ON-OFF Fluorescent Cyanine Dye Based on a Benzothiophenyl Rotor Enables Selective Illumination of G-Quadruplexes in Mitochondria.

Conventional cyanine dyes exist as "always-on" fluorescent probes leading to inevitable background signals which often limit their performance and scope of applications. To develop specific fluorescent probes with high sensitivity and robust OFF/ON switching for targeting G4s, we introduced aromatic heterocycles through conjugation with polymethine chains to construct a rotor-π system. Here, a universal strategy is presented to synthesize pentamethine cyanines with different aromatic heterocycle substituents on the meso-polymethine chain. In these probes, SN-Cy5-S is self-quenched in aqueous solution due to H-aggregation. The structure indicates that SN-Cy5-S with a flexible meso-benzothiophenyl rotor conjugated to the cyanine backbone matches adaptively with G-tetrad planes, enhancing π-π stacking and resulting in triggered fluorescence. This allows recognition of G-quadruplexes due to the synergy of disaggregation-induced emission (DIE) and inhibited twisted intramolecular charge-transfer effects. This combination leads to a robust lighting-up fluorescence response for c-myc G4 with superior fluorescence enhancement (98-fold), allowing for a low detection limit of 1.51 nM, which is much more sensitive than the previously reported DIE-based G4 probes (22-83.5 nM). In addition, the superior imaging properties and rapid internalization time (5 min) in mitochondria allow SN-Cy5-S to also have a high potential for mitochondrially targeting anti-cancer therapy.

A novel pomegranate-inspired bifunctional electrode materials design for acetylcholinesterase biosensor and methanol oxidation reaction.

A pomegranate-inspired bifunctional electrode material based on Ni/NiO nanoparticle embedded in nitrogen-doped, partially graphitized carbon framework (Ni/NiO@NPGC) was designed and prepared for the construction of novel electrochemical biosensor and methanol oxidation reaction (MOR). Profiting from itsspecialstructureandfunction, Ni/NiO@NPGC was employed as a matrix immobilizing acetylcholinesterase (AChE) for methyl parathion (MP) sensor. The developed biosensor was proved to have wide linear range (1.0 × 10-14-1.0 × 10-8 g mL-1), low detection limit (3.5 × 10-15 g mL-1), and good stability for the determination of MP in practical samples. In addition, the Ni/NiO@NPGC electrode exhibited high electrocatalytic activity (specific activity 73.1 mA cm-2) and durability for the MOR in alkaline medium. The results were mainly attributed to the pomegranate-like architecture structure with pyridinic N and carbon frame of Ni/NiO@NPGC, which ensured the electrochemical activities of all nanoparticles and immobilization of enzyme. In addition, the metal oxide was well dispersed to prevent from self-agglomeration and kept mass transfer paths. The work provides a reference for the development of high-performance bifunctional electrode material for the biosensor and MOR.

GJA1-20k attenuates Ang II-induced pathological cardiac hypertrophy by regulating gap junction formation and mitochondrial function.

Cardiac hypertrophy (CH) is characterized by an increase in cardiomyocyte size, and is the most common cause of cardiac-related sudden death. A decrease in gap junction (GJ) coupling and mitochondrial dysfunction are important features of CH, but the mechanisms of decreased coupling and energy impairment are poorly understood. It has been reported that GJA1-20k has a strong tropism for mitochondria and is required for the trafficking of connexin 43 (Cx43) to cell-cell borders. In this study, we investigated the effects of GJA1-20k on Cx43 GJ coupling and mitochondrial function in the pathogenesis of CH. We performed hematoxylin-eosin (HE) and Masson staining, and observed significant CH in 18-week-old male spontaneously hypertensive rats (SHRs) compared to age-matched normotensive Wistar-Kyoto (WKY) rats. In cardiomyocytes from SHRs, the levels of Cx43 at the intercalated disc (ID) and the expression of GJA1-20k were significantly reduced, whereas JAK-STAT signaling was activated. Furthermore, the SHR rats displayed suppressed mitochondrial GJA1-20k and mitochondrial biogenesis. Administration of valsartan (10 mg· [Formula: see text] d-1, i.g., for 8 weeks) prevented all of these changes. In neonatal rat cardiomyocytes (NRCMs), overexpression of GJA1-20k attenuated Ang II-induced cardiomyocyte hypertrophy and caused elevated levels of GJ coupling at the cell-cell borders. Pretreatment of NRCMs with the Jak2 inhibitor AG490 (10 µM) blocked Ang II-induced reduction in GJA1-20k expression and Cx43 gap junction formation; knockdown of Jak2 in NRCMs significantly lessened Ang II-induced cardiomyocyte hypertrophy and normalized GJA1-20k expression and Cx43 gap junction formation. Overexpression of GJA1-20k improved mitochondrial membrane potential and respiration and lowered ROS production in Ang II-induced cardiomyocyte hypertrophy. These results demonstrate the importance of GJA1-20k in regulating gap junction formation and mitochondrial function in Ang II-induced cardiomyocyte hypertrophy, thus providing a novel therapeutic strategy for patients with cardiomyocyte hypertrophy.

Oxygen reduction reaction electrocatalysis inducing Fenton-like processes with enhanced electrocatalytic performance based on mesoporous ZnO/CuO cathodes: Treatment of organic wastewater and catalytic principle.

To treat typical organic wastewater efficiently, a novel Fenton-like processes based on ZnO/CuO composite cathode induced by oxygen reduction reaction (ORR) electrocatalysis with enhanced electrocatalytic performance was established successfully. Electrochemical testing investigation indicated that the ZnO/CuO cathode possessed conspicuous redox peak and better conductivity than uncompounded electrodes. Additionally, the removal efficiency of methylene blue and its chemical oxygen demand (COD) reached 96.4% and 70.8% after 120 min, respectively. Next, the feasibility of the material in practical application was also discussed. Subsequently, electrocatalytic principle based on valence state changes of metal elements on the electrode surface were also studied by x-ray photoelectron spectroscopy (XPS). Redox reactions between the active species H2O2 and the species Cu+ promoting Fenton-like processes were deduced. Namely, the conversion of Cu(I) and Cu(II) on the electrode surface was accompanied by OH generation. The combination of ZnO and CuO improved the surface morphology, increasing the active site of ORR and the yield of H2O2, thus greatly enhanced the Fenton-like activity. Finally, the main intermediates were identified by Gas chromatography-mass spectrometer (GC-MS), and possible pathways for dye degradation were proposed. In short, the research of ZnO/CuO cathode provided great significance for heterogeneous Fenton-like degradation and also showed its application potential in water treatment and remediation.

Inhibition of ubiquitin­specific protease 14 promotes connexin 32 internalization and counteracts cisplatin cytotoxicity in human ovarian cancer cells.

Although cisplatin is one of the most accepted therapies for ovarian cancer, recurrence and drug resistance remain problematic. Both the ubiquitin­proteasome system (UPS) and connexin (Cx) are closely related to tumor progression. However, the role of ubiquitin­specific protease 14 (USP14) and Cx in mediating drug resistance remains unclear. In the present study, we aimed to determine whether USP14 is involved in cisplatin resistance and modulates the internalization of connexin 32 (Cx32) in ovarian cancer. The results of the deubiquitinase (DUB) trap assay and western blot analysis revealed that the expression and activity levels of USP14 were downregulated in A2780 cisplatin­resistant cells (A2780­CDDP) relative to these levels in A2780 cisplatin­sensitive cells (A2780). CCK­8 assay results showed that inhibition of USP14 by a specific inhibitor or siRNA decreased cisplatin cytotoxicity in A2780 cells. Additionally, USP14 inhibition increased the expression of Cx32 without changing its mRNA and ubiquitination levels, as showed by Real­time qPCR and immunoprecipitation assay respectively. Cisplatin resistance induced by USP14 inhibition was counteracted by Cx32 knockdown. Moreover, USP14 inhibition contributed to Cx32 internalization, as determined by western blot analysis and a reduction in gap junction intercellular communication (GJIC), as showed by parachute dye­coupling assay. Collectively, these data suggest that Cx32 internalization by USP14 inhibition modulates the cisplatin resistance in ovarian cancer cells, thus serving as a potential drug target to challenge chemotherapy failure. In addition, USP14 can also be used as a marker to monitor the development of cisplatin resistance in ovarian cancer treatment.

Cx32 exerts anti-apoptotic and pro-tumor effects via the epidermal growth factor receptor pathway in hepatocellular carcinoma.

BACKGROUND: Abnormal expression or distribution of connexin 32 (Cx32) is associated with hepatocarcinogenesis, but the role of Cx32 and the underlying mechanisms are still unclear. METHODS: The expression level of Cx32 in 96 hepatocellular carcinoma (HCC) specimens was determined using western blotting and immunohistochemistry. The correlation between Cx32 expression and clinicopathological parameters was analyzed. The cell apoptosis rate was examined using flow cytometry and western blotting. The role of Cx32 in the Src kinase and epidermal growth factor receptor (EGFR) signaling pathways was measured by quantitative real-time PCR, western blotting and coimmunoprecipitation (CO-IP). The effect of Cx32 overexpression on the streptonigrin (SN)-induced tumor growth suppression and apoptosis was assessed in nude mice. RESULTS: Our study showed that overexpressed Cx32 accumulated in the cytoplasm and that Cx32-containing gap junctions (GJs) were nearly absent in HCC specimens. Upregulated Cx32 expression was highly correlated with advanced tumor-node-metastasis (TNM) stage and poor tumor differentiation and was an independent predictive marker for poor prognosis in HCC. Overexpression of Cx32 significantly inhibited SN-induced apoptosis by activating the EGFR signaling pathway in vitro and in vivo. Moreover, the expression levels of Cx32 and EGFR were positively correlated in HCC specimens. The CO-IP experiments demonstrated that Cx32 could bind to Src kinase, and the western blotting results revealed that Cx32 increased the levels of EGFR and p-EGFR by upregulating Src expression. CONCLUSION: The present study demonstrated that overexpressed and internalized Cx32 was associated with advanced TNM stage and poor tumor differentiation and predicted poor prognosis in HCC. Cx32 facilitated HCC progression by blocking chemotherapy-induced apoptosis in vitro and in vivo via interacting with Src and thus promoting the phosphorylation of EGFR, subsequently activating the EGFR signaling pathway. Cx32 may be a potential biomarker and a new therapeutic target for HCC.

Involvement of UDP-glucuronosyltransferases in higenamine glucuronidation and the gender and species differences in liver.

OBJECTIVES: Higenamine (HG), an active ingredient of Aconite root in Chinese herbal medicine, is mainly metabolized by UDP-glucuronosyltransferases (UGT). However, the systematic glucuronidation of HG in humans remains unclear. The purpose of this study was to investigate the glucuronidation of HG. METHODS: 12 recombinant human UGT (rUGT) isozymes were used to characterize the HG glucuronidation. Liver microsomes from male and female mice, rats, guinea pigs, dogs, and humans were used to determine the species and gender differences using liquid chromatography-mass spectrometry. KEY FINDINGS: One monoglucuronide was detected in reactions catalyzed by rUGT1A6, rUGT1A8, rUGT1A9, also human and dog liver microsomes. UGT1A9 is the most important glucuronosyltransferase that metabolizes HG. Because carvacrol, a specific inhibitor of UGT1A9, can significantly decrease the glucuronidation of HG in Human liver microsomes and UGT1A9. HG metabolism by UGT1A9 described in Michaelis-Menten kinetics (Km=15.4 mM,Vmax=2.2 nmol/mg/min) and glucuronidation in liver microsomes were species dependent. Gender did not affect the kinetic parameters among species except in rats. CONCLUSIONS: UGT1A9 is a major isoenzyme responsible for the glucuronidation of HG in Human liver microsomes (HLMs). Dog may be an appropriate animal model to evaluate HG metabolism.