Use of EPR and FTIR to detect biological effects of ultrasound and microbubbles on a fibroblast cell line

Structural and functional effects of exposing murine fibroblasts (NIH 3T3) to therapeutic ultrasound at 1 MHz frequency are described. These bioeffects can be attributed to the formation of free radical species by sonolysis of water. When cavitation occurs, dissociation of water vapor into H atoms and OH radicals is observed; these H atoms and OH radicals combine to form H₂, H₂O₂, and HO₂. The radicals can chemically modify biomolecules, for example enzymes, DNA, and lipids. Generation of free radicals during exposure to ultrasound with or without encapsulated microbubbles (contrast agents) was studied by use of electron paramagnetic resonance with DMPO spin trapping. Recently the potential for possible use of these microbubbles in gene therapy has been investigated, because of the ability of the stabilized microbubbles to release their content when exposed to ultrasound. Structural changes were studied by Fourier-transform infrared spectroscopy, and induction of possible genotoxic damage by exposure of the cells to therapeutic ultrasound at 1 MHz frequency with our experimental device was verified by use of the cytokinesis-block micronucleus assay.     

Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn

Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the “know-how” of therapeutic modality by design.     

X-ray small angle scattering of the human transferrin protein aggregates. A fractal study.

X-ray small angle scattering experiments, using a pin hole SAXS camera with Synchrotron radiation source, have been performed to study the conformational changes of lyophilized samples of Apo-, Mono-, and Diferric- human transferrin. We report the experimental evidence that the analysis of the scattered intensity through the fractal theory may give information on the particle size and its variation upon iron binding.     

Heterogeneity of the isolated subunits of the fetal and adult human hemoglobin in solution, detected by XANES spectroscopy

Differences in the local structure of the heme in the isolated alpha-, beta- and gamma-chains of the adult and fetal human hemoglobin are detected by XANES (X-ray absorption near-edge structure) spectroscopy. The ligand bonding angle to the iron ion in the ligated forms and the displacement of the Fe respect to the porphyrin plane in the deoxy forms are found to be different for each chain.     

Distribution of G1m(1), G1m(2) and G3m(5) allotypes in Aragon.

The distribution of G1m(1), G1m(2) and G3m(5) allotypes was studied in 700 unrelated individuals from Aragon (North-East Spain). The Gm haplotype frequencies were similar to those reported in French areas next to Aragon.     

The Natural Occurrence of Eggplant Mottled Dwarf Rhabdovirus in Tobacco in Italy

The natural occurrence of eggplant mottled dwarf rhabdovirus (EMDV) in field-grown tobacco plants iti Italy is reported. The virus was identified by the differential reactions of herbaceous hosts, electron microscopy and serology. This seems to be the first record of natural itifection of tobacco by EMDV.