Photography of corneal contact lens optic sections and fluorescein patterns

Photography of corneal contact lens optic sections and fluorescein pattern

Probing the (H3-H4)(2) histone tetramer structure using pulsed EPR spectroscopy combined with site-directed spin labelling

The (H3-H4)2 histone tetramer forms the central core of nucleosomes and, as such, plays a prominent role in assembly, disassembly and positioning of nucleosomes. Despite its fundamental role in chromatin, the tetramer has received little structural investigation. Here, through the use of pulsed electron-electron double resonance spectroscopy coupled with site-directed spin labelling, we survey the structure of the tetramer in solution. We find that tetramer is structurally more heterogeneous on its own than when sequestered in the octamer or nucleosome. In particular, while the central region including the H3-H3′ interface retains a structure similar to that observed in nucleosomes, other regions such as the H3 αN helix display increased structural heterogeneity. Flexibility of the H3 αN helix in the free tetramer also illustrates the potential for post-translational modifications to alter the structure of this region and mediate interactions with histone chaperones. The approach described here promises to prove a powerful system for investigating the structure of additional assemblies of histones with other important factors in chromatin assembly/fluidity

The spatial effect of protein deuteration on nitroxide spin-label relaxation:implications for EPR distance measurement

This work was supported by a Wellcome Trust Senior Fellowship (095062) to T.O.-H. The Authors would also like to acknowledge funding from The MRC – United Kingdom, Grant G1100021.Pulsed electron-electron double resonance (PELDOR) coupled with site-directed spin labeling is a powerful technique for the elucidation of protein or nucleic acid, macromolecular structure and interactions. The intrinsic high sensitivity of electron paramagnetic resonance enables measurement on small quantities of bio-macromolecules, however short relaxation times impose a limit on the sensitivity and size of distances that can be measured using this technique. The persistence of the electron spin-echo, in the PELDOR experiment, is one of the most crucial limitations to distance measurement. At a temperature of around 50 K one of the predominant factors affecting persistence of an echo, and as such, the sensitivity and measurable distance between spin labels, is the electron spin echo dephasing time (Tm). It has become normal practice to use deuterated solvents to extend Tm and recently it has been demonstrated that deuteration of the underlying protein significantly extends Tm. Here we examine the spatial effect of segmental deuteration of the underlying protein, and also explore the concentration and temperature dependence of highly deuterated systems.Publisher PDFPeer reviewe

The histone chaperones Nap1 and Vps75 bind histones H3 and H4 in a tetrameric conformation

Histone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly

Effect of chlortetracycline on Salmonella and the fecal flora of swine

The goals of this study were to determine the impact of sub-therapeutic chlortetracycline in market swine diets on 1) the prevalence and antimicrobial resistance of Salmonella enterica 2) antimicrobial resistance of the aerobic Gram negative fecal flora. There was no significant difference in the prevalence or antimicrobial resistance of S. enterica isolates. For the gram-negative fecal flora, there was a statistically significant difference (p\u3c0.05) between treatment groups for the frequency of antimicrobial resistance in the gram negative flora with pigs receiving chlortetracycline having a greater frequency of isolates resistant tetracycline, gentamicin, and ceftriaxone, and a lesser proportion of isolates resistant to ampicillin

The Impact of Wage Bargaining Regime on Firm-Level Competitiveness and Wage Inequality: The Case of Ireland. ESRI WP266. December 2008

This paper uses a linked employer-employee dataset to analyse the impact of institutional wage bargaining regimes on levels of average labour costs and within firm wage dispersion in private sector companies in Ireland. The results show that while centralised bargaining reduced labour costs within both the indigenous and foreign-owned sectors, the relative advantage was greater among foreign-owned firms. The analysis suggests that there are potentially large competitiveness gains to multinational companies that choose to locate in countries implementing a centralised bargaining system. Furthermore, the results provide additional support to the view that collective bargaining reduces within firm wage inequality

Inhibition of Oncogenic Wnt Signaling through Direct Targeting of β-Catenin

Aberrant activation of signaling by the Wnt pathway is strongly implicated in the onset and progression of numerous types of cancer. Owing to the persistent dependence of these tumors on Wnt signaling for growth and survival, inhibition of this pathway is considered an attractive mechanism-based therapeutic approach. Oncogenic activation of Wnt signaling can ensue from a variety of distinct aberrations in the signaling pathway, but most share the common feature of causing increased cellular levels of β-catenin by interfering with its constitutive degradation. β-Catenin serves as a central hub in Wnt signaling by engaging in crucial protein–protein interactions with both negative and positive effectors of the pathway. Direct interference with these protein–protein interactions is a biologically compelling approach toward suppression of β-catenin hyperactivity, but such interactions have proven intransigent with respect to small-molecule targeting. Hence β-catenin remains an elusive target for translational cancer therapy. Here we report the discovery of a hydrocarbon-stapled peptide that directly targets β-catenin and interferes with its ability to serve as a transcriptional coactivator for T-cell factor (TCF) proteins, the downstream transcriptional regulators of the Wnt pathway.Chemistry and Chemical BiologyStem Cell and Regenerative Biolog

Modelling multi-protein complexes using PELDOR distance measurements for rigid body minimisation experiments using XPLOR-NIH

Crystallographic and NMR approaches have provided a wealth of structural information about protein domains. However, often these domains are found as components of larger multi domain polypeptides or complexes. Orienting domains within such contexts can provide powerful new insight into their function. The combination of site specific spin labelling and Pulsed Electron Double Resonance (PELDOR) provide a means of obtaining structural measurements that can be used to generate models describing how such domains are oriented. Here we describe a pipeline for modelling the location of thio-reactive nitroxyl spin locations to engineered sties on the histone chaperone Vps75. We then use a combination of experimentally determined measurements and symmetry constraints to model the orientation in which homodimers of Vps75 associate to form homotetramers using the XPLOR-NIH platform. This provides a working example of how PELDOR measurements can be used to generate a structural model

The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution

NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pulsed electron–electron double resonance approaches were used to show that both Vps75 and Nap1 adopt ring-shaped tetrameric conformations in solution. This suggests that the formation of homotetramers is a common feature of NAP-1 fold histone chaperones. The tetramerisation of NAP-1 fold histone chaperones may act to shield acidic surfaces in the absence of histone cargo thus providing a ‘self-chaperoning’ type mechanism

Photochemical Fingerprinting Is a Sensitive Probe for the Detection of Synthetic Cannabinoid Receptor Agonists; Toward Robust Point-of-Care Detection

With synthetic cannabinoid receptor agonist (SCRA) use still prevalent across Europe and structurally advanced generations emerging, it is imperative that drug detection methods advance in parallel. SCRAs are a chemically diverse and evolving group, which makes rapid detection challenging. We have previously shown that fluorescence spectral fingerprinting (FSF) has the potential to provide rapid assessment of SCRA presence directly from street material with minimal processing and in saliva. Enhancing the sensitivity and discriminatory ability of this approach has high potential to accelerate the delivery of a point-of-care technology that can be used confidently by a range of stakeholders, from medical to prison staff. We demonstrate that a range of structurally distinct SCRAs are photochemically active and give rise to distinct FSFs after irradiation. To explore this in detail, we have synthesized a model series of compounds which mimic specific structural features of AM-694. Our data show that FSFs are sensitive to chemically conservative changes, with evidence that this relates to shifts in the electronic structure and cross-conjugation. Crucially, we find that the photochemical degradation rate is sensitive to individual structures and gives rise to a specific major product, the mechanism and identification of which we elucidate through density-functional theory (DFT) and time-dependent DFT. We test the potential of our hybrid "photochemical fingerprinting"approach to discriminate SCRAs by demonstrating SCRA detection from a simulated smoking apparatus in saliva. Our study shows the potential of tracking photochemical reactivity via FSFs for enhanced discrimination of SCRAs, with successful integration into a portable device.</p