The influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine, whose use has been discontinued due to widespread drug resistance. Among the handful of drug-resistant mutants, S31N is found in more than 95% of the currently circulating viruses and shows greatly decreased inhibition by amantadine. The discovery of inhibitors of S31N has been hampered by the limited size, polarity, and dynamic nature of its amantadine-binding site. Nevertheless, we have discovered smallmolecule drugs that inhibit S31N with potencies greater than amantadine's potency against WT M2. Drug binding locks the protein into a well-defined conformation, and the NMR structure of the complex shows the drug bound in the homotetrameric channel, threaded between the side chains of Asn31. Unrestrained molecular dynamics simulations predicted the same binding site. This S31N inhibitor, like other potent M2 inhibitors, contains a charged ammonium group. The ammonium binds as a hydrate to one of three sites aligned along the central cavity that appear to be hotspots for inhibition. These sites might stabilize hydronium-like species formed as protons diffuse through the outer channel to the proton-shuttling residue His37 near the cytoplasmic end of the channel.M2-S31N mutant structure | membrane protein structure | M2-S31N inhibitorT he influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine (1-3), which bind directly to the pore of the channel (2-4). Although amantadine has been widely used for several decades, drug resistance has curtailed the use of this family of drugs. Many amantadineresistant influenza viruses can be selected in cell culture (5, 6). A subset of these mutations is found in infected patients undergoing treatment with amantadine (7), and reverse-engineered viruses harboring various pore-lining mutations are competent to replicate in the mouse (8). However, many of these mutations give rise to somewhat attenuated viruses that are less transmissible than WT virus, and they tend to revert in the absence of drug pressure (6, 9). Indeed, large-scale sequencing of transmissible viruses isolated as early as 1918 showed that mutations to pore-lining residues are allowed only within the first turn of the transmembrane (TM) helix at positions 26, 27, and 31 (10). S31N has long been the predominant amantadine-resistant mutation in M2 (11)(12)(13)(14). It predominated in 98-100% of the transmissible amantadineresistant H1N1, H5N1, and H3N2 strains isolated from humans, birds, and swine in the past decade. V27A and L26F are less frequent mutations (10,11,15). Extensive studies of point mutations to the pore-lining residues of M2 have been conducted to understand the paucity of natural variants (16,17). Numerous mutants in the N-terminal aqueous pore retained the ability to conduct protons selectively over other ions, although the magnitude and pH dependence of their conduction varied. However, only a few mutations at the most distal sites, V27A,...

The title concept involves the use of structurally modified RCM substrates that contain extender arms, terminating in a remote reactive alkene. Initiation of an RCM sequence at that reactive alkene is followed by rapid intramolecular relay of the metal center to an initially less reactive alkene in the parent substrate. This permits one to control the relative timing (or direction) of a metathesis sequence. For example, one can reverse the inherent tendency of an unsymmetrical alpha,omega-diene substrate to close, say, left-to-right, to that of right-to-left. Four distinct types of application of the RRCM concept are demonstrated. Among other things, they show the preparation of tetrasubstituted electron-deficient alkenes using G1 [(Cy3P)2(Cl2)Ru=CHPh], complementary control of directionality (endedness), auxiliary benefits (enzyme specificity) from the incorporation of additional steric bulk, the activation of otherwise ineffective substrates for RCM closure, the use of unorthodox alkenes as initiation sites for ring closure, and control of product olefin geometry.

The sea lamprey is an ancient, parasitic fish that invaded the Great Lakes a century ago, where it triggered the collapse of many fisheries. Like many fishes, this species relies on chemical cues to mediate key aspects of its life, including migration and reproduction. Here we report the discovery of a multicomponent steroidal pheromone that is released by stream-dwelling larval lamprey and guides adults to spawning streams. We isolated three compounds with pheromonal activity (in submilligram quantities from 8,000 l of larval holding water) and deduced their structures. The most important compound contains an unprecedented 1-(3-aminopropyl)pyrrolidin-2-one subunit and is related to squalamine, an antibiotic produced by sharks. We verified its structure by chemical synthesis; it attracts adult lamprey at very low (subpicomolar) concentrations. The second component is another new sulfated steroid and the third is petromyzonol sulfate, a known lamprey-specific bile acid derivative. This mixture is the first migratory pheromone identified in a vertebrate and is being investigated for use in lamprey control.

Plant bZIP transcription factors play crucial roles in multiple biological processes. However, little is known about the sorghum bZIP gene family although the sorghum genome has been completely sequenced. In this study, we have carried out a genome-wide identification and characterization of this gene family in sorghum. Our data show that the genome encodes at least 92 bZIP transcription factors. These bZIP genes have been expanded mainly by segmental duplication. Such an expansion mechanism has also been observed in rice, arabidopsis and many other plant organisms, suggesting a common expansion mode of this gene family in plants. Further investigation shows that most of the bZIP members have been present in the most recent common ancestor of sorghum and rice and the major expansion would occur before the sorghum-rice split era. Although these bZIP genes have been duplicated with a long history, they exhibited limited functional divergence as shown by nonsynonymous substitutions (Ka)/synonymous substitutions (Ks) analyses. Their retention was mainly due to the high percentages of expression divergence. Our data also showed that this gene family might play a role in multiple developmental stages and tissues and might be regarded as important regulators of various abiotic stresses and sugar signaling.

Anti-influenza drugs, amantadine and rimantadine, targeting the M2 channel from influenza A virus are no longer effective because of widespread drug resistance. S31N is the predominant and amantadine-resistant M2 mutant, present in almost all of the circulating influenza A strains as well as in the pandemic 2009 H1N1 and the highly pathogenic H5N1 flu strains. Thus, there is an urgent need to develop second-generation M2 inhibitors targeting the S31N mutant. However, the S31N mutant presents a huge challenge to drug discovery, and it has been considered undruggable for several decades. Using structural information, classical medicinal chemistry approaches, and M2-specific biological testing, we discovered benzyl-substituted amantadine derivatives with activity against both S31N and WT, among which 4-(adamantan-1-ylaminomethyl)-benzene-1,3-diol (44) is the most potent dual inhibitor. These inhibitors demonstrate that S31N is a druggable target and provide a new starting point to design novel M2 inhibitors that address the problem of drug-resistant influenza A infections.

Tip-enhanced Raman scattering (TERS) is a promising optical and analytical technique for chemical imaging and sensing at single molecule resolution. In particular, TERS signals generated by a gap-mode configuration where a silver tip is coupled with a gold substrate can resolve a single-stranded DNA (ssDNA) molecule with a spatial resolution below 1 nm. To demonstrate the proof of subnanometer resolution, we show direct nucleic acid sequencing using TERS of a phage ssDNA (M13mp18). M13mp18 provides a known sequence and, through our deposition strategy, can be stretched (uncoiled) and attached to the substrate by its phosphate groups, while exposing its nucleobases to the tip. After deposition, we scan the silver tip along the ssDNA and collect TERS signals with a step of 0.5 nm, comparable to the bond length between two adjacent DNA bases. By demonstrating the real-time profiling of a ssDNA configuration and furthermore, with unique TERS signals of monomeric units of other biopolymers, we anticipate that this technique can be extended to the high-resolution imaging of various nanostructures as well as the direct sequencing of other important biopolymers including RNA, polysaccharides, and polypeptides.

Gliomas are the most common form of malignant primary brain tumors with poor 5-year survival rate. Dysregulation of procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) was observed in gliomas, but the specific role and molecular mechanism of PLOD2 in glioma have not been reported yet. In this study, PLOD2 was found to be frequently up-regulated in glioma and could serve as an independent prognostic marker to identify patients with poor clinical outcome. Knockdown of PLOD2 inhibited proliferation, migration and invasion of glioma cells in vitro and in vivo. Mechanistically, inhibition of PLOD2 inactivated PI3K/AKT signaling pathway and thus regulated the expression of its downstream epithelial–mesenchymal transition (EMT)-associated regulators, including E-cadherin, vimentin, N-cadherin, β-catenin, snail and slug in glioma cells. Moreover, PLOD2 could be induced by hypoxia-inducible factor-1α (HIF-1α) via hypoxia, thereby promoting hypoxia-induced EMT in glioma cells. Our data suggests that PLOD2 may be a potential therapeutic target for patients with glioma.

(2012) The role of AKT1 and autophagy in the protective effect of hydrogen sulphide against hepatic ischemia/reperfusion injury in mice, Autophagy, 8:6, 954-962, DOI: 10.4161/auto.19927 To link to this article: https://doi.org/10.4161/auto.19927 Do not distribute.The role of AKT1 and autophagy in the protective effect of hydrogen sulphide against hepatic ischemia/reperfusion injury in mice Keywords: hydrogen sulphide, liver, ischemia-reperfusion injury, autophagy, mouseAbbreviations: I/R, ischemia-reperfusion; H 2 S, hydrogen sulphide; A/R, anoxia/reoxygenation; NaHS, sodium hydrosulfide; GPT, glutamic-pyruvate transaminase/alanine aminotransferase; GOT1, glutamic-oxaloacetic transaminase 1, soluble/aspartate aminotransferase; TNF, tumor necrosis factor(-a); IL6, interleukin 6; PtdIns3K, phosphatidylinositol 3-kinase; LC3, microtubule-associated protein 1 light chain 3; 3MA, 3-methyladenine; TUNEL, TdT-mediated dUTP nick-end labeling;ELISA, enzyme-linked immunosorbent assayHydrogen sulphide (H 2 S) exerts a protective effect in hepatic ischemia-reperfusion (I/R) injury. However, the exact mechanism of H 2 S action remains largely unknown. This study was designed to investigate the role of the PtdIns3K-AKT1 pathways and autophagy in the protective effect of H 2 S against hepatic I/R injury. Primary cultured mouse hepatocytes and livers with or without NaHS (a donor of H 2 S) preconditioning were exposed to anoxia/reoxygenation (A/R) and I/R, respectively. In certain groups, they were also pretreated with LY294002 (AKT1-specific inhibitor), 3-methyladenine (3MA, autophagy inhibitor) or rapamycin (autophagy enhancer), alone or simultaneously. Cell viability, expression of P-AKT1, T-AKT1, LC3 and BECN1 were examined. The severity of liver injury was measured by the levels of serum aminotransferase and inflammatory cytokine, apoptosis and histological examination. GFP-LC3 redistribution and transmission electron microscopy were used to test the activity of autophagy. H 2 S preconditioning activated PtdIns3K-AKT1 signaling in hepatocytes. LY294002 could abolish the AKT1 activation and attenuate the protective effect of H 2 S on hepatocytes A/R and hepatic I/R injuries. H 2 S suppressed hepatic autophagy in vitro and in vivo. Further reducing autophagy by 3MA also diminished the protective effect of H 2 S, while rapamycin could reverse the autophagy inhibitory effect and enhance the protective effect of H 2 S against hepatocytes A/R and hepatic I/R injuries, consequently. Taken together, H 2 S protects against hepatocytic A/R and hepatic I/R injuries, at least in part, through AKT1 activation but not autophagy. An autophagy agonist could be applied to potentiate this hepatoprotective effect by reversing the autophagy inhibition of H 2 S.