Low-energy X-radiation for prevention of restenosis results in localized inhibition of V79 fibroblast cell proliferation

Local delivery of high-energy ionizing radiation by using 3 or 3 emitters to injured vessels demonstrated inhibition of cell proliferation (CP) and neointima formation. Low-energy ('soft') X-radiation (LEXR) offers logistic and safety advantages over the use of disposable radioisotopes. This study evaluated the efficacy of LEXR in penetration and inhibition of CP at doses similar to those prescribed for the use of radioisotopes for prevention of restenosis. Serial measurements in an ion chamber detected the attenuation of LEXR using potentials of 17 and 40 kV at a distance of 17 cm of air through 0-10 mm depths of serum-containing tissue culture medium. The effect of inhibition on CP was determined by exposing V79 fibroblasts to a potential of 17 kV in order to deliver a prescribed dose of 13 Gy at a dose rate of 2.17 Gy/min to the surface of the cells. Complete inhibition of CP at a height of 0.00 mm occurred with 13 Gy; however, a 50% attenuation of the dose was measured at a medium depth of 1.22 mm and was associated with a reduction of 60% of the CP. LEXR demonstrated an ability to inhibit CP at doses equivalent to those used in techniques involving 3 and 3 irradiation. Under such conditions, the dose gradient is too high, especially for large vessels. However, a catheter-based LEXR that could be inserted into the artery with the capability of varying effective energy would be ideal for intravascular applications.     

Systematic Cell-Based Phenotyping of Missense Alleles Empowers Rare Variant Association Studies: A Case for LDLR and Myocardial Infarction

A fundamental challenge to contemporary genetics is to distinguish rare missense alleles that disrupt protein functions from the majority of alleles neutral on protein activities. High-throughput experimental tools to securely discriminate between disruptive and non-disruptive missense alleles are currently missing. Here we establish a scalable cell-based strategy to profile the biological effects and likely disease relevance of rare missense variants in vitro. We apply this strategy to systematically characterize missense alleles in the low-density lipoprotein receptor (LDLR) gene identified through exome sequencing of 3,235 individuals and exome-chip profiling of 39,186 individuals. Our strategy reliably identifies disruptive missense alleles, and disruptive-allele carriers have higher plasma LDL-cholesterol (LDL-C). Importantly, considering experimental data refined the risk of rare LDLR allele carriers from 4.5- to 25.3-fold for high LDL-C, and from 2.1- to 20-fold for early-onset myocardial infarction. Our study generates proof-of-concept that systematic functional variant profiling may empower rare variant-association studies by orders of magnitude.     

Expression,purification, phosphorylation and characterization of recombinant human statherin

This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria–Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47 mg per liter of cell culture, while 112 mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP–HPLC corresponded to 0.6 mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase.     

Elevation-induced variations of pollen assemblages in the North-western Alps: An analysis of their value as temperature indicators

Seventy-seven modern pollen samples from various elevations (350-2680 m a.s.l.) in two different areas of the north-western Alps (the Aosta Valley, Italy and the Taillefer Massif, France) were statistically analyzed to derive correlations between pollen assemblages, elevation and temperature at the sampled points. Numerical classifications were performed on pollen data to judge similarities between the two areas. The results show that a strong relationship exists between altitude and variations in pollen taxa percentages despite some floristic differences between the two areas. As a test, transfer functions from pollen percentages to elevation and temperature were calculated from pollen data. The reconstruction appears to be reliable, with a higher reliability at sites located over 1000m. This analysis aims to serve as a basis for further quantitative reconstruction of temperature changes during the Holocene based on fossil pollen data from sensitive regions that encompass a significant altitudinal gradient.     

Interaction between double helix DNA fragments and a new topopyrone acting as human topoisomerase I poison

A water soluble derivative (2) of topopyrones was selected for NMR studies directed to elucidate the mode of binding with specific oligonucleotides. Topopyrone 2 can intercalate into the CG base pairs, but the residence time into the double helix is very short and a fast chemical exchange averaging occurs at room temperature between the free and bound species. The equilibria involved become slow below room temperature, thus allowing to measure a mean lifetime of the complex of ca. 7 ms at 15 °C. Structural models of the complex with d(CGTACG)₂ were developed on the basis of DOSY, 2D NOESY and ³¹P NMR experiments. Topopyrone 2 presents a strong tendency to self-associate. In the presence of oligonucleotide a certain number of ligand molecules are found to externally stack to the double-helix, in addition to a small fraction of the same ligand intercalated. The external binding to the ionic surface of the phosphoribose chains may thus represents the first step of the intercalation process.     

Asymptotic behaviour of solutions to abstract logistic equations

We analyze the asymptotic behaviour of solutions of the abstract differential equation u'(t)=Au(t)-F(u(t))u(t)+f. Our results are applicable to models of structured population dynamics in which the state space consists of population densities with respect to the structure variables. In the equation the linear term A corresponds to internal processes independent of crowding, the nonlinear logistic term F corresponds to the influence of crowding, and the source term f corresponds to external effects. We analyze three separate cases and show that for each case the solutions stabilize in a way governed by the linear term. We illustrate the results with examples of models of structured population dynamics -- a model for the proliferation of cell lines with telomere shortening, a model of proliferating and quiescent cell populations, and a model for the growth of tumour cord cell populations.