The mechanisms and chemo- and regioselectivities of Ru(II)-catalyzed decarboxylative C-H alkenylation of aryl carboxylic acids with alkynes were investigated with density functional theory (DFT) calculations. The catalytic cycle involves sequential carboxylate-directed C-H activation, alkyne insertion, decarboxylation and protonation. The facile tether-assisted decarboxylation step directs the intermediate toward the desired decarboxylative alkenylation, instead of typical annulation and double alkenylation pathways. The decarboxylation barrier is very sensitive to the tether length, and only the seven-membered ring intermediate can selectively undergo the designed decarboxylation, suggesting a tether-dependent chemoselectivity. This tether-dependent chemoselectivity also applies to the alkyl tethers. In addition, the polarity of solvent is found to control the chemoselectivity between the decarboxylative alkenylation and [4 + 2] annulation. Solvent with low polarity (toluene) favors the decarboxylation pathway, leading to the decarboxylative alkenylation. Solvent with high polarity (methanol) favors the ionic stepwise C-O reductive elimination pathway, leading to the [4 + 2] annulation. To understand the origins of regioselectivity with asymmetric alkynes, the distortion/interaction analysis was applied to the alkyne insertion transition states, and led to a predictive frontier molecular orbital model. The asymmetric alkynes selectively use the terminal with the larger HOMO orbital coefficient to form the C-C bond in the insertion step.

The α3β2 and α3β4 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central and peripheral nervous systems, playing critical roles in various physiological processes and in such pathologies as addiction to nicotine and other drugs of abuse. α-Conotoxin LvIA, which we previously isolated from Conus lividus, modestly discriminates α3β2 and α3β4 rat nAChRs exhibiting a ∼17fold tighter binding to the former. Here, alanine scanning resulted in two more selective analogues [N9A]LvIA and [D11A]LvIA, the former having a >2000-fold higher selectivity for α3β2. The determined crystal structures of [N9A]LvIA and [D11A]LvIA bound to the acetylcholinebinding protein (AChBP) were followed by homologous modeling of the complexes with the α3β2 and α3β4 nAChRs and by receptor mutagenesis, which revealed Phe106, Ser108, Ser113, and Ser168 residues in the β2 subunit as essential for LvIA binding. These results may be useful for the design of novel compounds of therapeutic potential targeting α3β2 nAChRs.

Background: Circular RNAs (circRNAs) are considered as key regulators of cancer biology. Recently, cMTO1 (a circRNA derived from MTO1 gene, hsa_circ_0007874) has been demonstrated to act as a tumor suppressor in hepatocellular carcinoma (HCC). However, the roles of cMTO1 in liver fibrosis are largely unknown. Methods: Expressions and roles of cMTO1 were examined in vivo and in vitro during liver fibrosis. The interaction between microRNA-181b-5p (miR-181b-5p) and cMTO1 was analyzed by luciferase activity assays and pull down assays. Results: cMTO1 was shown to be reduced in the liver from patients with cirrhosis. In addition, cMTO1 was down-regulated in the mouse fibrotic livers as well as activated hepatic stellate cells (HSCs). Restoring of cMTO1 led to a reduction in HSC proliferation. Results of immunofluorescence analysis showed that cMTO1 suppressed the expressions of α-SMA and type I collagen. cMTO1 was found to be expressed in the cytoplasm of HSCs. Further studies confirmed that cMTO1 and miR-181b-5p were colocated in the cytoplasm. Interestingly, there was an interaction between cMTO1 and miR-181b-5p. Results of luciferase reporter assays and pull down assays confirmed that miR-181b-5p could bind to cMTO1. cMTO1-inhibited HSC activation was blocked down by miR-181b-5p or PTEN. Meanwhile, PTEN was a target of miR-181b-5p. Conclusion: cMTO1 inhibits HSC activation, at least in part, through miR-181b-5pmediated PTEN expression. Our results also suggest that cMTO1 may be a novel therapeutic target in liver fibrosis.

Based on the 13C chemical shift changes, the optimal monomer of MAA was selected and the rational binding sites were predicted. The resultant materials show good selectivity for erythromycin.

The α3* nAChRs, which are considered to be promising drug targets for problems such as pain, addiction, cardiovascular function, cognitive disorders etc., are found throughout the central and peripheral nervous system. The α-conotoxin (α-CTx) LvIA has been identified as the most selective inhibitor of α3β2 nAChRs known to date, and it can distinguish the α3β2 nAChR subtype from the α6/α3β2β3 and α3β4 nAChR subtypes. However, the mechanism of its selectivity towards α3β2, α6/α3β2β3, and α3β4 nAChRs remains elusive. Here we report the co-crystal structure of LvIA in complex with Aplysia californica acetylcholine binding protein (Ac-AChBP) at a resolution of 3.4 Å. Based on the structure of this complex, together with homology modeling based on other nAChR subtypes and binding affinity assays, we conclude that Asp-11 of LvIA plays an important role in the selectivity of LvIA towards α3β2 and α3/α6β2β3 nAChRs by making a salt bridge with Lys-155 of the rat α3 subunit. Asn-9 lies within a hydrophobic pocket that is formed by Met-36, Thr-59, and Phe-119 of the rat β2 subunit in the α3β2 nAChR model, revealing the reason for its more potent selectivity towards the α3β2 nAChR subtype. These results provide molecular insights that can be used to design ligands that selectively target α3β2 nAChRs, with significant implications for the design of new therapeutic α-CTxs.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-017-0426-2) contains supplementary material, which is available to authorized users.

Supercritical solution impregnation (SSI) was applied to prepare drug-loaded biodegradable films where poly(l-lactic acid) (PLLA) was used as the matrix and roxithromycin as the model drug. The effects of impregnation time, operating temperature, and pressure on drug loading capacity (DLC) of roxithromycin into PLLA matrix were investigated. With the extension of impregnation time, DLC increased gradually to an equilibrium value. DLC was also affected by impregnation temperature and pressure. At the optimal condition, i.e., impregnating at 70 °C and 300 bar for 2 h, the maximal DLC was approximately 10.5%. After SSI process, the PLLA film was still transparent. The SEM images showed that the morphologies of PLLA film did not change with the SSI process. The DSC data and XRD spectra demonstrated that roxithromycin molecules were dispersed into the PLLA film in an amorphous state and the SCCO2 processed PLLA film had a lower crystal degree than raw PLLA film. The residual dichloromethane due to the PLLA film preparation could be removed effectively during the SSI process and meet the Chinese Pharmacopoeia limit. In vitro release of roxithromycin consisted of two stages: initial rapid release and a following slow release. The SSI process is expected to be a promising technique to prepare a drug-loaded biodegradable polymer surface and matrix for antibacterial therapeutic implants.

This study aimed to evaluate the responses of human hepatocytes to azathioprine hepatotoxicity in comparison with the well-studied azathioprine hepatotoxicity in rat hepatocytes and the effects of protective agents to suppress azathioprine hepatotoxicity. Azathioprine presented its hepatotoxicity at clinically relevant concentrations (lower than 10 microm) in primary rat hepatocytes after 48 h of treatment as shown by a severe decrease in cell viability as well as intracellular GSH depletion. However, primary human hepatocytes exhibited only significant intracellular GSH depletion after treatment with azathioprine at these clinically relevant concentrations, while a reduction in cell viability by 29% was only evidenced after 48 h of treatment with azathioprine at the high concentration of 50 microm. In addition, a monolayer culture of primary rat hepatocytes was used as an in vitro model to examine the protective effects of antihepatotoxic drugs including glutathione (GSH), N-acetylcysteine (NAC, a GSH precursor), liquorice and glycyrrhizic acid (GA), a major bioactive component of liquorice, against hepatotoxicity of 1 microm azathioprine. It was found that both liquorice and GA showed substantial protection according to assays of cell viability and intracellular GSH, while neither GSH nor NAC had such a protective function. Similarly, GA protected human hepatocytes from intracellular GSH depletion on exposure to 1 microm azathioprine. These results implied that GA or liquorice could be considered as potent protection agents against azathioprine hepatotoxicity.

The α3β4 nicotinic acetylcholine receptor (nAChR) is an important target implicated in various disease states. α-Conotoxin TxID (1) is the most potent antagonist of α3β4 nAChR, but it also exhibits inhibition of α6/α3β4 nAChR. The results of alanine scanning of 1 suggested a vital role for Ser9 in the selectivity of the peptide. In this study, Ser9 was substituted with a series of 14 amino acids, including some non-natural amino acids, displaying different physicochemical characteristics to further improve the selectivity of 1 toward α3β4 nAChR. The pharmacological activities of the mutants were evaluated using an electrophysiological approach. The best selectivity was obtained with [S9K]TxID, 12, which inhibited α3β4 nAChR with an IC 50 of 6.9 nM and had no effects on other nAChRs. Molecular modeling suggested a possible explanation for the high selectivity of 12 toward α3β4 nAChR, providing deeper insight into the interaction between α-conotoxins and nAChRs as well as potential treatments for nAChR-related diseases.