Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation can be applied to any SNAP-tag substrate, improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic superresolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

Impermeable SNAP-tag substrates allow exclusive labelling of receptors on the cell membrane for nanoscopy, SiMPull and in vivo use.     

The kinetics and mechanism of the oxidation of DL ethionine and thiourea by potassium ferrate

The kinetics of the reaction between ethionine and thiourea were investigated under pseudo and non-pseudo-first-order conditions. Ethionine was oxidized to the sulfoxide within 500 s and thiourea was oxidized to urea within 10 s. Above a pH of ca. 8.5 the reaction was first-order in the concentration of the organosulfur compound, the hydrogen ions and the ferrate ions, whereas, below this pH, the kinetics were independent of the hydrogen ion concentration. A possible mechanism for both compounds is initial protonation of the ferrate ion followed by the two electron rate-determining step of addition of oxygen to the organosulfur compound. The kinetic parameters for ethionine compare favorably with those for similar compounds, whereas thiourea tends to be more active. The rate constant for the rate-determining step is 4.1 × 10² M^–1 s^–1 for ethionine and 4.1 × 10³ M^–1 s^–1 for thiourea.     

Student performance and attitudes in a collaborative and flipped linear algebra course

Flipped learning is gaining traction in K-12 for enhancing students’ problem-solving skills at an early age; however, there is relatively little large-scale research showing its effectiveness in promoting better learning outcomes in higher education, especially in mathematics classes. In this study, we examined the data compiled from both quantitative and qualitative measures such as item scores on a common final and attitude survey results between a flipped and a traditional Introductory Linear Algebra class taught by two individual instructors at a state university in California in Fall 2013. Students in the flipped class were asked to watch short video lectures made by the instructor and complete a short online quiz prior to each class attendance. The class time was completely devoted to problem solving in group settings where students were prompted to communicate their reasoning with proper mathematical terms and structured sentences verbally and in writing. Examination of the quality and depth of student responses from the common final exam showed that students in the flipped class produced more comprehensive and well-explained responses to the questions that required reasoning, creating examples, and more complex use of mathematical objects. Furthermore, students in the flipped class performed superiorly in the overall comprehension of the content with a 21% increase in the median final exam score. Overall, students felt more confident about their ability to learn mathematics independently, showed better retention of materials over time, and enjoyed the flipped experience.     

Insight into the Inhibition of Drug‐Resistant Mutants of the Receptor Tyrosine Kinase EGFR

Targeting acquired drug resistance represents the major challenge in the treatment of EGFR‐driven non‐small‐cell lung cancer (NSCLC). Herein, we describe the structure‐based design, synthesis, and biological evaluation of a novel class of covalent EGFR inhibitors that exhibit excellent inhibition of EGFR‐mutant drug‐resistant cells. Protein X‐ray crystallography combined with detailed kinetic studies led to a deeper understanding of the mode of inhibition of EGFR‐T790M and provided insight into the key principles for effective inhibition of the recently discovered tertiary mutation at EGFR‐C797S.     

From the Superatom Model to a Diverse Array of Super‐Elements: A Systematic Study of Dopant Influence on the Electronic Structure of Thiolate‐Protected Gold Clusters

The electronic properties of doped thiolate‐protected gold clusters are often referred to as tunable, but their study to date, conducted at different levels of theory, does not allow a systematic evaluation of this claim. Here, using density functional theory, the applicability of the superatomic model to these clusters is critically evaluated, and related to the degree of structural distortion and electronic inhomogeneity in the differently doped clusters, with dopant atoms Pd, Pt, Cu, and Ag. The effect of electron number is systematically evaluated by varying the charge on the overall cluster, and the nominal number of delocalized electrons, employed in the superatomic model, is compared to the numbers obtained from Bader analysis of individual atomic charges. We find that the superatomic model is highly applicable to all of these clusters, and is able to predict and explain the changing electronic structure as a function of charge. However, significant perturbations of the model arise due to doping, due to distortions of the core structure of the Au₁₃[RS(AuSR)₂]₆^? cluster. In addition, analysis of the electronic structure indicates that the superatomic character is distributed further across the ligand shell in the case of the doped clusters, which may have implications for the self‐assembly of these clusters into materials. The prediction of appropriate clusters for such superatomic solids relies critically on such quantitative analysis of the tunability of the electronic structure.     

Two‐Phase Electrospinning to Incorporate Polyelectrolyte Complexes and Growth Factors into Electrospun Chitosan Nanofibers

Growth factors are potent signaling proteins for tissue engineering, but they are susceptible to loss of activity when exposed to solvents used for polymer processing. This work explores preservation of fibroblast growth factor‐2 (FGF‐2) activity in chitosan nanofibers using two‐phase electrospinning via a compound coaxial needle and from a water‐in‐oil emulsion FGF‐2 in aqueous poly(vinyl alcohol) is added on either the inside (A/O) or the outside (O/A) of an organic chitosan phase, using the compound needle. FGF‐2 is further stabilized by complexation to heparin‐based nanoparticles. The emulsion method does not result in detectable incorporation of FGF‐2. The A/O fibers incorporate the highest amount of FGF‐2. Nanoparticle‐stabilized FGF‐2 in A/O nanofibers is most active toward bone‐marrow stromal cells.

     

Intermolecular hydrogen bonds in hetero-complexes of biologically active aromatic ligands: Monte Carlo simulations results

We report results of the Monte Carlo simulations of systems containing heterodimers of biological active ligands and water molecules. The study was designed to identify the possible formation of intermolecular hydrogen bonds in such systems in order to investigate the molecular mechanisms of hetero-association of aromatic ligands in aqueous solution. The geometry optimization and the calculation of the atomic charges of free ligands were carried out at DFT/B3LYP level of theory. Monte Carlo simulations with Metropolis algorithm were used to determine the low energy conformations of heterodimers in water clusters. The analysis of the Monte Carlo simulation results allows us to describe in detail the hydration properties of all investigated heterodimers and to determine the intermolecular hydrogen bonds between the functional donor–acceptor groups for some of hetero-associates under investigation. In the case of heterodimers without intermolecular hydrogen bonds, the additional stabilization of these hetero-complexes can be explained by the formation the water bridges between donor and acceptor groups of the ligands.     

A Disilene Base Adduct with a Dative Si–Si Single Bond

An experimental and theoretical study of the base‐stabilized disilene 1 is reported, which forms at low temperatures in the disproportionation reaction of Si₂Cl₆ or neo‐Si₅Cl₁₂ with equimolar amounts of NMe₂Et. Single‐crystal X‐ray diffraction and quantum‐chemical bonding analysis disclose an unprecedented structure in silicon chemistry featuring a dative Si→Si single bond between two silylene moieties, Me₂EtN→SiCl₂→Si(SiCl₃)₂. The central ambiphilic SiCl₂ group is linked by dative bonds to the amine donor and the bis(trichlorosilyl)silylene acceptor, which leads to push–pull stabilization. Based on experimental and theoretical examinations a formation mechanism is presented that involves an autocatalytic reaction of the intermediately formed anion Si(SiCl₃)₃^? with neo‐Si₅Cl₁₂ to yield 1 .     

Numerical study of light-emitting diode with injected current modulated by designed electrode

Numerical model and procedure are developed to study the output optical performance of light-emitting diode (LED) in which injected current is spatially modulated by mesh-like top metal electrode. The mesh strips have rectangular crossection as in realistic LEDs. The finite element method is applied to obtain three-dimensional distributions of electric potential which are incorporated in the equations for total output power. The numerical procedure is applied to evaluate LED’s total output optical power at different geometric parameters of the electrode: the mesh pitch, the width, and the height of the top mesh-like electrodes. Modeling results demonstrate the effect of mesh pitch variation on the output optical power. In particular, at a certain value of the mesh pitch maximum total output optical power is revealed. The presented approach can be used in the optimization of the LEDs with designed metal electrodes.