A novel synthetic chemistry approach to linkage-specific ubiquitin conjugation.

Ubiquitination is of great importance as the post-translational modification of proteins with ubiquitin, or ubiquitin chains, facilitates a number of vital cellular processes. Herein we present a facile method of preparing various ubiquitin conjugates under mild conditions using michael acceptors based on dibromo-maleimides and dibromo-pyridazinediones

Controlled delivery of membrane proteins to artificial lipid bilayers by nystatin-ergosterol modulated vesicle fusion

The study of ion channels and other membrane proteins and their potential use as biosensors and drug screening targets require their reconstitution in an artificial membrane. These applications would greatly benefit from microfabricated devices in which stable artificial lipid bilayers can be rapidly and reliably formed. However, the amount of protein delivered to the bilayer must be carefully controlled. A vesicle fusion technique is investigated where composite ion channels of the polyene antibiotic nystatin and the sterol ergosterol are employed to render protein-carrying vesicles fusogenic After fusion with an ergosterol-free artificial bilayer the nystatin-ergosterol channels do not dissociate immediately and thus cause a transient current signal that marks the vesicle fusion event. Experimental pitfalls of this method were identified, the influence of the nystatin and ergosterol concentration on the fusion rate and the shape of the fusion event marker was explored, and the number of different lipid was reduced. Under these conditions, the B-amyloid peptide could be delivered in a controlled manner to a standard planar bilayer. Additionally, the electrical recordings were obtained of vesicles fusing with a planar lipid bilayer in a microfabricated device, demonstrating the suitability of nystatin-ergosterol modulated vesicle fusion for protein delivery within microsystems

Brain activation in response to personalized behavioral and physiological feedback from self-monitoring technology: pilot study

Background: The recent surge in commercially available wearable technology has allowed real-time self-monitoring of behavior (eg, physical activity) and physiology (eg, glucose levels). However, there is limited neuroimaging work (ie, functional magnetic resonance imaging [fMRI]) to identify how people’s brains respond to receiving this personalized health feedback and how this impacts subsequent behavior. Objective: Identify regions of the brain activated and examine associations between activation and behavior. Methods: This was a pilot study to assess physical activity, sedentary time, and glucose levels over 14 days in 33 adults (aged 30 to 60 years). Extracted accelerometry, inclinometry, and interstitial glucose data informed the construction of personalized feedback messages (eg, average number of steps per day). These messages were subsequently presented visually to participants during fMRI. Participant physical activity levels and sedentary time were assessed again for 8 days following exposure to this personalized feedback. Results: Independent tests identified significant activations within the prefrontal cortex in response to glucose feedback compared with behavioral feedback (P<.001). Reductions in mean sedentary time (589.0 vs 560.0 minutes per day, P=.014) were observed. Activation in the subgyral area had a moderate correlation with minutes of moderate-to-vigorous physical activity (r=0.392, P=.043). Conclusion: Presenting personalized glucose feedback resulted in significantly more brain activation when compared with behavior. Participants reduced time spent sedentary at follow-up. Research on deploying behavioral and physiological feedback warrants further investigation

Metadata correction: Brain activation in response to personalized behavioral and physiological feedback from self-monitoring technology: pilot study

[This corrects the article DOI: 10.2196/jmir.8890.]

Challenges in the use of sortase and other peptide ligases for site-specific protein modification.

Site-specific protein modification is a widely-used biochemical tool. However, there are many challenges associated with the development of protein modification techniques, in particular, achieving site-specificity, reaction efficiency and versatility. The engineering of peptide ligases and their substrates has been used to address these challenges. This review will focus on sortase, peptidyl asparaginyl ligases (PALs) and variants of subtilisin; detailing how their inherent specificity has been utilised for site-specific protein modification. The review will explore how the engineering of these enzymes and substrates has led to increased reaction efficiency mainly due to enhanced catalytic activity and reduction of reversibility. It will also describe how engineering peptide ligases to broaden their substrate scope is opening up new opportunities to expand the biochemical toolkit, particularly through the development of techniques to conjugate multiple substrates site-specifically onto a protein using orthogonal peptide ligases

Release of mercury during contact metamorphism of shale: Implications for understanding the impacts of large igneous province volcanism

Elevated mercury (Hg) in sedimentary strata are a widely used tracer for assessing the relationship between large igneous province (LIP) activity and global environmental change. A key unknown in applying this proxy is the extent to which Hg was sourced from contact metamorphism of sedimentary rocks during sill intrusions versus gaseous emissions of the magmas themselves. Here, we investigate Hg behaviour during contact metamorphism of shales. We show loss of 80–99% of the sedimentary Hg in contact aureoles in four case studies covering the interactions around dykes, sills and plutons associated the High Arctic LIP (Sverdrup Basin, Canada), the Karoo LIP (South Africa) and the Skagerrak-centred LIP (Oslo Rift, Norway). A combination of geochemical data and thermal modelling around a dyke from the High Arctic LIP shows 33% Hg volatilization in the aureole at 265–300 °C. The other cases show similar behaviours with significant lowering of organic-bound Hg, more significantly in the innermost 60% of the contact aureoles. We hypothesize that gaseous Hg is transported out of aureoles during metamorphism, together with CH4 and CO2. Furthermore, we estimate the thermogenic Hg mobilization from Karoo LIP aureoles as 72–192 t per km3 of aureole, which is between 1–3 times the estimated volumetric Hg release from Karoo magmas. When scaling our results to the size of the shale portions of the Karoo Basin affected by the LIP and a timescale of 100 kyr of sill emplacement, the average Hg flux is calculated to have been 78–207 t/y with maximum values up to ∼300 t/y. The pulsed nature of intrusive volcanism suggests that this thermogenic Hg flux could have dominated LIP Hg emissions during periods of their life span. Our results demonstrate that the global Hg cycle can be significantly perturbed following LIP-scale sill emplacement into organic-rich sedimentary rocks and our quantification of the emissions based on source-rock analysis provides important information for independent interpretation of the sedimentary Hg record

Evaluating the use of Apo-neocarzinostatin as a cell penetrating protein.

Protein-ligand complex neocarzinostatin (NCS) is a small, thermostable protein-ligand complex that is able to deliver its ligand cargo into live mammalian cells where it induces DNA damage. Apo-NCS is able to functionally display complementarity determining regions loops, and has been hypothesised to act as a cell-penetrating protein, which would make it an ideal scaffold for cell targeting, and subsequent intracellular delivery of small-molecule drugs. In order to evaluate apo-NCS as a cell penetrating protein, we have evaluated the efficiency of its internalisation into live HeLa cells using matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry and fluorescence microscopy. Following incubation of cells with apo-NCS, we observed no evidence of internalisation