Aims: The potential receptor for hydrogen sulfide (H 2 S) remains unknown. Results: H 2 S could directly activate vascular endothelial growth factor receptor 2 (VEGFR2) and that a small interfering RNA (siRNA)-mediated knockdown of VEGFR2 inhibited H 2 S-induced migration of human vascular endothelial cells. H 2 S promoted angiogenesis in Matrigel plug assay in mice and this effect was attenuated by a VEGF receptor inhibitor. Using tandem mass spectrometry (MS), we identified a new disulfide complex located between Cys1045 and Cys1024 within VEGFR2 that was labile to H 2 S-mediated modification. Kinase activity of the mutant VEGFR2 (C1045A) devoid of the Cys1045-Cys1024 disulfide bond was significantly higher than wild-type VEGFR2. Transfection with vectors expressing VEGFR2 (C1045A) caused a significant increase in cell migration, while the migrationpromoting effect of H 2 S disappeared in the cells transfected with VEGFR2 (C1045A). Therefore, the Cys1045-Cys1024 disulfide bond serves as an intrinsic inhibitory motif and functions as a molecular switch for H 2 S. The formation of the Cys1045-Cys1024 disulfide bond disrupted the integrity of the active conformation of VEGFR2. Breaking the Cys1045-Cys1024 disulfide bond recovered the active conformation of VEGFR2. This motif was prone to a nucleophilic attack by H 2 S via an interaction of their frontier molecular orbitals. siRNA-mediated knockdown of cystathionine c-lyase attenuated migration of vascular endothelial cells induced by VEGF or moderate hypoxia.

This work presented an innovative and rationally engineered palindromic molecular beacon (PMB) based "Z-scheme" photoelectrochemical (PEC) biosensing protocol for the selective screening of kanamycin (Kana) through DNA hybridization-induced conformational conversion. Interestingly, the ingeniously designed PMB integrated the multifunctional elements including recognition region, primer-like palindromic fragment, and polymerization-nicking template. The cosensitized structures consisted of CdS quantum dot functionalized hairpin DNA2 (QD-HP2) and region-selectively deposited gold nanoparticles onto {001} facets of bismuth oxychloride (BiOCl-Au). Compared with BiOCl-Au alone, the attachment of CdS QDs onto BiOCl-Au (i.e., BiOCl-Au-CdS QDs) exhibited evidently enhanced photocurrent intensity thanks to the synergistic effect of Z-scheme BiOCl-Au-CdS QDs. After incubation with target Kana, Kana−aptamer binding could induce the exposure of PMB region for hairpin DNA1 (HP1). The exposed palindromic tails hybridized with each other (like a molecular machine) to consume the substrates (dNTPs) and fuels (enzyme) for the releasing of numerous nick fragments (Nick). The asgenerated nick fragments could specifically hybridize with the complementary region of QD-HP2, thus resulting in decreasing photocurrent because of the increasing spatial distance for electron transfer between two-type photosensitizers. Under optimum conditions, the PMB-based sensing system exhibited satisfying photocurrent responses toward target Kana within the working range from 50 to 5000 fM at a low detection limit of 29 fM. Impressively, the concept of a palindromic fragment-mediated primer-free biosensing strategy offers a new avenue for advanced development of efficient and convenient biodetection systems.

A novel photoelectrochemical (PEC) enzyme immunoassay was designed for the ultrasensitive detection of alpha-fetoprotein (AFP) based on near-infrared (NIR) light-excited core-core-shell UCNP@Au@CdS upconversion nanospheres. Plasmonic gold (Au) between the sandwiched layers was not only utilized as an energy harvester for the collection of the incident light but also acted as an energy conveyor to transfer the energy from upconversion NaYF:Yb,Er (UCNP) to semiconductor CdS, thus exciting the efficient separation of electron-hole pairs by the generated HO of enzyme immunoreaction under the irradiation of a 980 nm laser. By virtue of high catalytic activity of natural enzymes, gold nanoparticles heavily functionalized with glucose oxidase (GOx) and polyclonal anti-AFP antibody were utilized to generate HO. A sandwiched immunoreaction was first carried out in a monoclonal anti-AFP antibody-coated microplate by using an antibody-labeled gold nanoparticle as secondary antibody. Accompanying the gold nanoparticle, the carried GOx oxidized glucose in HO, thereby resulting in the enhanced photocurrent via capturing holes on the valence band of CdS to promote the separation of electron-hole pairs. Under optimum conditions, the NIR light-based PEC immunosensing system exhibited good photocurrent responses toward target AFP within the dynamic working range of 0.01-40 ng mL at a detection limit of 5.3 pg mL. Moreover, the NIR light-based sensing platform had good reproducibility and high selectivity. Importantly, good well-matched results obtained from NIR light-based PEC immunoassay were acquired for the analysis of human serum specimens by using AFP ELISA kit as the reference.

Pressure-based bioassays incorporating biomolecular recognition with a catalyzed gas-generation reaction have been developed for gas biosensors, but most involve poor sensitivity and are unsuitable for routine use. Herein we design an innovative gas pressure-based biosensing platform for the detection of Kanamycin (Kana) on polyaniline nanowires-functionalized reduced graphene oxide (PANI/rGO) framework by using platinum nanozyme-catalyzed gas generation. The signal was amplified by coupling with catalytic hairpin assembly (CHA) and strand-displacement amplification (SDA). Upon target Kana introduction, the analyte initially triggered a SDA reaction between hairpin DNA1 and hairpin DNA2, and then induced CHA conjugation between magnetic bead-labeled hairpin DNA3 (MB-H3) and platinum nanoparticle-labeled hairpin DNA4 (Pt-H4) to form a three-dimensional network. Numerous platinum nanoparticles (peroxidase-like nanozymes) were carried over with magnetic beads to reduce hydrogen peroxide into oxygen. The as-produced gas compressed PANI/rGO frameworks (modified to polyurethane sponge, used as the piezoelectric materials) in a homemade pressure-tight device, thus causing the increasing current of PANI/rGO sponge thanks to its deformation. The change in the current caused by the as-generated gas pressure was determined on an electrochemical workstation. Under optimum conditions, PANI/rGO sponge exhibited outstanding compressibility, stable signal-waveform output, fast response and recovery time (≈109 ms), and the current increased with the increasing Kana concentration within a dynamic working range of 0.2-50 pM at a detection limit of 0.063 pM. Good reproducibility, specificity, and acceptable precision were acquired for Kana analysis. In addition, the accuracy of this method was monitored to evaluate real milk samples with the well-matched results obtained by using the referenced Kana ELISA kit.

BACKGROUND AND PURPOSEFamitinib is a novel multi-targeted receptor tyrosine kinase inhibitor under development for cancer treatment. This study aims to characterize the metabolic and bioactivation pathways of famitinib. EXPERIMENTAL APPROACHThe metabolites in human plasma, urine and feces were identified via ultra-high performance liquid chromatographyquadrupole-time of flight-mass spectrometry and confirmed using synthetic standards. Biotransformation and bioactivation mechanisms were investigated using microsomes, recombinant metabolic enzymes and hepatocytes. KEY RESULTSFamitinib was extensively metabolized after repeated oral administrations. Unchanged famitinib was the major circulating material, followed by N-desethylfaminitib (M3), whose steady-state exposure represented 7.2 to 7.5% that of the parent drug. Metabolites in the excreta were mainly from oxidative deamination (M1), N-desethylation (M3), oxidative defluorination (M7), indolylidene hydroxylation (M9-1 and M9-5) and secondary phase-II conjugations. CYP3A4/5 was the major contributor to M3 formation, CYP3A4/5 and aldehyde dehydrogenase to M1 formation and CYP1A1/2 to M7, M9-1 and M9-5 formations. Minor cysteine conjugates were observed in the plasma, urine and feces, implying the formation of reactive intermediate(s). In vitro microsomal studies proved that famitinib was bioactivated through epoxidation at indolylidene by CYP1A1/2 and spontaneously defluorinated rearrangement to afford a quinone-imine species. A correlation between famitinib hepatotoxicity and its bioactivation was observed in the primary human hepatocytes. CONCLUSION AND IMPLICATIONSFamitinib is well absorbed and extensively metabolized in cancer patients. Multiple enzymes, mainly CYP3A4/5 and CYP1A1/2, are involved in famitinib metabolic clearance. The quinone-imine intermediate formed through bioactivation may be associated with famitinib hepatotoxicity. Co-administered CYP1A1/2 inducers or inhibitors may potentiate or suppress its hepatotoxicity. AbbreviationsABT, 1-aminobenzotriazole; BSO, L-buthionine-sulphoximine; CCK-8, cell counting kit-8; CE, collision energy; CYP, cytochrome P450; FMO, flavin mono-oxygenases; GSH, glutathione; HIM, human intestinal microsomes; HLM, human liver microsomes; HPM, human pulmonary microsomes; HRM, human renal microsomes; IS, internal standard; KET, ketoconazole; MEH, microsomal epoxide hydrolase; NADPH, b-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt; NQO1, recombinant human NAD(P)H : quinone oxidoreductase 1; RTK, receptor tyrosine kinase; UHPLC/Q-TOF MS, ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometer; a-NF, a-naphthoflavone BJP British Journal of Pharmacology