A functional link between housekeeping selenoproteins and phase II enzymes

Sec (selenocysteine) is biosynthesized on its tRNA and incorporated into selenium-containing proteins (selenoproteins) as the 21st amino acid residue. Selenoprotein synthesis is dependent on Sec tRNA and the expression of this class of proteins can be modulated by altering Sec tRNA expression. The gene encoding Sec tRNA (Trsp) is a single-copy gene and its targeted removal in liver demonstrated that selenoproteins are essential for proper function wherein their absence leads to necrosis and hepatocellular degeneration. In the present study, we found that the complete loss of selenoproteins in liver was compensated for by an enhanced expression of several phase II response genes and their corresponding gene products. The replacement of selenoprotein synthesis in mice carrying mutant Trsp transgenes, wherein housekeeping, but not stress-related selenoproteins are expressed, led to normal expression of phase II response genes. Thus the present study provides evidence for a functional link between housekeeping selenoproteins and phase II enzymes.     

Conformational States of ABC Transporter MsbA in a Lipid Environment Investigated by Small-Angle Scattering Using Stealth Carrier Nanodiscs

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Prp19/Pso4 Is an Autoinhibited Ubiquitin Ligase Activated by Stepwise Assembly of Three Splicing Factors

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Positioning of CCA-arms of the A- and the P-tRNAs towards the 28S rRNA in the human ribosome

Nucleotides of 28S rRNA involved in binding of the human 80S ribosome with acceptor ends of the A site and the P site tRNAs were determined using two complementary approaches, namely, cross-linking with application of tRNA^Asp analogues substituted with 4-thiouridine in position 75 or 76 and hydroxyl radical footprinting with the use of the full sized tRNA and the tRNA deprived of the 3′-terminal trinucleotide CCA. In general, these 28S rRNA nucleotides are located in ribosomal regions homologous to the A, P and E sites of the prokaryotic 50S subunit. However, none of the approaches used discovered interactions of the apex of the large rRNA helix 80 with the acceptor end of the P site tRNA typical with prokaryotic ribosomes. Application of the results obtained to available atomic models of 50S and 60S subunits led us to a conclusion that the A site tRNA is actually present in both A/A and A/P states and the P site tRNA in the P/P and P/E states. Thus, the present study gives a biochemical confirmation of the data on the structure and dynamics of the mammalian ribosomal pretranslocation complex obtained with application of cryo-electron microscopy and single-molecule FRET [Budkevich et al., 2011]. Moreover, in our study, particular sets of 28S rRNA nucleotides involved in oscillations of tRNAs CCA-termini between their alternative locations in the mammalian 80S ribosome are revealed.     

Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da. Using genome-wide linkage analyses, we discovered an association between nerve-injury-induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain.     

Social Media and Internet Driven Study Recruitment: Evaluating a New Model for Promoting Collaborator Engagement and Participation

Aims

A substantial challenge facing multicentre audit and research projects is timely recruitment of collaborators and their study centres. Cost-effective strategies are required and fee-free social media has previously been identified as a potential conduit. We investigated and evaluated the effectiveness of a novel multi-format social media and Internet strategy for targeted recruitment to a national multicentre cohort study.

Methods

Interventions involved a new Twitter account, including weekly live question-and-answer sessions, a new Facebook group page, online YouTube presentations and an information page on a national association website. Link tracking analysis was undertaken using Google Analytics, which was then related to subsequent registration. Social influence was calculated using the proprietary Klout score.

Results

Internet traffic analysis identified a total of 1562 unique registration site views, of which 285 originated from social media (18.2%). Some 528 unique registrations were received, with 96 via social media platforms (18.2%). Traffic source analysis identified a separate national association webpage as resulting in the majority of registration page views (15.8%), followed by Facebook (11.9%), Twitter (4.8%) and YouTube (1.5%). A combination of publicity through Facebook, Twitter and the dedicated national association webpage contributed to the greatest rise in registration traffic and accounted for 312 (48%) of the total registrations within a 2-week period. A Twitter ‘social influence’ (Klout) score of 42/100 was obtained during this period.

Conclusions

Targeted social media substantially aided study dissemination and collaborator recruitment. It acted as an adjunct to traditional methods, accounting for 18.2% of collaborator registration in a short time period with no associated financial costs. We provide a practical model for designing future recruitment campaigns, and recommend Facebook, Twitter and targeted websites as the most effective adjuncts for maximising cost-effective study recruitment.     

A Humanized Leucine Zipper-TRAIL Hybrid Induces Apoptosis of Tumors both In Vitro and In Vivo

Evidence suggests that stimulating apoptosis in malignant cells without inflicting collateral damage to the host''s normal tissues is a promising cancer therapy. Chemo- and radiation therapies that, especially if combined, induce apoptosis in tumor cells have been used for treating cancer patients for decades. These treatments, however, are limited in their ability to discriminate between malignant and non-malignant cells and, therefore, produce substantial healthy tissue damage and subsequent toxic side-effects. In addition, as a result of these therapies, many tumor types acquire an apoptosis-resistant phenotype and become more aggressive and metastatic. Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been considered a promising and reliable selective inducer of apoptosis in cancerous cells. TRAIL, however, is not uniformly effective in cancer and multiple cancer cell types are considered resistant to natural TRAIL. To overcome this deficiency of TRAIL, we have earlier constructed a yeast-human hybrid leucine zipper-TRAIL in which the yeast GCN4-pII leucine zipper was fused to human TRAIL (GCN4-TRAIL). This construct exhibited a significantly improved anti-tumor apoptotic activity and safety, but is potentially immunogenic in humans. Here, we report a novel, potent, and fully human ATF7 leucine zipper-TRAIL (ATF7-TRAIL) fusion construct that is expected to have substantially lower immunogenicity. In solution, ATF7-TRAIL exists solely as a trimer with a Tm of 80°C and is active against cancer cells both in vitro and in vivo, in a mouse tumor xenograft model. Our data suggest that our re-engineered TRAIL is a promising candidate for further evaluation as an antitumor agent.     

Asymmetric synthesis of enantiomerically and diastereoisomerically enriched 4-[F or Br]-substituted glutamic acids

A novel simple synthetic protocol for the preparation of both (2S,4R)- and (2S,4S)-FGlu, applying Michael addition of methyl α-fluoroacrylate to a Ni^II complex of glycine Schiff base with BPB, was elaborated. In addition, same reaction of mentioned complex with ethyl α-bromoacrylate leads to the Ni^II complex of the Schiff base of BPB with (2S,4R)-4-bromo-glutamic acid monoester, that can be transformed into the corresponding complexes of 1-aminocyclopropane-1,2-dicarboxylic acid. The decomposition of the diastereoisomerically pure complexes leads to corresponding enantiomerically enriched (ee > 98%) amino acids.     

Evidence That Mutation Is Universally Biased towards AT in Bacteria

Mutation is the engine that drives evolution and adaptation forward in that it generates the variation on which natural selection acts. Mutation is a random process that nevertheless occurs according to certain biases. Elucidating mutational biases and the way they vary across species and within genomes is crucial to understanding evolution and adaptation. Here we demonstrate that clonal pathogens that evolve under severely relaxed selection are uniquely suitable for studying mutational biases in bacteria. We estimate mutational patterns using sequence datasets from five such clonal pathogens belonging to four diverse bacterial clades that span most of the range of genomic nucleotide content. We demonstrate that across different types of sites and in all four clades mutation is consistently biased towards AT. This is true even in clades that have high genomic GC content. In all studied cases the mutational bias towards AT is primarily due to the high rate of C/G to T/A transitions. These results suggest that bacterial mutational biases are far less variable than previously thought. They further demonstrate that variation in nucleotide content cannot stem entirely from variation in mutational biases and that natural selection and/or a natural selection-like process such as biased gene conversion strongly affect nucleotide content.     

Cytochrome P450 from Photobacterium profundum SS9, a piezophilic bacterium, exhibits a tightened control of water access to the active site

We report cloning, expression in Escherichia coli, and purification of cytochrome P450 from a deep-sea bacterium Photobacterium profundum strain SS9 (P450-SS9). The enzyme, which is predominately high spin (86%) in the absence of any added ligand, binds fatty acids and their derivatives and exhibits the highest affinity for myristic acid. Binding of the majority of saturated fatty acids displaces the spin equilibrium further toward the high-spin state, whereas the interactions with unsaturated fatty acids and their derivatives (arachidonoylglycine) have the opposite effect. Pressure perturbation studies showed that increasing pressure fails to displace the spin equilibrium completely to the low-spin state in the ligand-free P450-SS9 or in the complexes with either myristic acid or arachidonoylglycine. Stabilization of high-spin P450-SS9 signifies a pressure-induced transition to a state with reduced accessibility of the active site. This transition, which is apparently associated with substantial hydration of the protein, is characterized by the reaction volume change (ΔV) around -100 to -200 mL/mol and P(1/2) of 300-800 bar, which is close to the pressure of habitation of P. profundum. The transition to a state with confined water accessibility is hypothesized to represent a common feature of cytochromes P450 that serves to coordinate heme pocket hydration with ligand binding and the redox state. Displacement of the conformational equilibrium toward the "closed" state in P450-SS9 (even ligand-free) may have evolved to allow the protein to adapt to enhanced protein hydration at high hydrostatic pressures.