An enantioselective synthetic route to cis-2,4-disubstituted and 2,4-bridged piperidines

A synthetic route to enantiopure cis-2,4-disubstituted and 2,4-bridged piperidines is reported, the key step being a stereoselective conjugate addition of an organocuprate to a phenylglycinol-derived unsaturated lactam bearing a substituent at the 8a-position.

Comparative genomics of Hedgehog loci in chordates and the origin of Shh regulatory novelties

The origin and evolution of the complex regulatory landscapes of some vertebrate developmental genes, often spanning hundreds of Kbp and including neighboring genes, remain poorly understood. The Sonic Hedgehog (Shh) genomic regulatory block (GRB) is one of the best functionally characterized examples, with several discrete enhancers reported within its introns, vast upstream gene-free region and neighboring genes (Lmbr1 and Rnf32). To investigate the origin and evolution of this GRB, we sequenced and characterized the Hedgehog (Hh) loci from three invertebrate chordate amphioxus species, which share several early expression domains with Shh. Using phylogenetic footprinting within and between chordate lineages, and reporter assays in zebrafish probing >30 Kbp of amphioxus Hh, we report large sequence and functional divergence between both groups. In addition, we show that the linkage of Shh to Lmbr1 and Rnf32, necessary for the unique gnatostomate-specific Shh limb expression, is a vertebrate novelty occurred between the two whole-genome duplications.

Divergent evolution and purifying selection of the flaA gene sequences in Aeromonas

BACKGROUND: The bacterial flagellum is the most important organelle of motility in bacteria and plays a key role in many bacterial lifestyles, including virulence. The flagellum also provides a paradigm of how hierarchical gene regulation, intricate protein-protein interactions and controlled protein secretion can result in the assembly of a complex multi-protein structure tightly orchestrated in time and space. As if to stress its importance, plants and animals produce receptors specifically dedicated to the recognition of flagella. Aside from motility, the flagellum also moonlights as an adhesion and has been adapted by humans as a tool for peptide display. Flagellar sequence variation constitutes a marker with widespread potential uses for studies of population genetics and phylogeny of bacterial species. RESULTS: We sequenced the complete flagellin gene (flaA) in 18 different species and subspecies of Aeromonas. Sequences ranged in size from 870 (A. allosaccharophila) to 921 nucleotides (A. popoffii). The multiple alignment displayed 924 sites, 66 of which presented alignment gaps. The phylogenetic tree revealed the existence of two groups of species exhibiting different FlaA flagellins (FlaA1 and FlaA2). Maximum likelihood models of codon substitution were used to analyze flaA sequences. Likelihood ratio tests suggested a low variation in selective pressure among lineages, with an omega ratio of less than 1 indicating the presence of purifying selection in almost all cases. Only one site under potential diversifying selection was identified (isoleucine in position 179). However, 17 amino acid positions were inferred as sites that are likely to be under positive selection using the branch-site model. Ancestral reconstruction revealed that these 17 amino acids were among the amino acid changes detected in the ancestral sequence. CONCLUSION: The models applied to our set of sequences allowed us to determine the possible evolutionary pathway followed by the flaA gene in Aeromonas, suggesting that this gene have probably been evolving independently in the two groups of Aeromonas species since the divergence of a distant common ancestor after one or several episodes of positive selection. REVIEWERS: This article was reviewed by Alexey Kondrashov, John Logsdon and Olivier Tenaillon (nominated by Laurence D Hurst).

Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice.

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19+CD1dhighCD5+ B cells (CD1dhighCD5+ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1dhighCD5+ Breg cells as compared to age-matched WT control mice. CD1dhighCD5+ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4+ T cells. Interestingly, CD1dhighCD5+ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops

Cytosine-and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34 -bases long cytosine-rich sequence and its complementary guanin e-rich strand corresponding to the first intron of the n-mycgene were studied. Both sequences , not yet studied, contain a 12 - base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively ...

The Political Communication of Independent Regulatory Agencies

Since independent regulatory agencies (IRAs) became key actors in European regulatory governance in the 1990s, a significant share of policy-making has been carried out by organizations that are neither democratically elected nor directly accountable to elected politicians. In this context, public communication plays an important role. On the one hand, regulatory agencies might try to use communication to raise their accountability and thereby to mitigate their democratic deficit. On the other hand, communication may be used with the intent to steer the behavior of the regulated industry when more coercive regulatory means are unfeasible or undesirable. However, empirical research focusing directly on how regulators communicate is virtually non-existent. To fill this gap, this paper examines the public communication of IRAs in four countries (the United Kingdom, Germany, Ireland, and Switzerland) and three sectors (financial services, telecommunications, and broadcasting). The empirical analysis, based on qualitative interviews and a quantitative content analysis, indicates that the organization of the communication function follows a national pattern approach while a policy sector approach is helpful for understanding the use of communication as a soft tool of regulation.

The role of MAR elements in DNA recombination

The untargeted integration of foreign DNA into the mammalian cell genome, extensively used in gene therapy and biotechnology, remains an incompletely understood process. It is believed to be based on cellular DNA double strand break (DSB) repair machinery and to involve two major steps: i) the formation of long gene arrays (concatemers), and ii) recombination of the resulting concatemer with the genome. The main DSB repair pathways in eukaryotes include non-homologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). However, it is still not clear, which of these pathways are responsible for transgene integration. Here, we show that NHEJ is not the primary pathway used by mammalian cells in the transgene integration process, while the components of the HR pathway seem to be important for genomic integration but not concatemerization. Instead, concatemer formation appears to be mediated by a subset of the MMEJ pathway, termed synthesis-dependent MMEJ (SD-MMEJ). This mechanism also seems to be preferentially used for plasmid integration into the genome, as confirmed by the analysis of plasmid-to-genome junction sequences, which were found to display an SD-MMEJ pattern. Therefore, we propose the existence of two distinct SD-MMEJ subpathways, relying on different subsets of enzymes. One of these mechanisms appears to be responsible for concatemerization, while the other mechanism, partially dependent in HR enzymes, seems to mediate recombination with the genome. Previous studies performed by our group suggested that matrix attachment regions (MARs), which are epigenetic regulatory DNA elements that participate in the formation of chromatin boundaries and augment transcription, may mediate increased plasmid integration into the genome of CHO cells by stimulating DNA recombination. In the present work, we demonstrate that MAR-mediated plasmid integration results from the enhanced SD-MMEJ pathway. Analysis of transgene integration loci and junction DNA sequences validated the prevalent use of this pathway by the MAR elements to target plasmid DNA into gene-rich areas of the CHO genome. We propose that this finding should in the future help to engineer cells for improved recombinant protein production. In addition to investigating the process of transgene integration, we designed recombination assays to better characterize the components of the MMEJ and SD-MMEJ pathways. We also used CHO cells expressing cycle-sensitive reporter genes to demonstrate a potential role of HR proteins in the cell cycle regulation.

An enantioselective entry to cis-perhydroisoquinolines

An enantioselective route to cis-perhydroisoquinolines, involving a cyclocondensation reaction of (R)-phenylglycinol with a racemic oxoester, a stereoselective conjugate addition to an unsaturated bicyclic lactam, and the closure of the carbocyclic ring by a ring-closing metathesis as the key steps is reported. This route allows the preparation of 3-cyano derivatives as well as cis-octahydroisoquinolines bearing a quaternary center at the C4-position.

Transnational private authority, regulatory space and workers' collective competences: Bringing local contexts and worker agency back in

The impact of transnational private regulation on labour standards remains in dispute. While studies have provided some limited evidence of positive effects on 'outcome standards' such as wages or occupational health and safety, the literature gives little reason to believe that there has been any significant effect on 'process rights' relating primarily to collective workers' voice and social dialogue. This paper probes this assumption by bringing local contexts and worker agency more fully into the picture. It outlines an analytical framework that emphasizes workers' potential to act collectively for change in the regulatory space surrounding the employment relationship. It argues that while transnational private regulation on labour standards may marginally improve workers access to regulatory spaces and their capacity to require the inclusion of enterprises in them, it does little to increase union leverage. The findings are based on empirical research work conducted in Sub-Saharan Africa.

When supply travels far beyond demand: Institutional and regulatory causes of oversupply in Spain’s transport infrastructure

Spain’s transport infrastructure policy has become a paradigmatic case of oversupply and of mismatch with demand. The massive expansion of the country’s transport infrastructure over the last decade has not been a response to demand bottlenecks or previously identified needs. For this reason, the intensity of use today on all interurban modes of transport in Spain falls well below that of other EU countries. This paper analyzes the institutional and regulatory factors that have permitted this policy, allowing us to draw lessons from the Spanish case that should help other countries avoid the pitfalls and shortcomings of Spanish policy. Based on our analysis, we also discuss policy remedies and suggest reforms in different regulatory areas, which could help improve the performance of Spain’s infrastructure policy.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes.

A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

A genotypic mutation system measuring mutations in restriction recognition sequences.

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of the human c-Ha-ras1 gene from a large excess of wild-type DNA. For this purpose we constructed a 272 bp fragment with 2 mutations in the PvuII recognition sequence 1727-1732 and studied the rescue by RFLP/PCR of a few copies of this 'PvuII mutant standard'. Following amplification with Taq-polymerase and cloning into lambda gt10, plaques containing wild-type sequence, PvuII mutant standard or Taq-polymerase induced bp changes were quantitated by hybridization with specific oligonucleotide probes. Our results indicate that 10 PvuII mutant standard copies can be rescued from 10(8) to 10(9) wild-type sequences. Taq polymerase errors originating from unrestricted, residual wild-type DNA were sequence dependent and consisted mostly of transversions originating at G.C bp. In contrast to a doubly mutated 'standard' the capacity to rescue single bp mutations by RFLP/PCR is limited by Taq-polymerase errors. Therefore, we assessed the capacity of our protocol to isolate a G to T transversion mutation at base pair 1698 of the MspI-site 1695-1698 of the c-Ha-ras1 gene from excess wild-type ras1 DNA. We found that 100 copies of the mutated ras1 fragment could be readily rescued from 10(8) copies of wild-type DNA.

3,4-Methylenedioxy-methamphetamine induces in vivo regional up-regulation of central nicotinic receptors in rats and potentiates the regulatory effects of nicotine on these receptors

Nicotine (NIC), the main psychostimulant compound of smoked tobacco, exerts its effects through activation of central nicotinic acetylcholine receptors (nAChR), which become up-regulated after chronic administration. Recent work has demonstrated that the recreational drug 3,4-methylenedioxymethamphetamine (MDMA) has affinity for nAChR and also induces up-regulation of nAChR in PC 12 cells. Tobacco and MDMA are often consumed together. In the present work we studied the in vivo effect of a classic chronic dosing schedule of MDMA in rats, alone or combined with a chronic schedule of NIC, on the density of nAChR and on serotonin reuptake transporters. MDMA induced significant decreases in [3H]paroxetine binding in the cortex and hippocampus measured 24 h after the last dose and these decreases were not modified by the association with NIC. In the prefrontal cortex, NIC and MDMA each induced significant increases in [3H]epibatidine binding (29.5 and 34.6%, respectively) with respect to saline-treated rats, and these increases were significantly potentiated (up to 72.1%) when the two drugs were associated. Also in this area, [3H]methyllycaconitine binding was increased a 42.1% with NIC + MDMA but not when they were given alone. In the hippocampus, MDMA potentiated the a7 regulatory effects of NIC (raising a 25.5% increase to 52.5%) but alone was devoid of effect. MDMA had no effect on heteromeric nAChR in striatum and a coronal section of the midbrain containing superior colliculi, geniculate nuclei, substantia nigra and ventral tegmental area. Specific immunoprecipitation of solubilised receptors suggests that the up-regulated heteromeric nAChRs contain a4 and b2 subunits. Western blots with specific a4 and a7 antibodies showed no significant differences between the groups, indicating that, as reported for nicotine, up-regulation caused by MDMA is due to post-translational events rather than increased receptor synthesis.

The Evolution of the European Regulatory Framework for Electronic Communications

The paper reviews the historical transformation of the European regulatory framework for electronic communications from the era dominated by state-owned enterprises to the presence of regulated competition. In the course of these developments, the vision of the roles of the public and private sectors in electronic communications changed in expected and unexpected ways. While the period is characterized by a shift toward less direct state intervention, the intensity of regulation has increased in many areas. Most recently, in the wake of the financial crisis, new forms of state intervention can be observed, including public investment in communications infrastructure and public-private partnerships. As a result of the reforms, Europe has been able to achieve major successes but it also suffered unanticipated setbacks compared to other regions. The European Union emerged as the global leader in mobile communications during the 1990s and was able to roll-out first-generation broadband access networks more rapidly than many of its peers. Recently, however, Europe as a whole has not performed as well in deploying next-generation networks and advanced mobile communications services. The paper offers a political-economic explanation for these developments and assesses their effects on the performance of the European electronic communications sector and the economy. From this analysis, the European model emerges as a unique institutional arrangement with peculiar advantages and disadvantages. Once these are recognized, sensible next steps to build the strengths while avoiding the weaknesses of the model can be seen more clearly.