Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB

The drug rapamycin has important uses in oncology, cardiology, and transplantation medicine, but its clinically relevant molecular effects are not understood. When bound to FKBP12, rapamycin interacts with and inhibits the kinase activity of a multiprotein complex composed of mTOR, mLST8, and raptor (mTORC1). The distinct complex of mTOR, mLST8, and rictor (mTORC2) does not interact with FKBP12-rapamycin and is not thought to be rapamycin sensitive. mTORC2 phosphorylates and activates Akt/PKB, a key regulator of cell survival. Here we show that rapamycin inhibits the assembly of mTORC2 and that, in many cell types, prolonged rapamycin treatment reduces the levels of mTORC2 below those needed to maintain Akt/PKB signaling. The proapoptotic and antitumor effects of rapamycin are suppressed in cells expressing an Akt/PKB mutant that is rapamycin resistant. Our work describes an unforeseen mechanism of action for rapamycin that suggests it can be used to inhibit Akt/PKB in certain cell types.     

Regeneration of soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merrill) through direct somatic embryogenesis from the immature embryonic shoot tip

We describe here a simple and efficient system of soybean (Glycine max L. Merrill) regeneration through direct somatic embryogenesis by using immature embryonic shoot tips (IEST) as explants. The cultivar Kaohsiung 10 (cv. K10) used in this study did not show embryogenic response either from mature seed-derived explants (cotyledon, embryonic tip, leaf, shoot and root) or immature cotyledons. However, it showed a high percentage (55.8%) of somatic embryo (SEm) formation from the IEST excised 2–3 wk after flowering, thus indicating the crucial roles of type and age of explants. The IEST put forth primary SEm after 2 mo of culturing on Murashige and Skoog (MS) medium supplemented with 6% sucrose, 164.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mM asparagine and 684 μM glutamine. Subsequently, secondary SEm were developed 1 mo after culturing on MS medium containing 123.6 μM 2,4-D and 3% sucrose. Cotyledonary embryos were induced on MS medium supplemented with 0.5% activated charcoal after 1 mo. The embryos were desiccated for 72–96 h on sterile Petri dishes and regenerated on hormone-free MS medium. Plantlets with well-developed shoots and roots were obtained within 5–6 mo of culturing of IEST. The SEm-derived plants were morphologically normal and fertile. Various parameters thought to be responsible for efficient regeneration of soybean through somatic embryogenesis are discussed. To our knowledge, this is the first report to employ IEST as explants for successful direct somatic embryogenesis in soybean.     

Caffeine and a selective adenosine A<Subscript>2A</Subscript> receptor antagonist induce sensitization and cross-sensitization behavior associated with increased striatal dopamine in mice

Background  

Caffeine, a nonselective adenosine A₁ and A_2A receptor antagonist, is the most widely used psychoactive substance in the world. Evidence demonstrates that caffeine and selective adenosine A_2A antagonists interact with the neuronal systems involved in drug reinforcement, locomotor sensitization, and therapeutic effect in Parkinson's disease (PD). Evidence also indicates that low doses of caffeine and a selective adenosine A_2A antagonist SCH58261 elicit locomotor stimulation whereas high doses of these drugs exert locomotor inhibition. Since these behavioral and therapeutic effects are mediated by the mesolimbic and nigrostriatal dopaminergic pathways which project to the striatum, we hypothesize that low doses of caffeine and SCH58261 may modulate the functions of dopaminergic neurons in the striatum.     

Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70

Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.     

Reticulon 3 interacts with NS4B of the hepatitis C virus and negatively regulates viral replication by disrupting NS4B self‐interaction

The non‐structural protein 4B (NS4B) of the hepatitis C virus (HCV) is an endoplasmic reticulum (ER) membrane protein comprising two consecutive amphipathic α‐helical domains (AH1 and AH2). Its self‐oligomerization via the AH2 domain is required for the formation of the membranous web that is necessary for viral replication. Previously, we reported that the host‐encoded ER‐associated reticulon 3 (RTN3) protein is involved in the formation of the replication‐associated membranes of (+)RNA enteroviruses during viral replication. In this study, we demonstrated that the second transmembrane region of RTN3 competed for, and bound to, the AH2 domain of NS4B, thus abolishing NS4B self‐interaction and leading to the downregulation of viral replication. This interaction was mediated by two crucial residues, lysine 52 and tyrosine 63, of AH2, and was regulated by the AH1 domain. The silencing of RTN3 in Huh7 and AVA5 cells harbouring an HCV replicon enhanced the replication of HCV, which was counteracted by the overexpression of recombinant RTN3. The synthesis of viral RNA was also increased in siRNA‐transfected human primary hepatocytes infected with HCV derived from cell culture. Our results demonstrated that RTN3 acted as a restriction factor to limit the replication of HCV.     

Integrative Functional Genomics of Hepatitis C Virus Infection Identifies Host Dependencies in Complete Viral Replication Cycle

Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets.     

Design and efficient synthesis of novel arylthiourea derivatives as potent hepatitis C virus inhibitors

A novel class of arylthiourea HCV inhibitors bearing various functionalities, such as cyclic urea, cyclic thiourea, urea, and thiourea, on the alkyl linker were designed and synthesized. Herein we report the synthesis and structure–activity relationships (SARs) of this novel class of arylthiourea derivatives that showed potent inhibitory activities against HCV in the cell-based subgenomic HCV replicon assay. Among compounds tested, the new carbazole derivative 64, which has an eight-carbon linkage between the phenyl and carbazole rings and a tolyl group at the N-9 position of carbazole, was found to possess strong anti-HCV activity (EC₅₀ = 0.031 μM), lower cytotoxicity (CC₅₀ >50 μM), and higher selectivity index (SI >1612) compared to its derivatives.     

Complementation between mutants of Phycomyces deficient with respect to carotenogenesis

Summary White and red mutants of Phycomyces, derived from two independent wild types (yellow) by mutagenesis using nitrosoguanidine, either in a single step (26 white, 5 red mutants), or in two steps (10 white mutants, from one of the red mutants) were studied with respect to complementation in heterokaryons. The tests clearly establish the involvement of three and only three genes, here named carA, carB, and carB. The carA and the carR mutants are white, the carA mutants do not accumulate phytoene, the carB mutants do. The carR mutants are red and accumulate lycopene. The two step mutants are either carA and carR, or carB and carR double mutants. A few of the white mutants obtained in a single mutagenization step are affected in carA and carR. They may be polar mutants in an operon or accidental double mutants.     

Associating somatic mutation with clinical outcomes through kernel regression and optimal transport

Somatic mutations in cancer patients are inherently sparse and potentially high dimensional. Cancer patients may share the same set of deregulated biological processes perturbed by different sets of somatically mutated genes. Therefore, when assessing the associations between somatic mutations and clinical outcomes, gene-by-gene analysis is often under-powered because it does not capture the complex disease mechanisms shared across cancer patients. Rather than testing genes one by one, an intuitive approach is to aggregate somatic mutation data of multiple genes to assess their joint association with clinical outcomes. The challenge is how to aggregate such information. Building on the optimal transport method, we propose a principled approach to estimate the similarity of somatic mutation profiles of multiple genes between tumor samples, while accounting for gene–gene similarities defined by gene annotations or empirical mutational patterns. Using such similarities, we can assess the associations between somatic mutations and clinical outcomes by kernel regression. We have applied our method to analyze somatic mutation data of 17 cancer types and identified at least five cancer types, where somatic mutations are associated with overall survival, progression-free interval, or cytolytic activity.     

Cardiolipin interacts with beta‐2‐glycoprotein I and forms an open conformation–Mechanisms analyzed using hydrogen/deuterium exchange

Beta‐2‐glycoprotein I (β₂GPI) is the major antigen for the antiphospholipid antibodies in the antiphospholipid syndrome. The exposed epitope in domain I of β₂GPI can be recognized by the anti‐β₂GPI antibody. Here, we prepared the anionic di‐oleoyl‐phosphatidylserine (DOPS) and cardiolipin (CL) liposomes to interact with the β₂GPI. The conformational changes of β₂GPI upon binding with the liposomes were analyzed using hydrogen/deuterium exchange mass spectrometry. The exchange level of sequences 21–27 significantly increased after β₂GPI had interacted with DOPS. This change indicated a reduced interaction between domain I and domain V, inferring to a protrusion of the sequences 21–27 from the ring conformation. After β₂GPI had interacted with CL for 30 min, the exchange levels in 4 of the 5 domains increased significantly. The deuteration levels of sequences 1–20, 21–27, 196–205, 273–279 and 297–306 increased, suggesting that these regions had become more exposed, and the domain I was no longer in contact with domain V. The increasing deuteration levels in sequences 70–86, 153–162, 191–198, 196–205 and 273–279 indicated β₂GPI undergoing conformational changes to expose these inner regions, suggesting a structural transition. Overall, DOPS and CL induced minor conformational changes of β₂GPI at sequences 21–27 and forms an intermediate conformation after 10 min of interaction. After a complete protein–lipid interaction, high negatively charged CL membrane induced a major conformation transition of β₂GPI.