Mammalian Target of Rapamycin Inhibition in Patients With ST-Segment Elevation Myocardial Infarction.

BACKGROUND: Early inflammation following acute ST-segment elevation myocardial infarction (STEMI) treated by primary percutaneous coronary intervention (PCI) affects myocardial infarct (MI) size and left ventricular remodeling. The mammalian target of rapamycin (mTOR) is involved in the enhanced inflammatory response and its inhibition has exerted beneficial effects on MI size in preclinical models of acute MI. OBJECTIVES: The CLEVER-ACS (Controlled Level Everolimus in Acute Coronary Syndromes) trial evaluated the effects of targeting inflammation by mTOR inhibition in patients with STEMI undergoing PCI. METHODS: CLEVER-ACS was a randomized, multicenter, international, double-blind, placebo-controlled trial. A total of 150 patients with STEMI undergoing PCI were randomly assigned to oral everolimus (days 1-3: 7.5 mg daily; days 4-5: 5.0 mg daily) or placebo for 5 days. The primary endpoint was the change in MI size. The secondary endpoint was the change in microvascular obstruction (MVO) from baseline (12 hours to 5 days after PCI) to 30 days as assessed by cardiac magnetic resonance imaging. RESULTS: The changes in MI size from baseline to 30 days, the primary endpoint, were -14.2 g (95% CI: -17.4 to -11.1 g) and -12.3 g (95% CI: -16.0 to -8.7 g) in the everolimus and placebo groups (P = 0.99). Corresponding changes in MVO were -4.8 g (95% CI: -6.7 to -2.9 g) and -6.3 g (95% CI: -8.7 to -4.0 g) in the everolimus and placebo groups (P = 0.14). Adverse events did not differ between the study groups. CONCLUSIONS: Among STEMI patients undergoing PCI, early mTOR inhibition with everolimus did not reduce MI size or MVO at 30 days. (CLEVER-ACS [Controlled Level Everolimus in Acute Coronary Syndromes; NCT01529554).

Adduct Fluorescence as a Tool to Decipher Sequence Impact on Frameshift Mutations Mediated by a C-Linked C8-Biphenyl-Guanine Lesion.

Aromatic chemicals can undergo metabolic activation to afford electrophilic species that react at the C8-site of 2'-deoxyguanosine (dG) to generate bulky C8-dG adducts as a basis of initiating carcinogenesis. These DNA lesions have served as models to understand the mechanism of frameshift mutagenesis, especially within CG-dinucleotide repeat sequences, such as NarI (5'-GGCXCC-3', where X = C8-dG adduct), however there is still limited capacity to predict the likelihood of mutation arising within particular contexts, and hence chemistry-based strategies are needed for probing relationships between nucleic acid sequence and structure with replication errors. In the NarI sequence, certain C8-dG adducts may trigger in the course of DNA synthesis the formation of a slipped mutagenic intermediate (SMI) that contains a two nucleotide (XC) bulge in the template strand that can form upstream of the polymerase active site. This distortion facilitates polymerization but affords a GC dinucleotide deletion product (-2 frameshift mutation). In the current study, incorporating the fluorescent C-linked 4-fluorobiphenyl-dG (FBP-dG) adduct into two 22-mer templates containing CG-dinucleotide repeats ( NarI: 3'-CXCGGC-5' and CG3: 3'-CXCGCG-5', X = FBP-dG) and performing primer extension reactions using DNA polymerase I, Klenow fragment exo- (Kf-) revealed a dramatic sequence-based difference in polymerase bypass efficiency. Primer extension past FBP-dG within the NarI sequence was strongly blocked, whereas Kf- extended the primer past FBP-dG within a CG3 template to afford a full-length product and the GC dinucleotide deletion. To model the nucleotide insertion steps in the fully paired (FP) versus the slipped mutagenic (SM) translesion pathways, adducted template:primer duplexes were constructed and characterized by UV thermal denaturation and fluorescence spectroscopy. The emission intensity of the FBP-dG lesion exhibits sensitivity to SMI formation (turn-on) versus a FP duplex (turn-off), permitting insight into adduct base-pairing within the template:primer duplexes. This fluorescence sensitivity provides a rationale for sequence impact on -2 frameshift mutations mediated by the C-linked FBP-dG lesion.

Conformational Preference and Fluorescence Response of a C-Linked C8-Biphenyl-Guanine Lesion in the NarI Mutational Hotspot: Evidence for Enhanced Syn Adduct Formation.

Aromatic chemical carcinogens can undergo enzymatic transformations to produce a range of electrophilic species that attach covalently to the C8-site of 2'-deoxyguanosine (dG) to afford C8-dG adducts. The most studied C8-dG adducts are formed from arylamines and contain a N-linkage separating the dG from the C8-aryl moiety. Other carcinogenic species result in direct aryl ring attachment to the dG moiety, resulting in C-linked adducts. The resulting C-linked adducts have reduced conformational flexibility compared to the corresponding N-linked C8-dG adducts, which can alter their orientation in the DNA duplex. Described herein are structural studies of a fluorescent C-linked 4-fluorobiphenyl-dG (FBP-dG) that has been incorporated into the reiterated G3-postion of the 12-mer NarI sequence and those containing other 5'-flanking nucleobases. FBP-dG displays a strong preference for adopting a syn conformation in the fully paired NarI duplex to produce an intercalated structure that exhibits stacking interactions between the C-linked biphenyl and the flanking bases. FBP-dG is also shown to significantly stabilize the slippage mutagenic intermediate (SMI) duplex containing the lesion and 5'-flanking base within a 2-base bulge. FBP-dG exhibits fluorescence sensitivity to SMI duplex formation that can readily distinguish it from the fully paired duplex. Molecular dynamics simulations and optical spectroscopy for the NarI oligonucleotides containing the C-linked FBP-dG predict increased rigidity of the biphenyl in the syn conformation. The greater propensity to generate the promutagenic syn conformation for the C-linked FBP-dG adduct compared to the N-linked 4-aminobiphenyl-dG adduct (ABP-dG) suggests greater mutagenicity for the C-linked analogue. These results highlight the effect of the adduct linkage type on the conformational properties of adducted DNA. The turn-on emission response of FBP-dG in the SMI duplex may be a powerful tool for monitoring SMI formation in the NarI sequence upon synthesis with DNA polymerases.

A Simple Molecular Rotor for Defining Nucleoside Environment within a DNA Aptamer-Protein Complex.

The simple 5-furyl-2'-deoxyuridine ((Fur)dU) nucleobase exhibits dual probing characteristics displaying emissive sensitivity to changes in microenvironment polarity and to changes in solvent rigidity due to its molecular rotor character. Here, we demonstrate its ability to define the microenvironment of the various thymidine (T) loop residues within the thrombin binding aptamer (TBA) upon antiparallel G-quadruplex (GQ) folding and thrombin binding. The emissive sensitivity of the (Fur)dU probe to microenvironment polarity provides a diagnostic handle to distinguish T bases that are solvent-exposed within the GQ structure compared with probe location in the apolar duplex. Its molecular rotor properties then provide a turn-on fluorescent switch to identify which T residues within the GQ bind specifically to the protein target (thrombin). The fluorescence sensing characteristics of (Fur)dU make it an attractive tool for mapping aptamer-protein interactions at the nucleoside level for further development of modified aptamers for a wide range of diagnostic and therapeutic applications.

[The challenges of the implementation of the European Directive 2001/20/CE in the main Member States]. / Les enjeux de la transposition de la Directive européenne 2001/20/CE dans les principaux Etats Membres.

Since the publication of Directive 2001/20/EC in the Official Journal of the European Communities on May 1, 2001, the Member States have been preparing its transposition into national legislation for application no later than May 2004. The discussions have proved to relate essentially to the organisation of the Ethics Committees, which must now give a single opinion per Member State, as well as to the time limits for obtaining this opinion and the authorization for a study from the competent authority. These considerations have led to proposals of shorter time limits for products at an early stage of development or involving certain diseases. Despite harmonization of the legislation, the application of the Directive does not eliminate all national differences.