O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry

The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, and neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer’s and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent, and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here, we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from <i>Clostridium perfringens</i>, <i>Cp</i>OGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize <i>Cp</i>OGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases

Towards the synthesis and characterization of metallocarbohedrenes

Metallocarbohedrene, Ti8C12, has been synthesised in the solid state by resistive evaporation of Ti-covered graphite electrodes in a dynamic helium atmosphere. It forms along with other Ti-containing clusters, all of which are air sensitive. Exposure of the soot to electron-rich molecules increases air stability. The clusters are evaporated thermally yielding chemical ionization mass spectra. The ions do not fragment significantly upon collisional activation. Vacuum evaporation of the soot yields a Ti-containing film. The infrared spectrum of the soot shows characteristic peaks attributable to molecular compounds which change rapidly upon exposure to air

PEG Branched Polymer for Functionalization of Nanomaterials with Ultralong Blood Circulation

Nanomaterials have been actively pursued for biological and medical applications in recent years. Here, we report the synthesis of several new poly(ethylene glycol) grafted branched-polymers for functionalization of various nanomaterials including carbon nanotubes, gold nanoparticles (NP) and gold nanorods (NRs), affording high aqueous solubility and stability for these materials. We synthesize different surfactant polymers based upon poly-(g-glutamic acid) (gPGA) and poly(maleic anhydride-alt-1-octadecene) (PMHC18). We use the abundant free carboxylic acid groups of gPGA for attaching lipophilic species such as pyrene or phospholipid, which bind to nanomaterials via robust physisorption. Additionally, the remaining carboxylic acids on gPGA or the amine-reactive anhydrides of PMHC18 are then PEGylated, providing extended hydrophilic groups, affording polymeric amphiphiles. We show that single-walled carbon nanotubes (SWNTs), Au NPs and NRs functionalized by the polymers exhibit high stability in aqueous solutions at different pHs, at elevated temperatures and in serum. Morever, the polymer-coated SWNTs exhibit remarkably long blood circulation (t1/2 22.1 h) upon intravenous injection into mice, far exceeding the previous record of 5.4 h. The ultra-long blood circulation time suggests greatly delayed clearance of nanomaterials by the reticuloendothelial system (RES) of mice, a highly desired property for in vivo applications of nanomaterials, including imaging and drug delivery

Morphology of supported polymer electrolyte ultra-thin films: a numerical study

Morphology of polymer electrolytes membranes (PEM), e.g., Nafion, inside PEM fuel cell catalyst layers has significant impact on the electrochemical activity and transport phenomena that determine cell performance. In those regions, Nafion can be found as an ultra-thin film, coating the catalyst and the catalyst support surfaces. The impact of the hydrophilic/hydrophobic character of these surfaces on the structural formation of the films has not been sufficiently explored yet. Here, we report about Molecular Dynamics simulation investigation of the substrate effects on the ionomer ultra-thin film morphology at different hydration levels. We use a mean-field-like model we introduced in previous publications for the interaction of the hydrated Nafion ionomer with a substrate, characterized by a tunable degree of hydrophilicity. We show that the affinity of the substrate with water plays a crucial role in the molecular rearrangement of the ionomer film, resulting in completely different morphologies. Detailed structural description in different regions of the film shows evidences of strongly heterogeneous behavior. A qualitative discussion of the implications of our observations on the PEMFC catalyst layer performance is finally proposed

Formulation Development and Characterization of Simvastatin Loaded Long Acting Microspheres

In this work only Physico-chemical characterization and in-vitro evaluation of Simvastatin with various class of polymers were performed. From the in-vitro and stability studies its concluded for a short‑term release requirement upto 16 hrs HPMC polymer loaded simvastatin microspheres can be recommended. For intermediate drug release over a period time of 24 hrs Ethyl cellulose loaded simvastatin microspheres can be recommended. For long term requirement of therapeutic need its promised with Carbopol 940 and PLGA microspheres (upto 48 hrs ).The controlled release behavior attained with the aid of sodium alginate which is incorporated to the formulation to provide the controlled release. The prepared formulation of microsphere is designed to administer via tablet by compression, capsule filling method and injectable formulation (with expected particle size). Based on the above mentioned reasons such microsphere drug delivery system is presently need for successful drug delivery in severe atherosclerotic /hyperlipidemic patients. Further studies can be performed to develop the product extreme consumption of commercial usage

Comparison of the accuracy of root ZX mini and Raypex 6 in detecting apical constriction in human permanent maxillary anterior teeth in the presence of various irrigants using Stereomicroscope: An In Vitro study

AIM OF THE STUDY: To compare the Accuracy of Root ZX mini (J.Morita Corp., Tokyo, Japan) and Raypex6 (VDW, Munich, Germany) apex locators in detecting the apical constriction in human permanent maxillary anterior teeth in the presence of 0.9% saline; 5% sodium hypochlorite; 2% chlorhexidine digluconate, as various intracanal irrigants. MATERIALS AND METHODS: Sixty extracted, straight, single rooted permanent human maxillary anterior teeth were randomly divided into two main groups according to the apex locators tested such as Group1 (n=30,Root ZX mini) Group 2 (n=30,Raypex6). Then each group is further divided into 3 subgroups according to the irrigants used such as Group 1A(n=10, Root ZX mini,0.9% normal saline), Group 1B(n=10,Root ZX mini,5% NaOCl),Group 1C(n=10,Root ZX mini,2% chlorhexidine digluconate), Group2A(n=10,Raypex6,0.9% normal saline), Group2B (n=10,Raypex6, 5% sodium hypochlorite), Group2C (n=10,Raypex6, 2% chlorhexidine digluconate). The teeth were decoronated at the level of cementoenamel junction and the actual length (AL) of each specimen was determined by introducing a size 10 or 15 K file into the canal until its tip emerged through the major apical foramen at ×10 magnification under a stereomicroscope. Each specimen was embedded in the gelatin model and the EALs were tested according to the manufacturer’s instructions. The Electronically measured canal length was recorded by using size 10 or 15 K file(EL). Then the K-files were fixed at the WL determined electronically with GIC. The apical 4 mm of the root was longitudinally sectioned and examined under Stereomicroscope with 30x magnification.The distance from the file tip to the minor diameter is calculated from the Stereomicroscopic images. Independent sample t test and Pearson Chi-Square test was used to statistically analyse the significance of irrigants on the accuracy of apex locators and to compare the accuracy of both apex locators. Significance was set at P<0.05. RESULTS: The overall accuracy of measurements within ±0.5 mm of AL by Root ZX mini was 93.33% and Raypex 6 was 90% respectively. CONCLUSION: Within the limitations of this in vitro study the two electronic apex locators, the Root ZX mini and the Raypex6 were found to have similar accuracy and the use of 5% NaOCl, 0.9% normal saline, or 2% Chlorhexidine as irrigation solutions did not affect the accuracy of the two apex locators in detecting the apical constriction

Lack of evidence for MHC-unrestricted (atypical) recognition of mucin by mucinous pancreatic tumour-reactive T-cells

Cytotoxic T-cells generated against heterologous, mucinous pancreatic tumour cells were shown to recognize mucin in a major histocombatibility complex (MHC)-unrestricted fashion. In contrast, the present study demonstrates a typical allogeneic response of heterologous cytotoxic T-cells established against mucin-expressing pancreatic tumour cells. Heterologous cytotoxic T cells lysed targets that were used as stimulators and other targets that shared human leucocyte antigen (HLA) with the stimulator. These cytotoxic T-cells lysed mucin-expressing stimulator cells but not autologous tumour cells in spite of expressing mucin on their surface. Likewise, tumour-infiltrating CD4+T-cells proliferated against its own tumour cell target, while such T-cells did not respond to heterologous, mucin-expressing pancreatic tumour cells. Culturing heterologous tumour-specific cytotoxic T-cells with purified pancreatic tumour cell-mucin rendered them unresponsive to their target cells. Furthermore, purified mucin did not produce a mucin-specific response in mucinous pancreatic tumour patients' primary T-cells even in the presence of antigen-presenting cells. Our study finds no evidence for MHC-unrestricted recognition of mucin by pancreatic cancer patients' T-cells. © 2000 Cancer Research Campaig

Machine learning for analysis of experimental scattering and spectroscopy data in materials chemistry

The rapid growth of materials chemistry data, driven by advancements in large-scale radiation facilities as well as laboratory instruments, has outpaced conventional data analysis and modelling methods, which can require enormous manual effort. To address this bottleneck, we investigate the application of supervised and unsupervised machine learning (ML) techniques for scattering and spectroscopy data analysis in materials chemistry research. Our perspective focuses on ML applications in powder diffraction (PD), pair distribution function (PDF), small-angle scattering (SAS), inelastic neutron scattering (INS), and X-ray absorption spectroscopy (XAS) data, but the lessons that we learn are generally applicable across materials chemistry. We review the ability of ML to identify physical and structural models and extract information efficiently and accurately from experimental data. Furthermore, we discuss the challenges associated with supervised ML and highlight how unsupervised ML can mitigate these limitations, thus enhancing experimental materials chemistry data analysis. Our perspective emphasises the transformative potential of ML in materials chemistry characterisation and identifies promising directions for future applications. The perspective aims to guide newcomers to ML-based experimental data analysis

A mutant O-GlcNAcase enriches Drosophila developmental regulators

YesProtein O-GlcNAcylation is a reversible post-translational modification of serines/threonines on nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is essential for early embryogenesis. Drosophila melanogaster OGT/supersex combs (sxc) is a polycomb gene, null mutants of which display homeotic transformations and die at the pharate adult stage. However, the identities of the O-GlcNAcylated proteins involved, and the underlying mechanisms linking these phenotypes to embryonic development, are poorly understood. Identification of O-GlcNAcylated proteins from biological samples is hampered by the low stoichiometry of this modification and limited enrichment tools. Using a catalytically inactive bacterial O-GlcNAcase mutant as a substrate trap, we have enriched the O-GlcNAc proteome of the developing Drosophila embryo, identifying, amongst others, known regulators of Hox genes as candidate conveyors of OGT function during embryonic development.Wellcome Trust Investigator Award (110061); MRC grant (MC_UU_12016/5); and Royal Society Research Grant