Normalization of clonal diversity in gene therapy studies using shape constrained splines.

Viral vectors are used to insert genetic material into semirandom genomic positions of hematopoietic stem cells which, after reinfusion into patients, regenerate the entire hematopoietic system. Hematopoietic cells originating from genetically modified stem cells will harbor insertions in specific genomic positions called integration sites, which represent unique genetic marks of clonal identity. Therefore, the analysis of vector integration sites present in the genomic DNA of circulating cells allows to determine the number of clones in the blood ecosystem. Shannon diversity index is adopted to evaluate the heterogeneity of the transduced population of gene corrected cells. However, this measure can be affected by several technical variables such as the DNA amount used and the sequencing depth of the library analyzed and therefore the comparison across samples may be affected by these confounding factors. We developed an advanced spline-regression approach that leverages on confounding effects to provide a normalized entropy index. Our proposed method was first validated and compared with two state of the art approaches in a specifically designed in vitro assay. Subsequently our approach allowed to observe the expected impact of vector genotoxicity on entropy level decay in an in vivo model of hematopoietic stem cell gene therapy based on tumor prone mice.

Structural and dynamical characterization of piroxicam by 1H- and 13C-NMR relaxation studies.

Carbon spin-lattice relaxation rates of an anti-inflammatory drug, piroxicam, have been measured. These results have been used in determining the reorientational rates of the proton carbon vectors. An analysis of internal motions within the pyridinyl moiety of piroxicam was carried out. Selective proton-carbon nuclear Overhauser effect (NOE) measurements were made in order to determine the solution structure of piroxicam. The effect of indirect NOE arising from exchangeable protons has been analyzed and considered.

Determination and identification of metabolites of the fungicides Iprodione and Procymidone in compost.

The main metabolites formed from Iprodione and Procymidone during the composting process have been isolated and identified by HPLC-DAD-MSD. After addition of the fungicides to the composting pile, we monitored the reaction of the two analytes and the formation of their degradation products for eight months. We verified the nature of the metabolites by comparison with those hypothesised in the literature and by comparison with the behaviour of an abiotic process in aqueous acetonitrile pH 6 and at 35 degrees C. After taking into account the different kinetic behaviours of the fungicides on degradation in compost and hydro-organic solution, breakdown pathways are proposed for biodegradation.

Determination of presence of fungicides by their common metabolite, 3,5-DCA, in compost.

This paper describes the determination of 3,5-DCA in commercial composts, a common metabolite in a class of fungicides, and dicarboximides (Vinclozolin, Chlozolinate, Iprodione, Procymidone) which are commonly used in agriculture. The extracts, obtained in acetonitrile by sonication, are analysed by HPLC/DAD without clean-up. This method has shown several advantages: reduced manipulation of samples, good recovery (80-90%) and good reproducibility (RSD% <7). The limit of detection (DL) of the analytical method has been estimated as 15 microg/kg for the common metabolite, and 35-145 microg/kg for the four fungicides in the matrices.

The "switch-off" mechanism of spontaneous resolution of acute gout attack.

Urate crystal size change and the modification of coated proteins by oxygen radicals released by stimulated polymorphonuclear cells (PMN) could represent a likely "switch-off" mechanism of the spontaneous resolution of acute gout attack. The absorption spectra and the uric acid, allantoin and urea concentrations were determined before and after in vitro exposition of monosodium urate (MSU) crystals to superoxide anion (O2) photochemically generated. The results showed a complete dissolution of MSU crystals after incubation under O2-, with decrease of uric acid and increase of allantoin and urea concentrations. Our results were confirmed by polarizing, electron microscopy and calorimetric techniques. The results obtained seem to confirm that the spontaneous resolution of acute gout attack could be attributed to the dissolving effect on urate crystals and the modification of coated proteins by O2- released by phagocytizing PMN.

[Pathogenesis of ankylosing spondylitis: relation between HLA-B 27 and Klebsiella aerogenes]. / Ricerche eziopatogenetiche sulla apondilite anchilosante: rapporti tra HLA-B 27 e Klebsiella aerogenes.

The Authors attested the evidence of a cross-reactivity among some Klebsiella antigens and HLA B 27 positive lymphocytes, suggesting the hypothesis that Klebsiella may be an initiating agent in ankylosing spondylitis (AS). The Authors determined also serum antibody response against Klebsiella antigens in AS patients. The results obtained are discussed.