUSIONS: Our study revealed aberrant aromatic amino acid metabolism and fatty acid oxidation might be associated with thymoma. The identified unique metabolic characteristics of thymoma may provide valuable information for study of the molecular mechanism of thymoma pathogenesis, and improvement of diagnosis and discovery of new therapeutic strategies for thymoma.

Association of serum Asprosin concentrations with heart failure.

BACKGROUND: To analyze the association of serum Asprosin concentrations with heart failure (HF). METHODS: A total of 103 patients with HF were included in the HF group, and 103 patients with health checkups were included in the non-HF group. The serum Asprosin levels of the two groups were measured, and relevant clinical data were collected for statistical analysis. RESULTS: Compared with the non-HF group, the serum Asprosin concentration was significantly higher in the HF group, and the difference was statistically significant (P < 0.001). According to the serum Asprosin levels, we divided all the subjects into three quartiles. We found that the prevalence of HF increased with increasing serum Asprosin levels in the three groups (P < 0.001). Serum Asprosin levels were positively correlated with NT-ProBNP (P < 0.05) and negatively correlated with LVEF (P < 0.001). Dichotomous logistic regression analysis found Asprosin and age to be independent risk factors for HF (OR = 1.010, 95% CI: 1.003-1.018; OR = 1.058, 95% CI:1.004-1.665, respectively). Combining Asprosin and NT-proBNP indicators to draw ROC curves can improve the specificity and sensitivity of HF diagnosis. CONCLUSIONS: Serum Asprosin levels were significantly elevated in HF patients. The serum Asprosin level is an independent risk factor for HF, and the combined detection of Asprosin and NT-proBNP levels can improve the accuracy of HF diagnosis.

Changes in Lipidomics, Metabolomics, and the Gut Microbiota in CDAA-Induced NAFLD Mice after Polyene Phosphatidylcholine Treatment.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in most parts of the world. Although there is no first-line drug approved for the treatment of NAFLD, polyene phosphatidylcholine (PPC) is used by clinicians to treat NAFLD patients. This study aimed to evaluate the efficacy of PPC on a mice model of NAFLD, and to study the PPC's mechanism of action. The mice were fed a choline-deficient, L-amino acid-defined (CDAA) diet to induce NAFLD and were subsequently treated with PPC. The treatment effects were evaluated by the liver index, histopathological examination, and routine blood chemistry analyses. Lipidomics and metabolomics analyses of 54 samples were carried out using ultraperformance liquid chromatography (UPLC) coupled to a mass spectrometer to select for changes in metabolites associated with CDAA diet-induced NAFLD and the effects of PPC treatment. The intestinal flora of mice were extracted for gene sequencing to find differences before and after the induction of NAFLD and PPC treatment. PPC significantly improved the CDAA diet-induced NAFLD condition in mice. A total of 19 metabolites including 5 polar metabolites and 14 lipids showed marked changes. In addition, significant differences in the abundance of Lactobacillus were associated with NAFLD. We inferred that the protective therapeutic effect of PPC on the liver was related to the supplement of phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin (PC, LPC, and SM, resectively) and acylcarnitine metabolism. This study developed a methodology for exploring the pathogenesis of NAFLD and can be extended to other therapeutic agents for treating NAFLD.

Biotransformation of Penindolone, an Influenza A Virus Inhibitor.

Penindolone (PND) is a novel broad-spectrum anti-Influenza A Virus (IAV) agent blocking hemagglutinin-mediated adsorption and membrane fusion. The goal of this work was to reveal the metabolic route of PND in rats. Ultra-high-performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS) was used for metabolite identification in rat bile, feces and urine after administration of PND. A total of 25 metabolites, including 9 phase I metabolites and 16 phase II metabolites, were characterized. The metabolic pathways were proposed, and metabolites were visualized via Global Natural Product Social Molecular Networking (GNPS). It was found that 65.24-80.44% of the PND presented in the formation of glucuronide conjugate products in bile, and more than 51% of prototype was excreted through feces. In in vitro metabolism of PND by rat, mouse and human liver microsomes (LMs) system, PND was discovered to be eliminated in LMs to different extents with significant species differences. The effects of chemical inhibitors of isozymes on the metabolism of PND in vitro indicated that CYP2E1/2C9/3A4 and UGT1A1/1A6/1A9 were the metabolic enzymes responsible for PND metabolism. PND metabolism in vivo could be blocked by UGTs inhibitor (ibrutinib) to a certain extent. These findings provided a basis for further research and development of PND.

Non-Small Cell Lung Cancer Detection and Subtyping by UPLC-HRMS-Based Tissue Metabolomics.

Non-small cell lung cancer (NSCLC) is the prevalent histological subtype of lung cancer. In this study, we performed ultraperformance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS)-based metabolic profiling of 227 tissue samples from 79 lung cancer patients with adenocarcinoma (AC) or squamous cell carcinoma (SCC). Orthogonal partial least squares-discriminant analysis (oPLS-DA) analyses showed that AC, SCC, and NSCLC tumors were discriminated from adjacent noncancerous tissue (ANT) and distant noncancerous tissue (DNT) samples with good accuracies (91.3, 100, and 88.3%), sensitivities (85.7, 100, and 83.9%), and specificities (94.3, 100, and 90.7%), using 12, 4, and 7 discriminant metabolites, respectively. The discriminant panel for AC detection included valine, sphingosine, glutamic acid γ-methyl ester, and lysophosphatidylcholine (LPC) (16:0), levels of which in tumor tissues were significantly altered. Valine, sphingosine, LPC (18:1), and leucine derivatives were used for SCC detection. The discrimination between AC and SCC had 96.8% accuracy, 98.2% sensitivity, and 85.7% specificity using a five-metabolite panel, of which valine and creatine had significant differences. The classification models were further verified with external validation sets, showing a promising prospect for NSCLC tissue detection and subtyping.

A label-free T4 polynucleotide kinase fluorescence sensor based on split dimeric G-quadruplex and ligation-induced dimeric G-quadruplex/thioflavin T conformation.

The phosphorylation process of DNA by T4 polynucleotide kinase (T4 PNK) plays a crucial role in DNA recombination, DNA replication, and DNA repair. Traditional monomeric G-quadruplex (G4) systems are always activated by single cation such as K+ or Na+. The conformation transformation caused by the coexistence of multiple cations may interfere with the signal readout and limit their applications in physiological system. In view of the stability of dimeric G4 in multiple cation solution, we reported a label-free T4 PNK fluorescence sensor based on split dimeric G4 and ligation-induced dimeric G4/thioflavin T (ThT) conformation. The dimeric G4 was divided into two independent pieces of one normal monomeric G4 and the other monomeric G4 fragment phosphorylated by T4 PNK in order to decrease the background signal. With the introduction of template DNA, DNA ligase, and invasive DNA, the dimeric G4 could be generated and liberated to combine with ThT to show obvious fluorescence signal. Using our strategy, the linear range from 0.005 to 0.5 U mL-1, and the detection limit of 0.0021 U mL-1 could be achieved without the consideration of interference caused by the coexistence of multiple cations. Additionally, research in real sample determination and inhibition effect investigations indicated its further potential application value in biochemical process research and clinic diagnostics.

In Vivo Anticoagulant and Antithrombic Activity of Depolymerized Glycosaminoglycan from Apostichopus japonicus and Dynamic Effect-Exposure Relationship in Rat Plasma.

Glycosaminoglycan from Apostichopus japonicus (AHG) and its depolymerized fragments (DAHGs) are anticoagulant fucosylated chondroitin sulfate. The aim of this study was to further evaluate the anticoagulant and antithrombic activity of AHG and DAHGs, as well as reveal the dynamic relationship between exposure and effect in vivo. The results demonstrated that AHG100 (Mw~100 kDa), DAHG50 (Mw~50 kDa), and DAHG10 (Mw~10 kDa) exhibited potent anticoagulant activity by inhibiting intrinsic factor Xase complex (FXase) as well as antithrombin-dependent factor IIa (FIIa) and factor Xa (FXa). These glycosaminoglycans markedly prevented thrombosis formation and thrombin-induced platelet aggregation in a dose- and molecular weight-dependent manner in vitro and in vivo. The further bleeding time measurement indicated that DAHG10 exhibited obviously lower hemorrhage risks than native AHG100. Following oral administration, DAHG10 could be absorbed into blood, further dose-dependently prolonging activated partial thromboplastin time (APTT) and thrombin time (TT) as well as inhibiting FXa and FIIa partially through FXase. Anticoagulant activity was positively associated with plasma concentration following oral administration of DAHG10. Our study proposed a new point of view to understand the correlation between effects and exposure of fucosylated chondroitin sulfate as an effective and safe oral antithrombotic agent.

Lipidomic-based investigation into the therapeutic effects of polyene phosphatidylcholine and Babao Dan on rats with non-alcoholic fatty liver disease.

In recent years, with the improvement of people's living standards, non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in the world. In this paper, the metabolic disorders in Sprague Dawley (SD) rats were induced by a choline-deficient, l-amino acid-defined (CDAA) diet. The therapeutic effects of polyene phosphatidylcholine (PPC) and Babao Dan (BBD) on NAFLD were observed. Lipidomic analysis was performed using ultra-high-performance liquid chromatography-Orbitrap MS, and data analysis and lipid identification were performed using the software LipidSearch. Both PPC and BBD can reduce lipid accumulation in the liver and improve abnormal biochemical indicators in rats, including reduction of triglycerides, total cholesterol, alanine transaminase and aspartate transaminase in serum. In addition, lipids in rat serum were systematically analyzed by lipidomics. The lipidomic results showed that the most obvious lipids with abnormal metabolism in CDAA diet-induced rats were glycerides (triglycerides and diacylglycerols), phospholipids and cholesterol esters. Both BBD and PPC partly reversed the disturbance to lipids induced by the CDAA diet. PPC may be more effective than BBD in alleviating NAFLD because it has a better effect on inhibiting the abnormal accumulation of lipids and reducing the inflammatory reaction in the body.

Pharmacokinetics and metabolism of penindolone in rat plasma using liquid chromatography-tandem mass spectrometry.

Penindolone (PND) is a novel influenza A virus dual inhibitor that blocks hemagglutinin-mediated adsorption and membrane fusion. A sensitive and specific ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated to determine PND in rat plasma. Plasma sample preparation was a simple deproteinization with acetonitrile followed by centrifugation. Chromatographic separation was performed on a C18 column with a gradient mobile phase of acetonitrile-water containing 0.1% formic acid. Detection was carried out by electrospray ionization in negative ion multiple reaction monitoring mode. Linear detection responses were obtained for PND ranging from 1 to 1,000 ng/ml. The intra- and inter-day precision (relative standard deviations, RSD) were within 6.5%, and accuracy (relative error, RE) was within ±11.0%. The extraction recovery data for PND and internal standard (IS) were >96.0%. PND was proved to be stable during the sample storage, preparation and analytic procedures. The validated method was successfully applied to pharmacokinetic and bioavailability studies for PND in rats. The results showed the existence of twin peaks, gender difference and nonlinear pharmacokinetics for PND. In addition, two oxidation metabolites and three glucuronidation metabolites of PND were detected by ultra-high-performance liquid chromatography-high resolution mass spectrometry.

A comprehensive metabolomic and lipidomic analysis reveals the effect of temperature on flounder (Paralichthys olivaceus).

Temperature is a considerable stressor in aquatic environments. The objective of this study was to investigate the changes in endogenous compounds of flounder (Paralichthys olivaceus) at different temperatures based on a holistic metabolomic and lipidomic analysis. Accordingly, this study involved reversed-phase chromatography and hydrophilic interaction chromatography coupled with a mass spectrometry detection platform, and using a new database, and multivariate statistical analysis. In total, 954 unique masses were obtained, including 495 metabolites and 459 lipids, and 53 qualified differential metabolites were filtered. Specifically, among the highlighted biomarkers, 2'-Hydroxy-5'-methylacetophenone and FFA (17:1) were the metabolites and lipids with the largest Log2FC, respectively. In addition, 18-Hydroxycorticosterone showed the greatest downregulation. Hypoxia-inducible factor 1 signaling pathway, biosynthesis of amino acids, glycerophospholipid metabolism, and choline metabolism in cancer were enriched and noteworthy metabolic pathways, which were closely related. Overall, this study provides potential serum biomarkers and metabolic pathways for flounder as well as a reference for further studies on the physiological functions of flounder under different stressors.

Dual Effect of Tryptamine on Prostate Cancer Cell Growth Regulation: A Pilot Study.

Abnormal tryptophan metabolism is linked to cancer and neurodegenerative diseases, and tryptophan metabolites have been reported as potential prostate cancer (PCa) biomarkers. However, little is known about the bioactivities of tryptophan metabolites on PCa cell growth. In this study, MTT and transwell assays were used to study the cytotoxicities of 13 major tryptophan metabolites on PCa and normal prostate epithelial cell lines. Ultraperformance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was used to analyze metabolic changes in cells treated with tryptamine. Flow cytometry, confocal imaging, and Western blot were used to test the apoptosis induced by tryptamine. It was shown that tryptamine had obvious inhibitory effects on PCa cell lines PC-3 and LNCaP, stronger than those on the normal prostate cell line RWPE-1. Tryptamine was further shown to induce apoptosis and inhibit PC-3 cell migration. Metabolic changes including amino acid metabolism related to cell proliferation and metastasis were found in PC-3 cells treated with tryptamine. Furthermore, a PC-3 xenograft mouse model was used to study the effect of tryptamine in vivo. The intratumoral injection of tryptamine was demonstrated to significantly reduce the tumor growth and tumor sizes in vivo; however, intraperitoneal treatment resulted in increased tumor growth. Such dual effects in vivo advanced our understanding of the bioactivity of tryptamine in regulating prostate tumor development, in addition to its major role as a neuromodulator.

Aztreonam/avibactam effect on pharmacodynamic indices for mutant selection of Escherichia coli and Klebsiella pneumoniae harbouring serine- and New Delhi metallo-ß-lactamases.

OBJECTIVES: Ceftazidime/avibactam is not active against MBL-producing bacteria. Combining ceftazidime/avibactam or avibactam with aztreonam can counter the resistance of MBL-producing Enterobacterales. The aim of this study was to evaluate whether the addition of avibactam could reduce or close the mutant selection window (MSW) of aztreonam in Escherichia coli and Klebsiella pneumoniae harbouring MBLs; MSW is a pharmacodynamic (PD) parameter for the selection of emergent resistant mutants. METHODS: In vitro susceptibility of 19 clinical isolates to ceftazidime/avibactam, aztreonam alone, and in co-administration (aztreonam/ceftazidime/avibactam and aztreonam/avibactam) was determined, as well as the mutant prevention concentration (MPC). The fraction of time within 24 h that the free drug concentration was within the MSW (fTMSW) and the fraction of time that the free drug concentration was above the MPC (fT>MPC) in both plasma and epithelial lining fluid (ELF) were determined from simulations of 10 000 profiles. The joint PTA was used to derive a joint cumulative fraction of response (CFR). RESULTS: All isolates were resistant to ceftazidime/avibactam or aztreonam. Combining aztreonam and avibactam or ceftazidime/avibactam resulted in synergistic bactericidal activities against all isolates. Synergism was primarily due to the aztreonam/avibactam combination. For aztreonam/avibactam dosing regimens evaluated in clinical trials, fT>MPC values were >90% and >80%, whereas fTMSW measures were <10% and <20% in plasma and ELF, respectively. The CFR was 100% for aztreonam/avibactam against the collection of clinical isolates. CONCLUSIONS: Effective antimicrobial combination optimized the PD parameters measuring selection for emergent mutants by increasing fT>MPC and reducing fTMSW.

Purlisin, a toxin-like defensin derived from clinical pathogenic fungus Purpureocillium lilacinum with both antimicrobial and potassium channel inhibitory activities.

Clinical fungal infections always cause a negative impact on human health. Moreover, during the interaction of pathogenic fungi with the environment and host, many biologically active substances are produced. Here, we report a new toxin-like defensin of purlisin derived from a clinical pathogenic isolate of Purpureocillium lilacinum. The analysis of its genomic and mRNA sequences revealed an open reading frame of 444 bp without introns. The deduced precursor peptide was composed of 147 amino acids, and the mature peptide were identified at protein level by LC-ESI-Q-TOF-MS/MS. After posttranslational processing, the precursor peptide of purlisin was split into two independent peptides. The two mature defensins, purlisin-NT and purlisin-CT, are consisting of 36 and 38 amino acid residues, which can form three and four intramolecular disulfide bonds, respectively. The results of circular dichroism and homology modeling revealed that they adopted a representative cysteine-stabilized α-helical and ß-sheet motif. The purlisin-NT showed a dose-dependent selective inhibition of immune-related hKv1.3 target channel with IC50 value of 0.2 ± 0.04 µM but no obvious antibacterial activity, while the purlisin-CT displayed antimicrobial activities against gram-positive bacteria as well as clinical isolates of MRSA and low affinities for potassium channels. Our findings suggest that purlisin-NT with immunosuppressive effects and purlisin-CT possessing antibacterial activities are adapted to the survival and pathogenicity of clinical P lilacinumis. Moreover, they can also be used as templates for the design of novel antibacterial peptide and immunosuppressive agents.

The prognostic value of general laboratory testing in patients with COVID-19.

BACKGROUND: Lymphocyte count (LYM) of peripheral blood and some indices of general biochemical analysis had diagnostic and prognostic value for coronavirus disease 2019 (COVID-19), and the value of other remaining indices is rare. METHODS: A total of 94 patients with COVID-19 were enrolled at Renmin Hospital of Wuhan University. According to the severity of COVID-19, the patients were divided into three groups (moderate 49, severe 35, and critical 10), and 40 healthy cases were enrolled in the same period as healthy controls. The diagnostic and prognostic value of indices in peripheral blood cell count and general biochemical analysis was analyzed. RESULTS: Compared with healthy cases, the value differences in peripheral blood analysis in patients with COVID-19 were statistically significant (p < 0.01), the differences in LYM, neutrophil count (Neu), platelet count (PLT), and white blood cell count (WBC) were statistically significant among different severity of COVID-19 (p < 0.05). Compared with healthy cases, the differences in general biochemical results in patients with COVID-19 were statistically significant (p < 0.01), the value differences in direct bilirubin (DBIL), low-density lipoprotein cholesterol (LDL-Ch), and nitrogen (urea) were statistically significant among different severity of COVID-19 (p < 0.05). Neutrophil/lymphocyte ratio (NLR) had higher sensitivity and specificity for COVID-19 diagnosis. CONCLUSIONS: Some indices of peripheral blood cell count and general biochemical analysis were valuable in discriminating COVID-19 and predicting severity and adverse outcome of patients with COVID-19. For clinician, it is better to use more economical and easy-to-get indices to diagnose and predict the prognosis of COVID-19.

Chronological Changes of Viral Shedding in Adult Inpatients With COVID-19 in Wuhan, China.

BACKGROUND: In December 2019, the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) broke out in Wuhan. Epidemiological and clinical characteristics of patients with COVID-19 have been reported, but the relationships between laboratory features and viral load has not been comprehensively described. METHODS: Adult inpatients (≥18 years old) with COVID-19 who underwent multiple (≥5 times) nucleic acid tests with nasal and pharyngeal swabs were recruited from Renmin Hospital of Wuhan University, including general patients (n = 70), severe patients (n = 195), and critical patients (n = 43). Laboratory data, demographic data, and clinical data were extracted from electronic medical records. The fitted polynomial curve was used to explore the association between serial viral loads and illness severity. RESULTS: Viral load of SARS-CoV-2 peaked within the first few days (2-4 days) after admission, then decreased rapidly along with virus rebound under treatment. Critical patients had the highest viral loads, in contrast to the general patients showing the lowest viral loads. The viral loads were higher in sputum compared with nasal and pharyngeal swab (P = .026). The positive rate of respiratory tract samples was significantly higher than that of gastrointestinal tract samples (P < .001). The SARS-CoV-2 viral load was negatively correlated with portion parameters of blood routine and lymphocyte subsets and was positively associated with laboratory features of cardiovascular system. CONCLUSIONS: The serial viral loads of patients revealed whole viral shedding during hospitalization and the resurgence of virus during the treatment, which could be used for early warning of illness severity, thus improve antiviral interventions.

Necessity for detection of SARS-CoV-2 RNA in multiple types of specimens for the discharge of the patients with COVID-19.

BACKGROUND: The SARS-CoV-2 RNA was detected positive again after discharged from hospital in some COVID-19 patients, with or without clinical symptoms such as fever or dry cough. METHODS: 1008 severe COVID-19 patients, with SARS-CoV-2 RNA positive detected with the mixed specimen of nasopharyngeal swab and oropharyngeal swab by real-time fluorescence quantitative PCR (RT-qPCR), were selected to monitor SARS-CoV-2 RNA with the 12 types of specimens by RT-qPCR during hospitalization. All of 20 discharged cases with COVID-19 were selected to detect SARS-CoV-2 RNA in isolation period with 7 types of specimens by RT-qPCR before releasing the isolation period. RESULTS: Of the enrolled 1008 severe patients, the nasopharyngeal swab specimens showed the highest positive rate of SARS-CoV-2 RNA (71.06%), followed by alveolar lavage fluid (66.67%), oropharyngeal swab (30.77%), sputum (28.53%), urine (16.30%), blood (12.5%), stool (12.21%), anal swab (11.22%) and corneal secretion (2.99%), and SARS-CoV-2 RNA couldn't be detected in other types of specimen in this study. Of the 20 discharged cases during the isolation period, the positive rate of SARS-CoV-2 RNA was 30% (6/20): 2 cases were positive in sputum at the eighth and ninth day after discharge, respectively, 1 case was positive in nasopharynx swab at the sixth day after discharge, 1 case was positive in anal swab at the eighth day after discharge, and 1 case was positive in 3 specimens (nasopharynx swab, oropharynx swab and sputum) simultaneously at the fourth day after discharge, and no positive SARS-CoV-2 RNA was detected in other specimens including stool, urine and blood at the discharged patients. CONCLUSIONS: SARS-CoV-2 RNA should be detected in multiple specimens, such as nasopharynx swab, oropharynx swab, sputum, and if necessary, stool and anal swab specimens should be performed simultaneously at discharge when the patients were considered for clinical cure and before releasing the isolation period.

Simultaneous determination of FL118 and W34 in rat Blood by LC-MS/MS: Application to pharmacokinetic studies.

W34 is a prodrug of FL118, and it can be converted to FL118 via a hydrolysis reaction. In this report, a highly sensitive LC-MS/MS method using a C18 column was validated and used for the simultaneous determination of W34 and FL118 in rat blood. A stepwise gradient elution with 0.1% formic acid in water and acetonitrile was employed. The assays were linear over a concentration range of 0.50-50.0 ng/ml for both W34 and FL118. The accuracy of the validation method ranged from 89.74 to 98.94% for W34 and from 88.61 to 94.60% for FL118. The precision was within 7.15% for W34 and 9.63% for FL118. Extraction recoveries of W34 were 94.56-100.49 and 87.67-106.32% for FL118. No significant matrix effects for both W34 and FL118 were observed in blood. The assay has been successfully applied to biological samples obtained from a stability and pharmacokinetic study of W34 and FL118.

Trichothecin Inhibits Cancer-Related Features in Colorectal Cancer Development by Targeting STAT3.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that contributes to cancer progression through multiple processes of cancer development, which makes it an attractive target for cancer therapy. The IL-6/STAT3 pathway is associated with an advanced stage in colorectal cancer patients. In this study, we identified trichothecin (TCN) as a novel STAT3 inhibitor. TCN was found to bind to the SH2 domain of STAT3 and inhibit STAT3 activation and dimerization, thereby blocking STAT3 nuclear translocation and transcriptional activity. TCN did not affect phosphorylation levels of STAT1. TCN significantly inhibited cell growth, arrested cell cycle at the G0/G1 phase, and induced apoptosis in HCT 116 cells. In addition, the capacities of colony formation, migration, and invasion of HCT 116 cells were impaired upon exposure to TCN with or without IL-6 stimulation. In addition, TCN treatment abolished the tube formation of HUVEC cells in vitro. Taken together, these results highlight that TCN inhibits various cancer-related features in colorectal cancer development in vitro by targeting STAT3, indicating that TCN is a promising STAT3 inhibitor that deserves further exploration in the future.

Radical Dehydroxymethylative Fluorination of Carbohydrates and Divergent Transformations of the Resulting Reverse Glycosyl Fluorides.

A mild and convenient method for the synthesis of reverse glycosyl fluorides (RGFs) has been developed that is based on the silver-promoted radical dehydroxymethylative fluorination of carbohydrates. A salient feature of the reaction is that furanoid and pyranoid carbohydrates furnish structurally diverse RGFs bearing a wide variety of functional groups in good to excellent yields. Intramolecular hydrogen atom transfer experiments revealed that the reaction involves an underexploited radical fluorination that proceeds via ß-fragmentation of sugar-derived primary alkoxyl radicals. Structurally divergent RGFs were obtained by catalytic C-F bond activation, and our method thus offers a concise and efficient strategy for the synthesis of reverse glycosides by late-stage diversification of RGFs. The potential of this method is showcased by the preparation and diversification of sotagliflozin, leading to the discovery of a promising SGLT2 inhibitor candidate.