CGRP-cleavage enzyme detection using a biotinylated hydroxymate affinity probe

Introduction: Neuropeptides contribute to the pathophysiology of peripheral inflammation and a neurogenic component has been described for many inflammatory diseases, including periodontitis. Neuropeptides are susceptible to cleavage by peptidases and therefore the exact location and level of expression of peptidases are major determinants of neuropeptide action. Previous studies by our research group suggested that levels of the neuropeptide calcitonin gene-related peptide (CGRP) may be regulated by peptidases present in gingival crevicular fluid (GCF). Objectives: The aim of this work was to purify and partially characterize the GCF enzyme responsible for CGRP degradation using a biotinylated hydroxymate affinity probe (based on the P1-P4 amino acid sequence of the observed cleavage site) which we previously showed to inhibit CGRP degradation. Methods: Pooled healthy and pooled periodontitis GCF samples were subject to a pre-clear step with magnetic streptavadin beads. Healthy and diseased samples were incubated with the biotinylated hydroxymate probe (20 uM) after which biotinylated proteins were purified from the sample using magnetic streptavadin beads. Bound proteins were subjected to SDS-PAGE and western blotting. Biotin incorporated proteins were disclosed using a streptavadin horse radish peroxidase conjugate. Results: A band was disclosed in the periodontitis pooled sample at a molecular weight of approximately 60 kDa. The band was absent in the pooled healthy samples. As expected, when periodontitis samples were pre-boiled to denature proteins before the addition of the hydroxymate probe, no biotin incorporated band was present. Conclusions: This work demonstrates the purification and disclosure of a protein found specifically in periodontitis which binds to the specific biotinylated hydroxymate affinity probe based on the cleavage site of CGRP only when in its native form. We intend to scale up the sample size thus allowing the identification of the putative CGRP degrading peptidase using MALDI-mass spectrometry.
Funded by an IADR/GlaxoSmithKline Innovation in Oral Care Award

Laboratory measurements of resistivity in warm dense plasmas relevant to the microphysics of brown dwarfs

Since the observation of the first brown dwarf in 1995, numerous studies have led to a better understanding of the structures of these objects. Here we present a method for studying material resistivity in warm dense plasmas in the laboratory, which we relate to the microphysics of brown dwarfs through viscosity and electron collisions. Here we use X-ray polarimetry to determine the resistivity of a sulphur-doped plastic target heated to Brown Dwarf conditions by an ultra-intense laser. The resistivity is determined by matching the plasma physics model to the atomic physics calculations of the measured large, positive, polarization. The inferred resistivity is larger than predicted using standard resistivity models, suggesting that these commonly used models will not adequately describe the resistivity of warm dense plasma related to the viscosity of brown dwarfs.

Disposable solid state probe for optical screening of chlorpromazine

We are presenting a simple, low-cost and rapid solid-state optical probe for screening chlorpromazine (CPZ) in aquacultures. The method exploits the colourimetric reaction between CPZ and Fe(III) ion that occurs at a solid/liquid interface, the solid layer consisting of ferric iron entrapped in a layer of plasticized PVC. If solutions containing CPZ are dropped onto such a layer, a colour change occurs from light yellow to dark pink or even light blue, depending on the concentration of CPZ. Visual inspection enables the concentration of CPZ to be estimated. The resulting colouration was also monitored by digital image collection for a more accurate quantification. The three coordinates of the hue, saturation and lightness system were obtained by standard image processing along with mathematical data treatment. The parameters affecting colour were assessed and optimized. Studies were conducted by visible spectrophotometry and digital image acquisition, respectively. The response of the optimized probe towards the concentration of CPZ was tested for several mathematical transformations of the colour coordinates, and a linear relation was found for the sum of hue and luminosity. The limit of detection is 50 μM (corresponding to about 16 μg per mL). The probe enables quick screening for CPZ in real water samples with prior sample treatment.

Pulsed laser deposition of YBa2Cu3O7-[delta]/PrBa2Cu3O7-[delta] /

The process of depositing thin films by the use of pulsed laser deposition (PLD) has become a more widely used technique for the growth of substances in a thin film form. Pulsed laser deposition allows for the stoichiometric film growth of the target which is of great significance in the deposition of High Temperature Superconducting materials. We will describe a system designed using an excimer laser and vaccum chamber in which thin films and superlattices of YBa2Cuj07_i, PrBa2Cu307_i, and YBajCujOr-j/ PrBajCusOr-^ were deposited on SrTiOs. Results of resistivity measurements using the four probe technique will be shown.

Optical properties of organic superconductor K-(BETS)2FeBr4 /

K-(BETS)2FeBr4 is a quasi-2D charge transfer organic metal with interesting electronic and magnetic properties. It undergoes a transition to an antiferromagnetic (AF) state at ambient pressure at the Neel temperature (T^^) = 2.5 K, as well as to a superconducting (SC) state at 1.1 K [1]. The temperature dependence of the electrical resistivity shows a small decrease at T;v indicating the resistivity drops as a result of the onset of the ordering of Fe'*''" spins. A sharp drop in the resistivity at 1.1 K is due to its superconducting transition. The temperature dependence of the susceptibility indicates an antiferromagnetic spin structure with the easy axis parallel to the a-axis. The specific heat at zero-field shows a large peak at about 2.4 K, which corresponds to the antiferromagnetic transition temperature (Tat) and no anomaly is observed around the superconducting transition temperature (1.1 K) demonstrating that the magnetically ordered state is not destroyed by the appearance of another phase transition (the superconducting transition) in the 7r-electron layers [1], [2]. This work presents an investigation of how the low frequency electromagnetic response is affected by the antiferromagnetic and superconducting states, as well as the onset of strong correlation. The location of the easy axis of three samples was determined and polarized thermal reflectance measurements of these «-(BETS)2FeBr4 samples oriented with their vertical axis along the a- and c axes were then carried out using a *He refrigerator cryostat and a Martin-Puplett type polarizing interferometer at various temperatures (T = 0.5 K, 1.4 K. 1.9 K, 2.8 K) above and below the superconducting state and/or antiferromagnetic state. Comparison of the SC state to the normal state along the o- and c-axes indicates a rising thermal reflectance at low frequencies (below 10 cm"' ) which may be a manifestation of the superconducting energy gap. A dip-Hke feature is detected at low frequencies (below 15 cm"') in the thermal reflectance plots which probe the antiferromagnetic state along the two axes, and may be due to the opening of a gap in the excitation spectrum as a result of the antiferromagnetism. In another set of experiments, thermal reflectance measurements carried out along the a- and c-axes at higher temperatures (10 K-80 K) show that the reflectivity decreases with increasing temperature to 60 K (the coherence temperature) above which it increases again. Comparison of the thermal reflectance plots along the a- and c-axes at higher temperatures reveals an anisotropy between these two axes. The Hagen-Rubens thermal reflectance plots corresponding to an average over the ac-plane were calculated using experimental hterature resistivity values. Comparison of the Hagen-Rubens plots with the experimental thermal reflectance along the a- and c-axes indicates that both exhibit the general trend of a decrease in thermal reflectance with increasing frequency, however the calculated Hagen-Rubens thermal reflectance at different temperatures is much lower than the experimental curves.

Electronic and magnetic properties of LaV1-xO3 /

A series of LaVi^xOs compounds (x=0.00, 0.02, 0.04, 0.06, 0.08) were prepeired using the standard solid reaction. The samples were chareicterized by X-ray diffraction (XRD), fourprobe resistivity, smd magnetic susceptibility studies. Powder X-ray diffraction analysis indicated the formation of a single-phase sample with a orthorhombic structure which was first found in GdFeOs (space group Pnma) . The Unit Cell program was used for calculating lattice peirameters from XFID data. The XRD spectnim could be indexed on a cubic lattice with Og = 2ap ~ (7.8578 to 7.9414 A). The lattice parameter was observed to increase as the Vanadium vacancy increased. Four-probe resistivity measurements exhibited semiconductor behavior for all sajnples from room temperature down to 19K. The resistivity of samples increased with increasing Vanadium vacancy. The resistivity of samples demonstrated activated conduction with an activation energy of approximately 0.2 eV. The activation energy increased with increasing lattice parameter. Field cool magnetic susceptibility measurements were performed with field of 500 G from 300 K to 5 K. These measurements indicated the presence of an antiferromagnetic transition at about 140 K. The data was fitted above Neel temperature to Ciurie-Weiss law yielding a negative parameignetic Curie temperature. This implies that antiferromagnetic ordering is present.

On the anharmonic, multi-phonon, Debye-Waller contributions to the phonon-limited resistivity of metals : applications to Na and K

The anharmonic, multi-phonon (MP), and Oebye-Waller factor (OW) contributions to the phonon limited resistivity (;0) of metals derived by Shukla and Muller (1979) by the doubletime temperature dependent Green function method have been numerically evaluated for Na and K in the high temperature limit. The anharmonic contributions arise from the cubic and quartic shift of phonons (CS, QS), and phonon width (W) and the interference term (1). The QS, MP and OW contributions to I' are also derived by the matrix element method and the results are in agreement with those of Shukla and Muller (1979). In the high temperature limit, the contributions to;O from each of the above mentioned terms are of the type BT2 For numerical calculations suitable expressions are derived for the anharmonic contributions to ~ in terms of the third and fourth rank tensors obtained by the Ewald procedure. The numerical calculation of the contributions to;O from the OW, MP term and the QS have been done exactly and from the CS, Wand I terms only approximately in the partial and total Einstein approximations (PEA, TEA), using a first principle approach (Shukla and Taylor (1976)). The results obtained indicate that there is a strong pairwise cancellation between the: OW and MP terms, the QS and CS and the Wand I terms. The sum total of these contributions to;O for Na and K amounts to 4 to 11% and 2 to 7%, respectively, in the PEA while in the TEA they amount to 3 to 7% and 1 to 4%, respectively, in the temperature range.

Selected topics in fast atom bombardment (FAB) mass spectrometry a)specially designed probe tips and ion generation, b)structures of vitamin B6 schiff base complexes

This thesis can be broken down into two sections. Section one is a study . of the ionization mechanisms and the ion source optimization for Fast Atom Bombardment (FAB) ionization. For this study, several specially designed probe tips were created and tested under various experimental conditions. The aIm of this section is to understand the operating characteristics of a FAB IOn source better. The second section involves the study of several Vitamin B6 Schiff Base complexes using both positive and negative ion FAB MS. This section is an exploration of the usefulness of FAB MS as a structure probe for the metalcoordination complexes of Vitamin B6.

The effects of moisture, cation concentration, temperature, density and composition of soils on their electrical resistivity

The effects. of moisture, cation concentration, dens ity , temper~ t ure and grai n si ze on the electrical resistivity of so il s are examined using laboratory prepared soils. An i nexpen si ve method for preparing soils of different compositions was developed by mixing various size fractions i n the laboratory. Moisture and cation c oncentration are related to soil resistivity by powe r functions, whereas soil resistiv ity and temperature, density, Yo gravel, sand , sil t, and clay are related by exponential functions . A total of 1066 cases (8528 data) from all the experiments were used in a step-wise multiple linear r egression to determine the effect of each variable on soil resistivity. Six variables out of the eight variables studied account for 92.57/. of the total variance in so il resistivity with a correlation coefficient of 0.96. The other two variables (silt and gravel) did not increase the · variance. Moisture content was found to be - the most important Yo clay. variable- affecting s oil res istivi ty followed by These two variables account for 90.81Yo of the total variance in soil resistivity with a correlation ~oefficient ·.of 0 . 95. Based on these results an equation to ' ~~ed{ ct soil r esist ivi ty using moisture and Yo clay is developed . To t est the predicted equation, resistivity measurements were made on natural soils both in s i tu a nd i n the laboratory. The data show that field and laboratory measurements are comparable. The predicted regression line c losely coinciqes with resistivity data from area A and area B soils ~clayey and silty~clayey sands). Resistivity data and the predicted regression line in the case of c layey soils (clays> 40%) do not coincide, especially a t l ess than 15% moisture. The regression equation overestimates the resistivity of so i l s from area C and underestimates for area D soils. Laboratory prepared high clay soils give similar trends. The deviations are probably caused by heterogeneous distribution of mo i sture and difference in the type o f cl ays present in these soils.

The Effects of Annealing URu2Si2 on the Resistivity and Meissner Effect

The enigmatic heavy fermion URu2Si2, which is the subject of this thesis, has attracted intensive theoretical and experimental research since 1984 when it was firstly reported by Schlabitz et al. at a conference [1]. The previous bulk property measurements clearly showed that one second order phase transition occurs at the Hidden Order temperature THO ≈ 17.5 K and another second order phase transition, the superconducting transition, occurs at Tc ≈ 1 K. Though twenty eight years have passed, the mechanisms behind these two phase transitions are still not clear to researchers. Perfect crystals do not exist. Different kinds of crystal defects can have considerable effects on the crystalline properties. Some of these defects can be eliminated, and hence the crystalline quality improved, by annealing. Previous publications showed that some bulk properties of URu2Si2 exhibited significant differences between as-grown samples and annealed samples. The present study shows that the annealing of URu2Si2 has some considerable effects on the resistivity and the DC magnetization. The effects of annealing on the resistivity are characterized by examining how the Residual Resistivity Ratio (RRR), the fitting parameters to an expression for the temperature dependence of the resistivity, the temperatures of the local maximum and local minimum of the resistivity at the Hidden Order phase transition and the Hidden Order Transition Width ∆THO change after annealing. The plots of one key fitting parameter, the onset temperature of the Hidden Order transition and ∆THO vs RRR are compared with those of Matsuda et al. [2]. Different media used to mount samples have some impact on how effectively the samples are cooled because the media have different thermal conductivity. The DC magnetization around the superconducting transition is presented for one unannealed sample under fields of 25 Oe and 50 Oe and one annealed sample under fields of 0 Oe and 25 Oe. The DC field dependent magnetization of the annealed Sample1-1 shows a typical field dependence of a Type-II superconductor. The lower critical field Hc1 is relatively high, which may be due to flux pinning by the crystal defects.

Design of Resistivity Instrumentation for a He3 Cryostat and its Application to the Charge Density Wave Superconductor CuxTiSe2

Fermi patches in quasi-two dimensional charge density waves (CDW) have not described the connection to superconductivity (SC) according to theory adequately at this point in time. The connection between CDW and SC in the quasi-two dimensional material CuxTiSe2 is an interesting one which might reveal mechanisms in unconventional superconductors. A previous Brock graduate student grew crystals of CuxTiSe2. The precise doping of the samples was not known. In order to determine the doping parameter x in CuxTiSe2, a sensitive resistivity measurement system was necessary. A new resistivity measurement system was designed and implemented utilizing an Infrared Labs HDL-10 He3 cryostat. By comparing with data from the literature, doping of two samples was investigated using the new measurement system and a Quantum Design Magnetic Property Measurement System (MPMS). Methods for determining the doping revealed that the old resistivity system would not be able to determine the CDW transition temperature of highly doped samples or doping for elongated samples due to electronic noise. Doping in one sample was found to be between x=0.06 and x=0.065. Values of doping in the second sample had a discrepancy but could be explained by incorrect sample orientation.

Probe-level linear model fitting and mixture modeling results in high accuracy detection of differential gene expression

Affiliation: Institut de recherche en immunologie et en cancérologie, Université de Montréal

Laser Induced Fluorescence Based Optical Fiber Probe for Analyzing Bacteria

Optical fiber based laser induced fluorescence (LIF) measurements were carried out using Rhodamine B to analyze two different species of bacteria , a Gram-positive bacteria namely Bacillus smithii , and fibrin alginolvticus, a Gram- negative bacteria . The fiber sensor was clearly able to distinguish between the two species of bacteria . Quenching effect of the dye Rhodamine B by Bacillus smithii was observed . The effect of dye on the samples was also studied in detail.

Characterization of radio frequency plasma using Langmuir probe and optical emission spectroscopy

The radio frequency plasma generated during the sputtering of Indium Tin Oxide target using Argon was analyzed by Langmuir probe and optical-emission spectroscopy. The basic plasma parameters such as electron temperature and ion density were evaluated. These studies were carried out by varying the RF power from 20 to 50 W. A linear increase in ion density and an exponential decrease in electron temperature with rf power were observed. The measured plasma parameters were then correlated with the properties of ITO thin films deposited under similar plasma conditions.

Laser induced fluorescence based optical fiber probe for analyzing bacteria

Optical fiber based laser induced fluorescence (LIF) measurements were carried out using Rhodamine B to analyze two different species of bacteria , a Gram-positive bacteria namely Bacillus .cmithii , and fibrin alginolvticus, a Gram-' negative bacteria . The fiber sensor was clearly able to distinguish between the two species of bacteria . Quenching effect of the dye Rhodamine B by Bacillus smitltii was observed . The effect of dye on the samples was also studied in detail.