Microscopic localisation of protoporphyrin IX in normal mouse skin after topical application of 5-aminolevulinic acid or methyl 5-aminolevulinate

Light fractionation does not enhance the response to photodynamic therapy (PDT) after topical methyl-aminolevulinate (MAL) application, whereas it is after topical 5-aminolevulinic acid (ALA). The differences in biophysical and biochemical characteristics between MAL and ALA may result in differences in localisation that cause the differences in response to PDT. We therefore investigated the spatial distribution of protoporphyrin IX (PpIX) fluorescence in normal mouse skin using fluorescence microscopy and correlated that with the PDT response histologically observed at 2.5, 24 and 48h after PDT. As expected high fluorescence intensities were observed in the epidermis and pilosebaceous units and no fluorescence in the cutaneous musculature after both MAL and ALA application. The dermis showed localised fluorescence that corresponds to the cytoplasma of dermal cells like fibroblast and mast cells. Spectral analysis showed a typical PpIX fluorescence spectrum confirming that it is PpIX fluorescence. There was no clear difference in the depth and spatial distribution of PpIX fluorescence between the two precursors in these normal mouse skin samples. This result combined with the conclusion of Moan et al. that ALA but not MAL is systemically distributed after topical application on mouse skin [Moan et al., Pharmacology of protoporphyrin IX in nude mice after application of ALA and ALA esters, Int. J. Cancer 103 (2003) 132-135] suggests that endothelial cells are involved in increased response of tissues to ALA-PDT using light fractionation. Histological analysis 2.5h after PDT showed more edema formation after ALA-PDT compared to MAL-PDT that was not accompanied by a difference in the inflammatory response. This suggests that endothelial cells respond differently to ALA and MAL-PDT. Further investigation is needed to determine the role of endothelial cells in ALA-PDT and the underlying mechanism behind the increased effectiveness of light fractionation using a dark interval of 2h found after ALA but not after MAL-PDT.     

Highly accurate first-principles benchmark data sets for the parametrization and validation of density functional and other approximate methods. Derivation of a robust, generally applicable, double-hybrid functional for thermochemistry and thermochemical kinetics

We present a number of near-exact, nonrelativistic, Born-Oppenheimer reference data sets for the parametrization of more approximate methods (such as DFT functionals). The data were obtained by means of the W4 ab initio computational thermochemistry protocol, which has a 95% confidence interval well below 1 kJ/mol. Our data sets include W4-08, which are total atomization energies of over 100 small molecules that cover varying degrees of nondynamical correlations, and DBH24-W4, which are W4 theory values for Truhlar's set of 24 representative barrier heights. The usual procedure of comparing calculated DFT values with experimental atomization energies is hampered by comparatively large experimental uncertainties in many experimental values and compounds errors due to deficiencies in the DFT functional with those resulting from neglect of relativity and finite nuclear mass. Comparison with accurate, explicitly nonrelativistic, ab initio data avoids these issues. We then proceed to explore the performance of B2x-PLYP-type double hybrid functionals for atomization energies and barrier heights. We find that the optimum hybrids for hydrogen-transfer reactions, heavy-atoms transfers, nucleophilic substitutions, and unimolecular and recombination reactions are quite different from one another: out of these subsets, the heavy-atom transfer reactions are by far the most sensitive to the percentages of Hartree-Fock-type exchange y and MP2-type correlation x in an (x, y) double hybrid. The (42,72) hybrid B2K-PLYP, as reported in a preliminary communication, represents the best compromise between thermochemistry and hydrogen-transfer barriers, while also yielding excellent performance for nucleophilic substitutions. By optimizing for best overall performance on both thermochemistry and the DBH24-W4 data set, however, we find a new (36,65) hybrid which we term B2GP-PLYP. At a slight expense in performance for hydrogen-transfer barrier heights and nucleophilic substitutions, we obtain substantially better performance for the other reaction types. Although both B2K-PLYP and B2GP-PLYP are capable of 2 kcal/mol quality thermochemistry, B2GP-PLYP appears to be the more robust toward nondynamical correlation and strongly polar character. We additionally find that double-hybrid functionals display excellent performance for such problems as hydrogen bonding, prototype late transition metal reactions, pericyclic reactions, prototype cumulene-polyacetylene system, and weak interactions.     

High pressure ionic and molecular crystals of ammonia monohydrate within density functional theory

A combination of first-principles density functional theory calculations and a search over structures is used to predict the stability of a proton-transfer modification of ammonia monohydrate with space group P4∕nmm. The phase diagram is calculated with the Perdew-Burke-Ernzerhof (PBE) density functional, and the effects of a semi-empirical dispersion correction, zero point motion, and finite temperature are investigated. Comparison with MP2 and coupled cluster calculations shows that the PBE functional over-stabilizes proton transfer phases because too much electronic charge moves with the proton. This over-binding is partially corrected by using the PBE0 hybrid exchange-correlation functional, which increases the enthalpy of P4∕nmm by about 0.6 eV per formula unit relative to phase I of ammonia monohydrate and shifts the transition to the proton transfer phase from the PBE pressure of 2.8 GPa to about 10 GPa. This is consistent with experiment as proton transfer phases have not been observed at pressures up to ～9 GPa, while higher pressures have not yet been explored experimentally.     

Optimisation of Batch Culture Conditions for Cell-Envelope-Associated Proteinase Production from Lactobacillus delbrueckii subsp. lactis ATCC? 7830?

Using a combination of conventional sequential techniques, the batch growth conditions for the production of cell-envelope-associated proteinases have for the first time been studied and optimised in Lactobacillus delbrueckii subsp. lactis 313 (ATCC 7830; LDL 313). Concentrations of inoculum (0.1?<?X?<?10?% vol/vol), agitation speed (0?<?S?<?200?rpm), varying incubation temperature (30?<?T?<?50?°C), starting pH (4.5?<?pH?<?7) and carbon/nitrogen ratio of production medium (0.2?<?r?<?5) had an individual effect on proteinase yield (p?<?0.01). Optimal conditions for proteinase production included an initial pH of 6.0, 45?°C incubation temperature, 2?% (v/v) inoculum size of OD₅₆₀?=?1, 150?rpm agitation speed, and growth medium carbon/nitrogen ratio of 1.0. Maximum proteinase activity obtained for whole cells was 0.99 U/ml after 8?h of incubation. The variables studied are very relevant due to their significance in improving the productivity of proteinase synthesis from LDL 313, under process and, likely, economic optimum conditions.     

Dye Bonding to TiO(2): In Situ Attenuated Total Reflection Infrared Spectroscopy Study, Simulations, and Correlation with Dye-Sensitized Solar Cell Characteristics

Processing dye-sensitized solar cells gains more and more importance as interest in industrial applications grows daily. For large-scale processing and optimizing manufacturing in terms of environmental acceptability as well as time and material saving, a detailed knowledge of certain process steps is crucial. In this paper we concentrate on the sensitizing step of production, i.e., the anchoring of the dye molecules onto the TiO(2) semiconductor. A vacuum-tight attentuated total reflection infrared (ATR-IR) flow-through cell was developed, thus allowing measurements using a vacuum spectrometer to monitor infiltration of dye molecules into the porous TiO(2) film in situ at high sensitivity. In particular, the influence of the anchor and backbone of perylene dye molecules as well as the influence of solvents on the adsorption process was investigated. The experiments clearly show that an anhydride group reacts much slower than an acid group. A significantly lower amount of anhydride dye can be adsorbed on the films. Ex situ transmission experiments furthermore indicate that the availability of OH groups on the TiO(2) surface may limit dye adsorption. Also the backbone and base frame of the dye can influence the adsorption time drastically. Electrical cell characteristics correlate with the amount of adsorbed dye molecules determined by in situ ATR-IR measurements. The latter is also sensitive toward the diffusion of the dye through the porous layer. To gain a deeper understanding of the interplay between diffusion and adsorption, simulations were performed that allowed us to extract diffusion and adsorption constants. Again it was demonstrated that the anchoring group has a strong effect on the adsorption rate. The influence of the solvent was also studied, and it was found that both adsorption and desorption are affected by the solvent. Protic polar solvents are able to remove bound dye molecules, which is a possible pathway of cell degradation. Most importantly, the analysis shows the potential of this approach for the evaluation of molecules or additives concerning their characteristics important for cell processing.     

Effect of gamma radiation on the physico-chemical properties of alginate-based films and beads

Alginate solution (3%, w/v) was prepared using deionized water from its powder. Then the solution was exposed to gamma radiation (0.1?25 kGy). The alginate films were prepared by solution casting. It was found that gamma radiation has strong effect on alginate solution. At low doses, mechanical strength of the alginate films improved but after 5 kGy dose, the strength started to decrease. The mechanism of alginate radiolysis in aqueous solution is discussed. Film formation was not possible from alginate solution at doses >5 kGy. The mechanical properties such as puncture strength (PS), puncture deformation (PD), viscoelasticity (Y) coefficient of the un-irradiated films were investigated. The values of PS, PD and Y coefficient of the films were 333 N/mm, 3.20 mm and 27%, respectively. Alginate beads were prepared from 3% alginate solution (w/v) by ionotropic gelation method in 5% CaCl₂ solution. The rate of gel swelling improved in irradiated alginate-based beads at low doses (up to 0.5 kGy).     

O(3P) + CO2 collisions at hyperthermal energies: dynamics of nonreactive scattering, oxygen isotope exchange, and oxygen-atom abstraction

The dynamics of O((3)P) + CO(2) collisions at hyperthermal energies were investigated experimentally and theoretically. Crossed-molecular-beams experiments at = 98.8 kcal mol(-1) were performed with isotopically labeled (12)C(18)O(2) to distinguish products of nonreactive scattering from those of reactive scattering. The following product channels were observed: elastic and inelastic scattering ((16)O((3)P) + (12)C(18)O(2)), isotope exchange ((18)O + (16)O(12)C(18)O), and oxygen-atom abstraction ((18)O(16)O + (12)C(18)O). Stationary points on the two lowest triplet potential energy surfaces of the O((3)P) + CO(2) system were characterized at the CCSD(T)/aug-cc-pVTZ level of theory and by means of W4 theory, which represents an approximation to the relativistic basis set limit, full-configuration-interaction (FCI) energy. The calculations predict a planar CO(3)(C(2v), (3)A') intermediate that lies 16.3 kcal mol(-1) (W4 FCI excluding zero point energy) above reactants and is approached by a C(2v) transition state with energy 24.08 kcal mol(-1). Quasi-classical trajectory (QCT) calculations with collision energies in the range 23-150 kcal mol(-1) were performed at the B3LYP/6-311G(d) and BMK/6-311G(d) levels. Both reactive channels observed in the experiment were predicted by these calculations. In the isotope exchange reaction, the experimental center-of-mass (c.m.) angular distribution, T(θ(c.m.)), of the (16)O(12)C(18)O products peaked along the initial CO(2) direction (backward relative to the direction of the reagent O atoms), with a smaller isotropic component. The product translational energy distribution, P(E(T)), had a relatively low average of = 35 kcal mol(-1), indicating that the (16)O(12)C(18)O products were formed with substantial internal energy. The QCT calculations give c.m. P(E(T)) and T(θ(c.m.)) distributions and a relative product yield that agree qualitatively with the experimental results, and the trajectories indicate that exchange occurs through a short-lived CO(3)* intermediate. A low yield for the abstraction reaction was seen in both the experiment and the theory. Experimentally, a fast and weak (16)O(18)O product signal from an abstraction reaction was observed, which could only be detected in the forward direction. A small number of QCT trajectories leading to abstraction were observed to occur primarily via a transient CO(3) intermediate, albeit only at high collision energies (149 kcal mol(-1)). The oxygen isotope exchange mechanism for CO(2) in collisions with ground state O atoms is a newly discovered pathway through which oxygen isotopes may be cycled in the upper atmosphere, where O((3)P) atoms with hyperthermal translational energies can be generated by photodissociation of O(3) and O(2).