Chemical synthesis, DNA incorporation and biological study of a new photocleavable 2′-deoxyadenosine mimic

The phototriggered cleavage of chemical bonds has found numerous applications in biology, particularly in the field of gene sequencing through photoinduced DNA strand scission. However, only a small number of modified nucleosides that are able to cleave DNA at selected positions have been reported in the literature. Herein, we show that a new photoactivable deoxyadenosine analogue, 3-nitro-3-deaza-2′-deoxyadenosine (d(3-NiA)), was able to induce DNA backbone breakage upon irradiation (λ > 320 nm). The d(3-NiA) nucleoside was chemically incorporated at desired positions into 40-mer oligonucleotides as a phosphoramidite monomer and subsequent hybridization studies confirmed that the resulting modified duplexes display a behaviour that is close to that of the related natural sequence. Enzymatic action of the Klenow fragment exonuclease free revealed the preferential incorporation of dAMP opposite the 3-NiA base. On the other hand, incorporation of the analogous 3-NiA triphosphate to a primer revealed high enzyme efficiency and selectivity for insertion opposite thymine. Furthermore, only the enzymatically synthesized base pair 3-NiA:T was a substrate for further extension by the enzyme. All the hybridization and enzymatic data indicate that this new photoactivable 3-NiA triphosphate can be considered as a photochemically cleavable dATP analogue

Linear and Branched Glyco-Lipopeptide Vaccines Follow Distinct Cross-Presentation Pathways and Generate Different Magnitudes of Antitumor Immunity

Glyco-lipopeptides, a form of lipid-tailed glyco-peptide, are currently under intense investigation as B- and T-cell based vaccine immunotherapy for many cancers. However, the cellular and molecular mechanisms of glyco-lipopeptides (GLPs) immunogenicity and the position of the lipid moiety on immunogenicity and protective efficacy of GLPs remain to be determined.We have constructed two structural analogues of HER-2 glyco-lipopeptide (HER-GLP) by synthesizing a chimeric peptide made of one universal CD4(+) epitope (PADRE) and one HER-2 CD8(+) T-cell epitope (HER(420-429)). The C-terminal end of the resulting CD4-CD8 chimeric peptide was coupled to a tumor carbohydrate B-cell epitope, based on a regioselectively addressable functionalized templates (RAFT), made of four alpha-GalNAc molecules. The resulting HER glyco-peptide (HER-GP) was then linked to a palmitic acid moiety, attached either at the N-terminal end (linear HER-GLP-1) or in the middle between the CD4+ and CD8+ T cell epitopes (branched HER-GLP-2). We have investigated the uptake, processing and cross-presentation pathways of the two HER-GLP vaccine constructs, and assessed whether the position of linkage of the lipid moiety would affect the B- and T-cell immunogenicity and protective efficacy. Immunization of mice revealed that the linear HER-GLP-1 induced a stronger and longer lasting HER(420-429)-specific IFN-gamma producing CD8(+) T cell response, while the branched HER-GLP-2 induced a stronger tumor-specific IgG response. The linear HER-GLP-1 was taken up easily by dendritic cells (DCs), induced stronger DCs maturation and produced a potent TLR- 2-dependent T-cell activation. The linear and branched HER-GLP molecules appeared to follow two different cross-presentation pathways. While regression of established tumors was induced by both linear HER-GLP-1 and branched HER-GLP-2, the inhibition of tumor growth was significantly higher in HER-GLP-1 immunized mice (p<0.005).These findings have important implications for the development of effective GLP based immunotherapeutic strategies against cancers

Influence of cis-trans isomerisation on pentapeptide cyclisation

Cyclisation reactions on pentapeptides containing the turn promoting residues Gly and Pro are investigated. Although the cyclisation reaction is fast, solvent-dependent mixtures of cyclic penta- and decapeptides are observed. NMR studies suggest a strong dependence of the monomer/dimer ratio on the cis-trans isomerisation found in linear and cyclic peptides. The critical influence on ring closure of a cis Xaa-Pro peptide bond conformation is demonstrated by using the cis peptide bond-inducing residue 2,2-dimethyl-1,3-thiazolidine-4 carboxylic acid (Dmt, pseudo-proline) as proline substitute. The results shed new light on the role of cis-trans isomerisation in pentapeptide cyclisation. (C) 1999 Elsevier Science Ltd. All rights reserved

Minimal Access Spinal Technologies (Mast) Fusion Procedures For The Treatment Of The Degenerative Lumbar Spine (A Part Of Multicentral Prospective Study)

A prospective multicentral observational study of minimally invasive fusion to treat degenerative lumbar disorders, and to report outcomes of one or two level minimally invasive posterior lumbar interbody fusion (MLIF) for degenerative lumbar disorders in a multi-center 1-year prospective study. We prospectively studied a group of 32 patients, mostly female 24 ( 75% female ), and 8 males ( 25%). They underwent minimally invasive transforaminal lumbar interbody fusion (mTLIF), 21 of them monosegmental and 11 bisegmental. Patients demographics, intraoperative data and complications were recorded. Time to first ambulation, time to study-defined recovery, surgical duration, blood loss, fluoroscopy time and adverse events were recorded. Visual analogue scale (VAS) of back and legs pain, Oswestry disability index (ODI) and health-related questionnaire (EQ-5D) were assessed preoperatively and at defined time points through 12 months postoperatively. Mean surgical duration, blood loss and intraoperative fluoroscopy time were 125 vs.175 minutes, 150 vs. 170 ml, and 105 vs. 145 seconds in one- and twolevel segments, respectively. Mean preoperative VAS back (6.5) and VAS leg (7.9) scores dropped significantly (p<0.0001) to 3.5 (2.6) and 2.1 (2.0) at discharge (6 weeks). At the end, this is the largest prospective multi-center observational study of MLIF to date, following routine local standard of practice and, MLIF demonstrated favourable clinical results with early and sustained improvement in patient reported outcomes and low major perioperative morbidity