Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs

The link between rejection sensitivity and borderline personality disorder:A systematic review and meta-analysis

OBJECTIVE: People with Borderline Personality Disorder (BPD) may experience heightened rejection sensitivity (RS), a disposition developing from repeated childhood rejecting experiences. It is not known whether the full RS model accounts for the cognitive-affective experiences common in BPD. This systematic review extends upon previous reviews, firstly by assessing the link between childhood rejecting experiences and adult RS, and secondly by considering the link between BPD and RS in both non-clinical and clinical samples.METHOD: Two research questions were devised, and searches based on predetermined criteria were conducted using PsycNET, PubMed, SCOPUS, and Web of Science. Data were extracted by one researcher and 20% was inter-rated, with high levels of agreement. Forty-three papers were systematically reviewed, and 31 included in meta-analysis and meta-regression.RESULTS: Studies assessing the link between childhood rejection and RS are limited; however, emotional abuse and neglect appears linked with RS. Pooled effect sizes suggest RS is linked with BPD (r = .326), with strong effect sizes when comparing clinical and control samples (r = .655). Qualitative synthesis suggests this may be mediated by executive control, although further research is required. The small number of studies considering the full RS model with regard to BPD suggests the interaction between emotional abuse and neglect affects rejection sensitivity; however, outcomes are inconsistent.CONCLUSIONS: Childhood rejection, particularly emotional abuse and neglect, appears to be linked to rejection sensitivity, and rejection sensitivity is linked to BPD. However, this may not be linear. Implications for clinical practice and research are discussed.PRACTITIONER POINTS: Rejection sensitivity is consistently linked with BPD, in clinical and non-clinical samples. Supporting mentalization or improved theory of mind may offer a therapeutic target for this disposition. Considering the causes and effects of rejection sensitivity may offer a non-blaming explanation of interpersonal difficulties in BPD and could be utilized as part of formulation and the therapeutic relationship. However, the possible interaction between emotional abuse and neglect and rejection sensitivity suggests rejection sensitivity is not always apparent for people with BPD. Idiosyncratic formulation should consider this. The literature included in the review is limited to Western populations with a high proportion of females, which may limit generalizability. Measures of rejection sensitivity included in the review were restricted to self-report, which may be subject to bias. Furthermore, measures of childhood rejection were retrospective in nature due to the exclusion of child samples. Further research should consider longitudinal and observational study designs.</p

Towards sustainable polymeric nano-carriers and surfactants: facile low temperature enzymatic synthesis of bio-based amphiphilic copolymers in scCO2

We demonstrate that useful bio-based amphiphilic polymers can be produced enzymatically at a mild temperature, in a solvent-free system and using renewably sourced monomers, by exploiting the unique properties of supercritical CO2 (scCO2). We present the use of a novel near-ambient temperature approach to prepare renewable amphiphilic ABA copolymers in scCO2. Bio-based commercially available monomers have been polymerised to prepare chains with targeted molecular weight. The amphiphilic materials were prepared by end-capping the synthesised polymers with methoxy poly(ethylene glycol) (MPEG) chains in a one-pot high pressure reaction utilising Candida Antarctica Lipase B (CaLB) as a catalyst at a temperature as low as 35 °C. The block copolymers are characterised by 1H-NMR, GPC and DSC in order to carefully assess their structural and thermal properties. These polymers form self-assembled aggregates in aqueous environment and these nanostructures are studied through DLS, TEM and UV-Vis. Highly hydrophobic Coumarin-6 was used as a model to prove dispersion in water of lipophilic molecules. Maximum bubble pressure tests demonstrate the reduction in surface tension of these polymers and comparisons are made directly to commercial polymeric non-ionic surfactants

High-pressure rheological analysis of CO2-induced melting point depression and viscosity reduction of poly(ε-caprolactone)

High-pressure rheology has been used to assess the effects of supercritical carbon dioxide (scCO2) on the melting point (Tm) and viscosity of poly (ε-caprolactone) (PCL) over a range of temperatures and pressures up to 300 bar over a wide range of shear rates. Plots of the storage and loss moduli against temperature show a significant shift of Tm to lower temperatures in the presence of CO2, indicating that the polymer crystals melt at temperatures much lower than the ambient pressure Tm. Furthermore, a significant decrease in the viscosity of two PCL grades with different molecular weight (Mn ~ 10 kDa and 80 kDa) was also detected upon increasing the CO2 pressure to 300 bar. Experimental viscosity data were fitted to the Carreau model to quantify the extent of the plasticising effects on the zero-shear viscosity and relaxation time under different conditions. Similar analyses were conducted under high-pressure nitrogen, to compare the effects obtained in the presence of a non-plasticising gas

Assembly and structural analysis of a covalently closed nano-scale DNA cage

The inherent properties of DNA as a stable polymer with unique affinity for partner molecules determined by the specific Watson–Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA-interacting enzymes. More recently, strategies for synthesis of more complex two-dimensional (2D) and 3D DNA structures have emerged. However, the building of such structures is still in progress and more experiences from different research groups and different fields of expertise are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnology. Here we present the design, construction and structural analysis of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double-stranded DNA helices that assembles from eight oligonucleotides with a yield of ∼30%. As demonstrated by Small Angle X-ray Scattering and cryo-Transmission Electron Microscopy analyses the eight-stranded DNA structure has a central cavity larger than the apertures in the surrounding DNA lattice and can be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-molecules to enable their investigation in certain harmful environments or even allow their organization into higher order structures

The ATP-Binding Cassette Proteins of the Deep-Branching Protozoan Parasite Trichomonas vaginalis

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters

Applying electric field modeling to TMS motor mapping

Realistic field calculations in transcranial neurostimulation promise a better insight into the position and extent of the affected brain areas and improve the spatial specificity of stimulation. This is underlined by recent work that demonstrated a strong influence of individual gyral geometry on the strength and distribution of the induced electric field [1]. The field calculations are based on fundamental laws of electrodynamics and rely on conductivity model of individual heads, which are based on segmented structural MR images. For the wider application and the general acceptance of electric field modeling for neurostimulation, two steps seem essential: – a demonstration that the simulated fields correlate with observable effects of neurostimulation (e.g. behavioral or electrophysiological). That is, the simulated fields contribute accurate and relevant information to the experiments; integration electric field modeling into regular TMS experiments in a user-friendly way. Methods and results.– We demonstrate the integration of the Simulation for Non-Invasive Brain Stimulation (SimNIBS, www.simnibs.de) software package with neuronavigation tools for TMS (VISOR from ANT). The coil position and orientation for each TMS pulse is saved by VISOR. These coil positions are automatically converted into the SimNIBS format and used to carry out electric field simulations for each coil position. First results from TMS-motor mapping show how the simulated electric fields can be correlated with the motor evoked potentials of individual muscles. Conclusion.– These results demonstrate that advanced electric field simulations can be applied routinely in experiments involving TMS. In addition, the application to TMS-motor mapping allows validating these simulations in a brain system that is well characterized

One-step purification of the NADH dehydrogenase fragment of the Escherichia coli complex I by means of strep-tag affinity chromatography

AbstractThe proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Complex I of Escherichia coli can be split into three fragments. One of these fragments, the soluble NADH dehydrogenase fragment, represents the electron input part of complex I. It comprises the subunits NuoE, F and G and harbors one flavin mononucleotide and up to six iron-sulfur clusters. Here, we report the one-step purification of this fragment by means of affinity chromatography on StrepTactin. This was achieved by fusing the Strep-tag II peptide to the C-terminus of NuoF or NuoG. Fusion of this peptide to the N-terminus of either NuoE or NuoF disturbed the assembly of the NADH dehydrogenase fragment

Where does TMS Stimulate the Motor Cortex? Combining Electrophysiological Measurements and Realistic Field Estimates to Reveal the Affected Cortex Position

Much of our knowledge on the physiological mechanisms of transcranial magnetic stimulation (TMS) stems from studies which targeted the human motor cortex. However, it is still unclear which part of the motor cortex is predominantly affected by TMS. Considering that the motor cortex consists of functionally and histologically distinct subareas, this also renders the hypotheses on the physiological TMS effects uncertain. We use the finite element method (FEM) and magnetic resonance image-based individual head models to get realistic estimates of the electric field induced by TMS. The field changes in different subparts of the motor cortex are compared with electrophysiological threshold changes of 2 hand muscles when systematically varying the coil orientation in measurements. We demonstrate that TMS stimulates the region around the gyral crown and that the maximal electric field strength in this region is significantly related to the electrophysiological response. Our study is one of the most extensive comparisons between FEM-based field calculations and physiological TMS effects so far, being based on data for 2 hand muscles in 9 subjects. The results help to improve our understanding of the basic mechanisms of TMS. They also pave the way for a systematic exploration of realistic field estimates for dosage control in TMS