Calibration of MTT assay in proton beams using radiochromic films

PurposeThis study provides methodology of calibrating as well as controlling the output for an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay irradiated in a low energy proton beam using EBT3-model GAFCHROMIC^TM film, without correcting for quenching effect.MethodsA calibrated Markus ionization chamber was used to measure the depth dose and beam output for 26.5 MeV protons produced by a CS30 cyclotron. A time-controlled aluminum cylinder was added in front of the horizontal beam-exit serving as a radiation shutter. Following the TRS-398 reference dosimetry protocol for proton beams, the output was calibrated in water at a reference depth of 3 mm. EBT3 film was calibrated for doses up to 8 Gy at the same depth. To verify the dose distribution for each 96-well MTT assay plate, EBT3 film was placed at the reference depth during irradiation and cell doses were scaled by measured percent depth dose (PDD) data.ResultsThe radiochromic film dosimetry system in this study provides dose measurements with an uncertainty better than 3.3% for doses higher than 1 Gy. From a single exposure and utilizing the Gaussian shape of the beam, multiple dose points can be obtained within different wells of the same plate ranging from 6.9 Gy (sigma ∼4%) in the central well, and 2 Gy (sigma ∼8%) for wells positioned closer to the periphery.ConclusionsWe described a methodology for radiochromic film-based dose monitoring system, using low-energy protons, which can be used for the MTT assay in any proton beam, except within Bragg peak region.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

Development of a multivariate statistical model to predict peroxisome proliferation in the rat,based on urinary 1H-NMR spectral patterns

A previous report of this work (Ringeissen et al. 2003) described the use of nuclear magnetic resonance (NMR) spectroscopy coupled with multivariate statistical data analysis (MVDA) to identify novel biomarkers of peroxisome proliferation (PP) in Wistar Han rats. Two potential biomarkers of peroxisome proliferation in the rat were described, N-methylnicotinamide (NMN) and N-methyl-4-pyridone-3-carboxamide (4PY). The inference from these results was that the tryptophan-nicotinamide adenine dinucleotide (NAD⁺) pathway was altered in correlation with peroxisome proliferation, a hypothesis subsequently confirmed by TaqMan® analysis of the relevant genes encoding two key enzymes in the pathway, aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) and quinolinate phosphoribosyltransferase (EC 2.4.2.19). The objective of the present study was to investigate these data further and identify other metabolites in the NMR spectrum correlating equally with PP. MVDA Partial Least Squares (PLS) models were constructed that provided a better prediction of PP in Wistar Han rats than levels of 4PY and NMN alone. The resulting Wistar Han rat predictive models were then used to predict PP in a test group of Sprague Dawley rats following administration of fenofibrate. The models predicted the presence or absence of PP (above on arbitrary threshold of >2-fold mean control) in all Sprague Dawley rats in the test group.     

Switching substrate specificity of AMT/MEP/ Rh proteins

In organisms from all kingdoms of life, ammonia and its conjugated ion ammonium are transported across membranes by proteins of the AMT/Rh family. Efficient and successful growth often depends on sufficient ammonium nutrition. The proteins mediating this transport, the so called Ammonium Transporter (AMT) or Rhesus like (Rh) proteins, share a very similar trimeric overall structure and a high sequence similarity even throughout the kingdoms. Even though structural components of the transport mechanism, like an external substrate recruitment site, an essential twin histidine pore motif, a phenylalanine gate and the hydrophobic pore are strongly conserved and have been analyzed in detail by molecular dynamic simulations and mutational studies, the substrate(s), which pass the central pores of the AMT/Rh subunits, NH₄⁺, NH₃ + H⁺, NH₄⁺ + H⁺ or NH₃, are still a matter of debate for most proteins, including the best characterized AmtB protein from Escherichia coli. The lack of a robust expression system for functional analysis has hampered proof of structural and mutational studies, although the NH₃ transport function for Rh-like proteins is rarely disputed. In plant transporters belonging to the subfamily AMT1, transport is associated with electrical currents, while some plant transporters, notably of the AMT2 type, were suggested to transport NH₃ across the membrane, without associated ionic currents. Here we summarize data in favor of each substrate for the distinct AMT/Rh classes, discuss mutants and how they differ in structure and functionality. A common mechanism with deprotonation and subsequent NH₃ transport through the central subunit pore is suggested.     

Precursor Decomposition,Microstructure, and Porosity of Yttria Stabilized Zirconia Thin Films Prepared by Aerosol‐Assisted Chemical Vapor Deposition

Microstructures of yttria stabilized zirconia thin films deposited by aerosol assisted chemical vapor deposition (AA‐CVD) are correlated with the thermal decomposition behavior of the corresponding metal precursors, zirconium and yttrium 2,4‐pentanedionate. Process conditions of AA‐CVD are investigated with the aim of producing dense and compact YSZ thin films for applications as gas‐tight electrolyte. Based on systematic cross sectional scanning transmission electron microscopy (STEM) investigations and conductivity measurements, the development of percolating nanoporosity is observed in samples prepared at temperatures between 350 °C and 600 °C at standard solution throughput. Compact columnar thin films with bulk conductivity are obtained at 600 °C by reducing the metal content of the precursor solution and at 450 °C by reducing the solution throughput.     

Excited state intramolecular proton transfer in 3-hydroxychromone: a DFT-based computational study

Potential energy (PE) curves for intramolecular proton transfer in the ground (GSIPT) and intramolecular proton transfer in the excited (ESIPT) states of 3-hydroxychromone (3HC) have been studied using DFT-B3LYP/6-31G(d,p) and TD-DFT/6-31G(d,p) level of theory, respectively. Our calculations suggest the non-viability of GSIPT in 3HC. Excited states PE calculations show the existence of ESIPT process in 3HC. ESIPT in 3HC has also been explained in terms of HOMO and LUMO electron densities of the enol and keto tautomers of 3HC.     

Substrate-mediated proton relay mechanism for the religation reaction in topoisomerase II

The DNA religation reaction of yeast type II topoisomerase (topo II) was investigated to elucidate its metal-dependent general acid/base catalysis. Quantum mechanical/molecular mechanical calculations were performed for the topo II religation reaction, and the proton transfer pathway was examined. We found a substrate-mediated proton transfer of the topo II religation reaction, which involves the 3′ OH nucleophile, the reactive phosphate, water, Arg781, and Tyr782. Metal A stabilizes the transition states, which is consistent with a two-metal mechanism in topo II. This pathway may be required for the cleavage/religation reaction of topo IA and II and will provide a general explanation for the catalytic mechanism in the topo IA and II.     

Effects of Delipidation on Proton Translocation and ATPase Activity in Beef Heart Electron Transport Particles

Delipidation of beef heart electron transport particles with phospholipase A₂ has been examined. When the particles were treated with the lipase and subjected to a low bovine serum albumin wash, ATPase activity was unaffected as was the lipid/protein ratio of the particles. However, energisation by ATP/Mg²⁺ was abolished. Furthermore, unsaturated but not saturated fatty acids discharged the steady-state ATP-driven membrane potential of control samples. When the phospholipase A₂ hydrolysis products were removed, inhibition of energy-linked reactions in the lipid-depleted particles was still observed and was interpreted in terms of non-specific leaks in the vesicle membranes, and ‘specific’ leaks through impaired H⁺-ATPase complexes. ATPase activity was less susceptible to delipidation than energisation but was, nevertheless, strongly inhibited at 50 percent lipid depletion.

Spin label studies indicated a decrease in the fluidity of particle membranes accompanying delipidation. Moreover, the discontinuity seen in Arrhenius plots of ATPase activity was shifted from 17°C (control) to 22°C at 50 percent phospholipid depletion. The data are consistent with a release of unsaturated fatty acids by phospholipase A₂ rendering the transport particles both leakier and the membranes less fluid than controls.     

Characterisation of H+ and Cu2+ binding to humic and fulvic acids

Abstract

The nature of H⁺ and Cu²⁺ binding by soil-derived humic (HA) and fulvic (FA) acid was characterised using potentiometric titrations. The experimental data obtained showed that the derived proton balance equation was valid and capable of describing proton consumption by both polyelectrolytes. HA was found to be more acidic and more reactive as shown by its lower equivalent weight compared to FA. Acid consumption by HA during titrations was little affected at ionic strength (μ) up to 0.1 M although it was enhanced at higher μ. Displacement of protons by Cu²⁺ resulted in a nonlinear sigmoidal pattern suggesting the formation of different Cu-HA chelates, or existence of sites that differed in their affinities for Cu on the ligand. Different concentrations of added Cu appeared to favour one or both mechanisms, although the titration method could not differentiate which of the probable mechanisms was more dominant at a specific level of Cu added. Similar values were obtained for conditional stability constants using either the equation of Scatchard or Ruzic.