Active site labeling of cysteine cathepsins by a straightforward diazomethylketone probe derived from the N-terminus of human cystatin C

We designed a straightforward biotinylated probe using the N-terminal substrate-like region of the inhibitory site of human cystatin C as a scaffold, linked to the thiol-specific reagent diazomethylketone group as a covalent warhead (i.e. Biot-(PEG)2-Ahx-LeuValGly-DMK). The irreversible activity-based probe bound readily to cysteine cathepsins B, L, S and K. Moreover affinity labeling is sensitive since active cathepsins were detected in the nM range using an ExtrAvidin®-peroxidase conjugate for disclosure. Biot-(PEG)2-Ahx-LeuValGly-DMK allowed a slightly more pronounced labeling for cathepsin S with a compelling second-order rate constant for association (kass = 2,320,000 M−1 s−1). Labeling of the active site is dose-dependent as observed using 6-cyclohexylamine-4-piperazinyl-1,3,5-triazine-2-carbonitrile, as competitive inhibitor of cathepsins. Finally we showed that Biot-(PEG)2-Ahx-LeuValGly-DMK may be a simple and convenient tool to label secreted and intracellular active cathepsins using a myelomonocytic cell line (THP-1 cells) as model.

Structure and dynamics of aqueous 2-propanol: A THz-TDS, NMR and neutron diffraction study

Aqueous liquid mixtures, in particular, those involving amphiphilic species, play an important role in many physical, chemical and biological processes. Of particular interest are alcohol/water mixtures; however, the structural dynamics of such systems are still not fully understood. Herein, a combination of terahertz time-domain spectroscopy (THz-TDS) and NMR relaxation time analysis has been applied to investigate 2-propanol/water mixtures across the entire composition range; while neutron diffraction studies have been carried out at two specific concentrations. Excellent agreement is seen between the techniques with a maximum in both the relative absorption coefficient and the activation energy to molecular motion occurring at ∼90 mol% H₂O. Furthermore, this is the same value at which well-established excess thermodynamic functions exhibit a maximum/minimum. Additionally, both neutron diffraction and THz-TDS have been used to provide estimates of the size of the hydration shell around 2-propanol in solution. Both methods determine that between 4 and 5 H₂O molecules per 2-propanol are found in the 2-propanol/water clusters at 90 mol% H₂O. Based on the acquired data, a description of the structure of 2-propanol/water across the composition range is presented.

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

Laser-driven x-ray and neutron source development for industrial applications of plasma accelerators

Pulsed beams of energetic x-rays and neutrons from intense laser interactions with solid foils are promising for applications where bright, small emission area sources, capable of multi-modal delivery are ideal. Possible end users of laser-driven multi-modal sources are those requiring advanced non-destructive inspection techniques in industry sectors of high value commerce such as aerospace, nuclear and advanced manufacturing. We report on experimental work that demonstrates multi-modal operation of high power laser-solid interactions for neutron and x-ray beam generation. Measurements and Monte Carlo radiation transport simulations show that neutron yield is increased by a factor ∼2 when a 1 mm copper foil is placed behind a 2 mm lithium foil, compared to using a 2 cm block of lithium only. We explore x-ray generation with a 10 picosecond drive pulse in order to tailor the spectral content for radiography with medium density alloy metals. The impact of using >1 ps pulse duration on laser-accelerated electron beam generation and transport is discussed alongside the optimisation of subsequent bremsstrahlung emission in thin, high atomic number target foils. X-ray spectra are deconvolved from spectrometer measurements and simulation data generated using the GEANT4 Monte Carlo code. We also demonstrate the unique capability of laser-driven x-rays in being able to deliver single pulse high spatial resolution projection imaging of thick metallic objects. Active detector radiographic imaging of industrially relevant sample objects with a 10 ps drive pulse is presented for the first time, demonstrating that features of 200 μm size are resolved when projected at high magnification.

Beamed neutron emission driven by laser accelerated light ions

Highly anisotropic, beam-like neutron emission with peak flux of the order of 10^9 n/sr was obtained from light nuclei reactions in a pitcher–catcher scenario, by employing MeV ions driven by subpetawatt laser. The spatial profile of the neutron beam, fully captured for the first time by employing a CR39 nuclear track detector, shows a FWHMdivergence angle of ~70 deg, with a peak flux nearly an order of magnitude higher than the isotropic component elsewhere. The observed beamed flux of neutrons is highly favourable for a wide range of applications, and indeed for further transport and moderation to thermal energies. A systematic study employing various combinations of pitcher–catcher materials indicates the dominant reactions being d(p, n+p)1Hand d(d,n)3He. Albeit insufficient cross-section data are available for modelling, the observed anisotropy in the neutrons’ spatial and spectral profiles are most likely related to the directionality and high energy of the projectile ions.

Neutron Scattering of Aromatic and Aliphatic Liquids

Organic solvents, such as cyclohexane, cyclohexene, methylcyclohexane, benzene and toluene, are widely used as both reagents and solvents in industrial processes. Despite the ubiquity of these liquids, the local structures that govern the chemical properties have not been studied extensively. Herein, we report neutron diffraction measurements on liquid cyclohexane, cyclohexene, methylcyclohexane, benzene and toluene at 298 K to obtain a detailed description of the local structure in these compounds. The radial distribution functions of the centres of the molecules, as well as the partial distribution functions for the double bond for cyclohexene and methyl group for methylcyclohexane and toluene have been calculated. Additionally, probability density functions and angular radial distribution functions were extracted to provide a full description of the local structure within the chosen liquids. Structural motifs are discussed and compared for all liquids, referring specifically to the functional group and aromaticity present in the different liquids.