Roger B. Gregory

Free‐volume and pore size distributions determined by numerical Laplace inversion of positron annihilation lifetime data

Positron annihilation lifetime spectroscopy is a powerful method for characterizing free volumes in a variety of materials. Correlations between positron annihilation rates and the size of free‐volume regions in which o‐Ps localizes are well described. Unfortunately, difficulties in the analysis of positron annihilation lifetime data have limited the approach to the determination of average lifetimes and average free volumes. Recent advances in the development of numerical integral transform methods now make it possible to extract continuous distributions of positron annihilation rates. The application of these methods to the determination of free‐volume distributions is described. The variable transformations required to convert positron annihilation rate probability density functions (PDF) to the corresponding lifetime, radius, and free‐volume PDFs are given and the approach is illustrated by application to positron annihilation lifetime data for amorphous polytetrafluoroethylene.

Analysis of positron annihilation lifetime data by numerical laplace inversion with the program CONTIN

Abstract The performance of the program CONTIN [Stephen W. Provencher, Comput. Phys. Commun. 27 (1982) 229], modified to solve Fredholm integral equations with convoluted kernels of the type that occur in the deconvolution and analysis of positron annihilation lifetime data, is investigated with computer-simulated test data. The method avoids direct determination of the instrument resolution function by employing the decay curve of a reference material with a well-known single lifetime. CONTIN employs a constrained, regularized least-squares analysis to calculate a continuous annihilation-rate probability density function (pdf) which is the most parsimonious solution that is consistent with the experimental data and prior knowledge. The performance of the algorithm for extracting positron annihilation lifetime information was evaluated by using several measures of the information content of the data described by Schrader and Usmar [in: Positron Annihilation Studies of Fluids, ed. S. Sharma (World Scientific, Singapore, 1988) p. 215]. The quality of the CONTIN reconstruction of the annihilation-rate pdf is strongly dependent on the information content of the data and is greatly improved as the total number of counts in the data set is increased. Nevertheless, the method provides excellent estimates of the intensities and mean lifetimes of peaks in the annihilation-rate pdf, even when the total counts in the data set are relatively low (105–106). The sensitivity of the algorithm to systematic errors in the data, including errors in the instrument resolution function, shifts in the positron of the zero-time channel of the sample and reference data and contamination of the reference decay by additional lifetime components was also evaluated. Errors in the FWHM of the instrument resolution function and shifts in the zero time channel as small as 1 10 to 1 5 of the channel width of the instrument generate additional spurious peaks in the annihilation-rate pdf and introduce errors in the lifetime parameters of the short-lived components.

Analysis of the mitochondrial proteome in multiple sclerosis cortex

Mitochondrial dysfunction has been proposed to play a role in the neuropathology of multiple sclerosis (MS). Previously, we reported significant alterations in the transcription of nuclear-encoded electron transport chain genes in MS and confirmed translational alterations for components of Complexes I and III that resulted in reductions in their activity. To more thoroughly and efficiently elucidate potential alterations in the expression of mitochondrial and related proteins, we have characterized the mitochondrial proteome in postmortem MS and control cortex using Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS). Using principal component analysis (PCA) and hierarchical clustering techniques we were able to analyze the differential patterns of SELDI-TOF spectra to reveal clusters of peaks which distinguished MS from control samples. Four proteins in particular were responsible for distinguishing disease from control. Peptide fingerprint mapping unambiguously identified these differentially expressed proteins. Three proteins identified are involved in respiration including cytochrome c oxidase subunit 5b (COX5b), the brain specific isozyme of creatine kinase, and hemoglobin β-chain. The fourth protein identified was myelin basic protein (MBP). We then investigated whether these alterations were consistent in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We found that MBP was similarly altered in EAE but the respiratory proteins were not. These data indicate that while the EAE mouse model may mimic aspects of MS neuropathology which result from inflammatory demyelinating events, there is another distinct mechanism involved in mitochondrial dysfunction in gray matter in MS which is not modeled in EAE.

Analysis of positron annihilation lifetime data by numerical Laplace inversion: Corrections for source terms and zero-time shift errors

Abstract We have recently described modifications to the program CONTIN [S.W. Provencher, Comput. Phys. Commun. 27 (1982) 229] for the solution of Fredholm integral equations with convoluted kernels of the type that occur in the analysis of positron annihilation lifetime data [R.B. Gregory and Yongkang Zhu, Nucl. Instr. and Meth. A290 (1990) 172]. In this article, modifications to the program to correct for source terms in the sample and reference decay curves and for shifts in the position of the zero-time channel of the sample and reference data are described. Unwanted source components, expressed as a discrete sum of exponentials, may be removed from both the sample and reference data by modification of the sample data alone, without the need for direct knowledge of the instrument resolution function. Shifts in the position of the zero-time channel of up to half the channel width of the multichannel analyzer can be corrected. Analyses of computer-simulated test data indicate that the quality of the reconstructed annihilation rate probability density functions is improved by employing a reference material with a short lifetime and indicate that reference materials which generate free positrons by quenching positronium formation (i.e. strong oxidizing agents) have lifetimes that are too long (400–450 ps) to provide reliable estimates of the lifetime parameters for the shortlived components with the methods described here. Well-annealed single crystals of metals with lifetimes less than 200 ps, such as molybdenum (123 ps) and aluminum (166 ps) do not introduce significant errors in estimates of the lifetime parameters and are to be preferred as reference materials. The performance of our modified version of CONTIN is illustrated by application to positron annihilation in polytetrafluoroethylene.

Neuronal Hemoglobin Expression and Its Relevance to Multiple Sclerosis Neuropathology

Multiple sclerosis (MS) is characterized by demyelination and progressive neurological disability. Previous studies have reported defects to mitochondria in MS including decreased expression of nuclear encoded electron transport chain subunit genes and inhibition of respiratory complexes. We previously reported increased levels of the hemoglobin β subunit (Hbb) in mitochondrial fractions isolated from postmortem MS cortex compared to controls. In the present study, we performed immunohistochemistry to determine the distribution of Hbb in postmortem MS cortex and identified proteins which interact with Hbb by liquid chromatography tandem mass spectrometry (LC-MS/MS). We found that Hbb was enriched in pyramidal neurons in internal layers of the cortex and interacts with subunits of ATP synthase, histones, and a histone lysine demethylase. We also found that Hbb is present in the nucleus and that expression of Hbb in SH-SY5Y neuroblastoma cells increased trimethylation of histone H3 on lysine 4 (H3K4me3), a histone mark that regulates cellular metabolism. These data suggest that Hbb may be a part of a mechanism linking neuronal energetics with epigenetic changes to histones in the nucleus and may provide neuroprotection in MS by supporting neuronal metabolism.

Characterization and multi-mode liquid chromatographic application of 4-propylaminomethyl benzoic acid bonded silica—A zwitterionic stationary phase

4-Propylaminomethyl benzoic acid bonded silica (4-PAMBA-silica) was synthesized by reacting aminopropyl modified silica with 4-carboxybenzaldehyde and reducing the resulting Schiff base with sodium cyanoborohydride in situ. The structure of this bonded phase was confirmed by (13)C cross polarization magic angle spinning ((13)C CP MAS) NMR. Elemental analysis indicated a coupling efficiency of about 79%. Chromatographic characterization of a 4-PAMBA-silica stationary phase revealed that at a mobile phase pH of 3.0, basic compounds were unresolved and co-eluted near the void volume, while aromatic sulfonates were retained and were well-resolved. By contrast, at a mobile phase pH of 7.0, the aromatic sulfonates were unresolved and eluted at the void volume, while basic compounds were retained and were well-resolved. To further understand the chromatographic retention mechanism the retention factors for a series of cationic and anionic compounds were measured at pH 7.0 and 3.0 as a function of the charge and concentration of competing ions in the mobile phase. A plot of the logarithm of the retention factor versus the logarithm of the eluent ion concentration was linear with a negative slope that is equal to the ratio of effective charges of the solutes and the eluent ions. This indicates that an ion exchange mechanism contributes to the separation of both cations and anions at pH 7.0 and pH 3.0, respectively. The increase in retention of alkanoic acids with their number of carbons at a mobile phase pH of 7.0 and exclusion of alkanoic acids at a mobile phase pH of 3.0 suggests that an ion exclusion mode and hydrophobic interaction mode are also operational with 4-PAMBA-silica. The amino acids, L-arginine, L-phenylalanine, and L-tyrosine were retained and well-resolved with a mobile phase containing a high concentration of organic solvent. This behavior was further studied by measuring the retention factors of polar and charged compounds as a function of the organic solvent content in the aqueous mobile phase. An increase in retention with decreasing water content in the mobile phase was observed, consistent with a hydrophilic interaction liquid chromatographic (HILIC) mode of separation.

Analysis of positron annihilation lifetime data presented as a sum of convoluted exponentials with the program “SPLMOD”

Abstract The reliability of a least squares analysis of positron annihilation lifetime data represented as a sum of convoluted exponentials is investigated with computer-stimulated test data. The method of analysis, which is available as a computer program called “SPLMOD” [R.H. Vogel, Technical Report EMBL-DA08, European Molecular Biology Laboratory, Heidelberg, FRG, (1986)], avoids direct determination of the instrument resolution function by employing the decay curve of a reference material with a well-known single lifetime. The performance of the algorithm for extracting positron annihilation lifetime information was evaluated by using several measures of the information content of the decay curve described recently by Schrader and Usmar [in: Positron Annihilation Studies of Fluids, ed. S. Sharma (World Scientific, Singapore, 1988)]. The sensitivity of the algorithm to systematic errors including errors in the resolution function, shifts in the zero time channel and contamination of the reference decay curves by additional lifetime components was evaluated. The method provides excellent estimates of lifetime parameters when the reference decay curve accurately reflects the resolution function of the sample data. However, the algorithm is extremely sensitive to errors in the instrument resolution function.

Monte Carlo calculation of positron implantation in multilayered materials

Abstract A simple Monte Carlo algorithm for the calculation of positron implantation in multilayered materials is described. Given estimates of the linear positron absorption and backscattering coefficients for each material in a multilayer the algorithm calculates the fraction of implanted positrons in each layer. The algorithm can be applied to systems with an arbitrary number and arrangement of layers, including systems which give rise to multiple backscattering. Application of the method to the calculation of source corrections and positron implantation in coatings is described. Monte Carlo calculation of positron implantation in aluminum/molybdenum multilayers, representing typical single and double foil source/sample arrangements gives results that are indentical to those calculated with exact analytical expressions for those systems where such expressions are available. The inclusion of nonabsorbing, void regions between layers, as occurs in nonbouded multilayers, can change the pattern of backscattering and lead to significant changes in positron implantation fractions. Positron implantation in coatings and thin films is particularly sensitive to backscattering effects. For certain positron source/sample geometries, backscattering can increase the fraction of implanted positrons in the source as the coating thickness decreases.

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns

BackgroundExperimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined.ResultsVariability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue.ConclusionsSELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights.

Multinuclear nuclear magnetic resonance spectroscopic and high-performance liquid chromatographic characterization of silica, grafted with specifically deuterated 4-((propylamino)methyl)benzoic acid

Specifically deuterated 4-((propylamino)methyl)benzoic acid-grafted silica (PAMBA-silica) was prepared by benzylation of propylamino-grafted silica (PA-silica) by either in situ reduction by sodium cyanoborodeuteride (NaCNBD3) of the Schiff base, formed by the reaction between PA-silica and 4-formylbenzoic acid, or by NaCNBD3 reduction of the isolated Schiff base. The PAMBA-silicas, which contain amine and carboxylic acid functionalities, were characterized by elemental analysis, (13)C, (29)Si, and (2)H solid state NMR, and HPLC. Solid state (13)C NMR revealed that PAMBA-silica prepared by the in situ method consists of di-benzylated, mono-benzylated, and unreacted amino-groups while PAMBA-silica prepared by the two-step synthesis consists of only mono-benzylated and unreacted amino-groups. (29)Si solid-state NMR spectra indicated that no significant loss of propylamine groups had occurred during benzylation. Nearly ideal uniaxial rigid-limit (2)H NMR spectra of grafted 4-PAMBA ligands indicates that they form a rigid structure, which provides effective electrostatic screening of inner positive charges when the ligands are in zwitterionic form. HPLC columns packed with PAMBA-silica and PA-silica were evaluated for ionic solutes at different pH of the mobile phase. Retention times increased for cations and decreased for anions at increasing pH. These trends show that PAMBA-silicas act as cation and anion exchangers at high and low pH, respectively. The pKa values of grafted carboxylic acid, determined from HPLC of weakly retaining solutes, are close to pKa of the solution PAMBA.