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The photoelectrochemical immunoassay of glycogen phosphorylase BB (GPBB) was studied. A methyl orange/TiO 2 nanorod heterojunction was constructed on a fluorine-doped tin oxide electrode by hydrothermal synthesis, calcination, and chemical adsorption. A sandwich immune structure consisting of GPBB as the first antibody, GPBB, and a CdS@mesoporous silica-ascorbic acid (AA)-GPBB as secondary antibody composite was constructed on each of the selected well surfaces of a 96-well microplate. By adding mercaptoethylamine to structurally destroy the secondary antibody composite and release the electron donor AA, the amplification of photocurrent, and thus the "off-on" photoelectrochemical biosensing of GPBB were realized. The use of the 96-well microplate provides good reproducibility of the assembled immune structures and eliminates the possible effect of the photogenerated hole-induced protein oxidation on the photocurrent. The relevant electrodes and materials were characterized by electrochemistry, UV-vis diffuse reflectance spectra, Fourier transform infrared spectroscopy, X-ray diffractometer, scanning electron microscopy/energy dispersive spectroscopy, transmission electron microscopy and BET method. Under the optimal conditions, the photocurrent was linear with the logarithm of GPBB concentration from 0.005 to 200 ng mL -1 and with a limit of detection of 1.7 pg mL -1 (S/N = 3). Satisfactory results were obtained in the analysis of real serum samples. A sandwich immune structure consisting of GPBB first antibody, GPBB, and a CdS@mesoporous silica-ascorbic acid (AA)-GPBB secondary antibody composite was constructed on each of the selected well surfaces of a 96-well microplate. By adding mercaptoethylamine to structurally destroy the secondary antibody composite and release the electron donor AA, the amplification of photocurrent, and thus the "off-on" photoelectrochemical biosensing of GPBB were realized., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background: Panax notoginseng is a highly valued medicinal herb used widely in China and many Asian countries. Its root and rhizome have long been used for the treatment of cardiovascular and hematological diseases. Imaging the spatial distributions and dynamics of metabolites in heterogeneous plant tissues is significant for characterizing the metabolic networks of Panax notoginseng , and this will also provide a highly informative approach to understand the complex molecular changes in the processing of Panax notoginseng ., Methods: Here, a high-sensitive MALDI-MS imaging method was developed and adopted to visualize the spatial distributions and spatiotemporal changes of metabolites in different botanical parts of Panax notoginseng ., Results: A wide spectrum of metabolites including notoginsenosides, ginsenosides, amino acids, dencichine, gluconic acid, and low-molecular-weight organic acids were imaged in Panax notoginseng rhizome and root tissues for the first time. Moreover, the spatiotemporal alterations of metabolites during the steaming of Panax notoginseng root were also characterized in this study. And, a series of metabolites such as dencichine, arginine and glutamine that changed with the steaming of Panax notoginseng were successfully screened out and imaged., Conclusion: These spatially-resolved metabolite data not only enhance our understanding of the Panax notoginseng metabolic networks, but also provide direct evidence that a serious of metabolic alterations occurred during the steaming of Panax notoginseng ., Competing Interests: The authors declare no conflict of interest., (© 2021 The Korean Society of Ginseng. Publishing services by Elsevier B.V.)

The application of mass spectrometry imaging (MSI) for the study of spatiotemporal alterations of the metabolites in tumors has brought a number of significant biological results. At present, metabolite profiling based on MSI is typically performed on frozen tissue sections; however, the majority of clinical specimens need to be fixed in tissue fixative to avoid autolysis and to preserve antigenicity. In this study, we present the global impacts of different fixatives on the MS imaging of gastric cancer tissue metabolites. The MSI performances of 17 kinds of metabolites, such as amino acids, polyamines, cholines, organic acids, polypeptides, nucleotides, nucleosides, nitrogen bases, cholesterols, fatty acids, and phospholipids, in untreated, 10% formalin-, 4% paraformaldehyde-, acetone-, and 95% ethanol-fixed gastric cancer tissues were thoroughly explored for the first time. Furthermore, we also investigated the spatial expressions of 6 metabolic enzymes, namely, GLS, FASN, CHKA, PLD2, cPLA2, and EGFR, closely related to tumor-associated metabolites. Immunohistochemical staining carried out on the same tissue sections' which have undergone MSI analysis' suggests that enzymatic characterization is feasible after metabolite imaging. Combining the spatial signatures of metabolites and pathway-related metabolic enzymes in heterogeneous tumor tissues offers an insight to understand the complex tumor metabolism. Graphical abstract.

Characterization of tumor metabolism with spatial information contributes to our understanding of complex cancer metabolic reprogramming, facilitating the discovery of potential metabolic vulnerabilities that might be targeted for tumor therapy. However, given the metabolic variability and flexibility of tumors, it is still challenging to characterize global metabolic alterations in heterogeneous cancer. Here, we propose a spatially resolved metabolomics approach to discover tumor-associated metabolites and metabolic enzymes directly in their native state. A variety of metabolites localized in different metabolic pathways were mapped by airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) in tissues from 256 esophageal cancer patients. In combination with in situ metabolomics analysis, this method provided clues into tumor-associated metabolic pathways, including proline biosynthesis, glutamine metabolism, uridine metabolism, histidine metabolism, fatty acid biosynthesis, and polyamine biosynthesis. Six abnormally expressed metabolic enzymes that are closely associated with the altered metabolic pathways were further discovered in esophageal squamous cell carcinoma (ESCC). Notably, pyrroline-5-carboxylate reductase 2 (PYCR2) and uridine phosphorylase 1 (UPase1) were found to be altered in ESCC. The spatially resolved metabolomics reveal what occurs in cancer at the molecular level, from metabolites to enzymes, and thus provide insights into the understanding of cancer metabolic reprogramming., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Most cells are believed to be capable of producing type I interferons (IFN I) as part of an innate immune response against, for instance, viral infections. In macrophages, IFN I is potently induced upon cytoplasmic exposure to foreign nucleic acids. Infection of these cells with herpesviruses leads to triggering of the DNA sensors interferon-inducible protein 16 (IFI16) and cyclic GMP-AMP (cGAMP) synthase (cGAS). Thereby, the stimulator of interferon genes (STING) and the downstream molecules TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) are sequentially activated culminating in IFN I secretion. Human gamma-herpesviruses, such as Epstein-Barr virus (EBV), exploit B cells as a reservoir for persistent infection. In this study, we investigated whether human B cells, similar to macrophages, engage the cytoplasmic DNA sensing pathway to induce an innate immune response. We found that the B cells fail to secrete IFN I upon cytoplasmic DNA exposure, although they express the DNA sensors cGAS and IFI16 and the signaling components TBK1 and IRF3. In primary human B lymphocytes and EBV-negative B cell lines, this deficiency is explained by a lack of detectable levels of the central adaptor protein STING. In contrast, EBV-transformed B cell lines did express STING, yet both these lines as well as STING-reconstituted EBV-negative B cells did not produce IFN I upon dsDNA or cGAMP stimulation. Our combined data show that the cytoplasmic DNA sensing pathway is dysfunctional in human B cells. This exemplifies that certain cell types cannot induce IFN I in response to cytoplasmic DNA exposure providing a potential niche for viral persistence., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Huoxue-Tongluo lyophilized powder for injection (HTLPI), a traditional Chinese medicine preparation, is a compound of Persicae semen and Paeoniae Radix Rubra that is used mainly for treating blood-stasis obstruction syndrome in the acute stage of cerebral ischemic stroke. Amygdalin (AD) and paeoniflorin (PF) are 2 typical bioactive components in HTLPI and were selected as indicators for this pharmacokinetic study of HTLPI. The objective of this study was to investigate the safety profile, tolerability, and pharmacokinetic properties of AD and PF after single and multiple intravenous infusions of HTLPI in healthy Chinese volunteers., Methods: Twenty-one healthy Chinese subjects were recruited for this open-label, single ascending-dose (3, 6, and 9 g) and multiple-dose (6 g, once daily) study. Safety profile was assessed by adverse events and physical examination throughout the study. Serial plasma and urine samples were analyzed by HPLC-MS/MS. Pharmacokinetic parameters of AD and PF were calculated using noncompartmental analysis., Findings: In the single-dose phase of the study, the mean maximum plasma concentration and the mean area under the plasma concentration-time curve of AD and PF increased proportionally with each dose escalation. In the multiple-dose phase, the steady state was achieved by day 4 after multiple-dose administration of 6 g HTLPI. Mean pharmacokinetic parameters achieved on day 1 were similar to those on day 7. No significant accumulation was observed after repeat doses of 6 g HTLPI. Approximately 79.6% of the administered AD and 48.4% of the administered PF were excreted unchanged in urine within 24 hours. No serious adverse events were observed during the entire study., Implications: The pharmacokinetic properties of AD and PF were linear after a single intravenous infusion of HTLPI in the dose range of 3-9 g. No systemic accumulation was observed with repeat doses of HTLPI. Sex had no significant effect on the pharmacokinetic properties of AD and PF. Intravenous infusion of HTLPI was well tolerated in healthy Chinese subjects., (Copyright © 2016 Elsevier HS Journals, Inc. All rights reserved.)

Lidocaine hydrochloride ophthalmic gel is a novel ophthalmic preparation for topical ocular anesthesia. The study is aimed at evaluating the ocular and systemic pharmacokinetics of lidocaine hydrochloride 3.5 % ophthalmic gel in rabbits after ocular topical administration. Thirty-six rabbits were randomly placed in 12 groups (3 rabbits per group). The rabbits were quickly killed according to their groups at 0 (predose), 0.0833, 0.167, 0.333, 0.667, 1, 1.5, 2, 3, 4, 6, and 8 h postdose and then the ocular tissue and plasma samples were collected. All the samples were analyzed by a validated LC-MS/MS method. The test result showed that the maximum concentration (C max) of lidocaine in different ocular tissues and plasma were all achieved within 20 min after drug administration, and the data of C max were (2,987 ± 1814) μg/g, (44.67 ± 12.91) μg/g, (26.26 ± 7.19) μg/g, (11,046 ± 2,734) ng/mL, and (160.3 ± 61.0) ng/mL for tear fluid, cornea, conjunctiva, aqueous humor, and plasma, respectively. The data of the elimination half-life in these tissues were 1.5, 3.2, 3.5, 1.9, and 1.7 h for tear fluid, cornea, conjunctiva, aqueous humor, and plasma, respectively. The intraocular lidocaine levels were significantly higher than that in plasma, and the elimination half-life of lidocaine in cornea, conjunctiva, and aqueous humor was relatively longer than that in tear fluid and plasma. The high intraocular penetration, low systemic exposure, and long duration in the ocular tissues suggested lidocaine hydrochloride 3.5 % ophthalmic gel as an effective local anesthetic for ocular anesthesia during ophthalmic procedures.

The rabies virus (RABV) glycoprotein (G) is responsible for inducing neutralizing antibodies against rabies virus. Development of recombinant vaccines using the G genes from attenuated strains rather than street viruses is a regular practice. In contrast to this scenario, we generated three human adenovirus type 5 recombinants using the G genes from the vaccine strains SRV9 and Flury-LEP, and the street RABV strain BD06 (nrAd5-SRV9-G, nrAd5-Flury-LEP-G, and nrAd5-BD06-G). These recombinants were non-replicative, but could grow up to ~10(8) TCID50/ml in helper HEK293AD cells. Expression of the G protein was verified by immunostaining, quantitative PCR and cytometry. Animal experiments revealed that immunization with nrAd5-BD06-G can induce a higher seroconversion rate, a higher neutralizing antibody level, and a longer survival time after rabies virus challenge in mice when compared with the other two recombinants. Moreover, the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly higher in mice immunized with nrAd5-BD06-G, which might also contribute to the increased protection. These results show that the use of street RABV G for non-replicative systems may be an alternative for developing effective recombinant rabies vaccines.

Purpose: Multiple myeloma (MM) is a malignant and incurable neoplasm of plasma cells that accumulate in the bone marrow. Bendamustine, an antitumor agent including double property of alkylating agent and purine analogues, displayed clinical antitumor activity in patients with MM. However, the precise mechanism of action of bendamustine has not been completely elucidated., Methods: In this study, we established the cell model of bendamustine-induced MM RPMI8226 cell apoptosis, and used two dimensional differential in-gel electrophoresis (2D-DIGE) proteomics to analyze the bendamustine-induced protein alterations., Results: Our results revealed that compared with control group, bendamustine significantly inhibited the proliferation of RPMI8226 cells in a concentration-dependent and time-dependent manner. Proteomic approach was performed to identify 30 differentially expressed proteins in RPMI8226 cells upon bendamustine treatment, which included 15 up-regulated and 15 down-regulated proteins. Of these, protein disulfide isomerase A3 (PDIA3) and cytokine- induced apoptosis inhibitor 1 (CPIN1), were selected for further studies., Conclusion: These results implicate PDIA3 and CPIN1 as potential molecular targets for drug intervention in MM and thus provide novel insights into the mechanisms of antitumor activity of bendamustine.