Protein expression dynamics during replicative senescence of endothelial cells studied by 2-D difference in-gel electrophoresis

Endothelial senescence contributes to endothelium dysfunctionality and is thereby linked to vascular aging. A dynamic proteomic study on human umbilical vein endothelial cells, isolated from three umbilical cords, was performed. The cells were cultured towards replicative senescence and whole cell lysates were subjected to 2-D difference gel electrophoresis (DIGE). Despite the biological variability of the three independent isolations, a set of proteins was found that showed senescence-dependent expression patterns in all isolations. We focused on those proteins that showed significant changes, with a paired analysis of variance (RM-ANOVA) p-value of < or =0.05. Thirty-five proteins were identified with LC-Fourier transform MS, and functional annotation revealed that endothelial replicative senescence is accompanied by increased cellular stress, protein biosynthesis and reduction in DNA repair and maintenance. Nuclear integrity becomes affected and cytoskeletal structure is also changed. Such important changes in the cell infrastructure might accelerate endothelium dysfunctionality. This study provides biological information that will initiate studies to further unravel endothelial senescence and gain more knowledge about the consequences of this process in the in vivo situation.     

Ultralow hysteresis superhydrophobic surfaces by excimer laser modification of SU-8

We present a new and simple method to produce superhydrophobic surfaces with ultralow hysteresis. The method involves surface modification of SU-8 using an excimer laser treatment. The modified surface is coated with a hydrophobic plasma-polymerized hexafluoropropene layer. The advancing and receding water contact angles were measured to be approximately 165 degrees . The achieved water contact angle hysteresis was below the measurement limit. This low hysteresis can be ascribed to nanoscale debris generated during the excimer laser process.     

Aqueous Copper‐Mediated Living Radical Polymerisation of N‐Acryloylmorpholine,SET‐LRP in Water

The polymerisation of N‐acryloylmorpholine in water is reported utilising Cu(0)‐mediated living radical polymerisation (SET‐LRP). The inherent instability of [Cu^I(Me₆‐Tren)Br] in aqueous solution is exploited via rapid disproportionation to prepare Cu(0) particles and [Cu^II(Me₆‐Tren)Br₂] in situ prior to addition of monomer and initiator. Quantitative conversion is attained within 30 min for various degrees of polymerisation (DP_n = 20–640) with SEC showing symmetrical narrow molecular weight distributions (Đ < 1.18) in all cases. Optimised conditions are subsequently applied for the preparation of a diblock copolymer poly(NIPAm)‐b‐(N‐acryloylmorpholine), illustrating the versatility of this approach.

     

Synthesis of thiol-modified peptide nucleic acids designed for post-assembly conjugation reactions

Two orthogonally protected PNA monomers were prepared having the mercaptomethyl moiety attached to the PNA backbone. These building blocks were employed in solid-phase PNA synthesis and it was shown that Boc/S-p-methoxybenzyl protection scheme was only satisfactory for the introduction of N-terminal thiol modification while the Fmoc/S-butylthio protected monomer proved to be amenable to elongation. The mercaptomethyl modification did not influence the thermal stability of a PNA/RNA duplex. The feasibility of PNA-PNA native ligation was demonstrated.     

Hydrogen Bonding Directed Supramolecular Polymerisation of Oligo(Phenylene-Ethynylene)s: Cooperative Mechanism, Core Symmetry Effect and Chiral Amplification

The design of supramolecular motifs with tuneable stability and adjustable supramolecular polymerisation mechanisms is of crucial importance to precisely control the properties of supramolecular assemblies. This report focuses on constructing π-conjugated oligo(phenylene ethynylene) (OPE)-based one-dimensional helical supramolecular polymers that show a cooperative growth mechanism. Thus, a novel set of discotic molecules comprising a rigid OPE core, three amide groups, and peripheral solubilising wedge groups featuring C(3) and C(2) core symmetry was designed and synthesised. All of the discotic molecules are crystalline compounds and lack a columnar mesophase in the solid state. In dilute methylcyclohexane solution, one-dimensional supramolecular polymers are formed stabilised by threefold intermolecular hydrogen bonding and π-π interactions, as evidenced by (1) H?NMR measurements. Small-angle X-ray and light scattering measurements reveal significant size differences between the columnar aggregates of C(3) - and C(2) -symmetrical discotics, that is, the core symmetry strongly influences the nature of the supramolecular polymerisation process. Temperature-dependent CD measurements show a highly cooperative polymerisation process for the C(3) -symmetrical discotics. In contrast, the self-assembly of C(2) -symmetrical discotics shows a smaller enthalpy release upon aggregation and decreased cooperativity. In all cases, the peripheral stereogenic centres induce a preferred handedness in the columnar helical aggregates. Moreover, one stereogenic centre suffices to fully bias the helicity in the C(2) -symmetrical discotics. Finally, chiral amplification studies with the C(3) -symmetrical discotics were performed by mixing chiral and achiral discotics (sergeants-and-soldiers experiment) and discotics of opposite chirality (majority-rules experiment). The results demonstrate a very strong sergeants-and-soldiers effect and a rather weak majority-rules effect.     

Real time quantitative amplification detection on a microarray: towards high multiplex quantitative PCR

Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that allows quantitative amplification detection at higher multiplexing by the integration of amplification in solution and monitoring via hybridization to a microarray in real-time. This method does not require any manipulation of the PCR product and runs in a single closed chamber. Employing labeled primers, one of the main challenges is to measure surface signals against a high fluorescence background from solution. A compact, confocal scanner is employed, based on miniaturized optics from DVD technology and combined with a flat thermocycler for simultaneous scanning and heating. The feasibility of this method is demonstrated in singleplex with an analytical sensitivity comparable to routine qrtPCR.