Catalytic hyperbranched polymers as enzyme mimics; exploiting the principles of encapsulation and supramolecular chemistry

Nature uses the principles of encapsulation and supramolecular chemistry to bind and orientate substrates within active catalytic sites. Over the years, synthetic chemistry has generated a number of small molecule active site mimics capable of catalysing reactions involving bound substrates. Another approach uses larger molecules that better represent an enzymes globular structure. These molecules mimic an enzymes structure by incorporating binding/catalytic sites within the globular structure of the polymer. As such, the electronic and steric properties around the binding/catalytic site(s) can be controlled and fine-tuned. One class of polymer that is particularly adept at mimicking the globular structure of enzymes are dendritic polymers. This review will concentrate on the use of hyperbranched polymers as synthetic enzyme mimics.     

Targeted Polymersome Delivery of siRNA Induces Cell Death of Breast Cancer Cells Dependent upon Orai3 Protein Expression

Polymersomes, polymeric vesicles that self-assemble in aqueous solutions from block copolymers, have been avidly investigated in recent years as potential drug delivery agents. Past work has highlighted peptide-functionalized polymersomes as a highly promising targeted delivery system. However, few reports have investigated the ability of polymersomes to operate as gene delivery agents. In this study, we report on the encapsulation and delivery of siRNA inside of peptide-functionalized polymersomes composed of poly(1,2-butadiene)-b-poly(ethylene oxide). In particular, PR_b peptide-functionalized polymer vesicles are shown to be a promising system for siRNA delivery. PR_b is a fibronectin mimetic peptide targeting specifically the α(5)β(1) integrin. The Orai3 gene was targeted for siRNA knockdown, and PR_b-functionalized polymer vesicles encapsulating siRNA were found to specifically decrease cell viability of T47D breast cancer cells to a certain extent, while preserving viability of noncancerous MCF10A breast cells. siRNA delivery by PR_b-functionalized polymer vesicles was compared to that of a current commercial siRNA transfection agent, and produced less dramatic decreases in cancer cell viability, but compared favorably in regards to the relative toxicity of the delivery systems. Finally, delivery and vesicle release of a fluorescent encapsulate by PR_b-functionalized polymer vesicles was visualized by confocal microscopy, and colocalization with cellular endosomes and lysosomes was assessed by organelle staining. Polymersomes were observed to primarily release their encapsulate in the early endosomal intracellular compartments, and data may suggest some escape to the cytosol. These results represent a promising first generation model system for targeted delivery of siRNA.     

Corylucinine, a new alkaloid from Corydalis cava (Fumariaceae), and its cholinesterase activity

A new benzyldihydroisoquinoline alkaloid (1) was isolated from the tubers of Corydalis cava and named corylucinine. Additionally, 8-trichloromethyl-7,8-dihydropalmatine (2), an isolation artifact of tetrahydropalmatine, was obtained. The structures were established by spectroscopic (including 2D NMR and optical rotation) and HR-ESI-MS methods. Both compounds were tested for human blood acetylcholinesterase (HuAChE) and human plasma butyrylcholinesterase (HuBuChE) inhibitory activity. In comparison with the used standards, both compounds showed only moderate inhibitory activity against HuAChE (IC50,. HuAChE = 127.6 +/- 5.2 microM for 1, and IC50, HuAChE = 82.9 +/- 3.9 microM for 2) and none against HuBuChE.     

Increased imaging speed and force sensitivity for bio-applications with small cantilevers using a conventional AFM setup

In this study, we demonstrate the increased performance in speed and sensitivity achieved by the use of small AFM cantilevers on a standard AFM system. For this, small rectangular silicon oxynitride cantilevers were utilized to arrive at faster atomic force microscopy (AFM) imaging times and more sensitive molecular recognition force spectroscopy (MRFS) experiments. The cantilevers we used had lengths between 13 and 46μm, a width of about 11μm, and a thickness between 150 and 600nm. They were coated with chromium and gold on the backside for a better laser reflection. We characterized these small cantilevers through their frequency spectrum and with electron microscopy. Due to their small size and high resonance frequency we were able to increase the imaging speed by a factor of 10 without any loss in resolution for images from several μm scansize down to the nanometer scale. This was shown on bacterial surface layers (s-layer) with tapping mode under aqueous, near physiological conditions and on nuclear membranes in contact mode in ambient environment. In addition, we showed that single molecular forces can be measured with an up to 5 times higher force sensitivity in comparison to conventional cantilevers with similar spring constants.     

Probing electrostatic interactions and structural changes in highly charged protein polyanions by conformer-selective photoelectron spectroscopy

We have recorded the first conformer-selective photoelectron spectra of a protein polyanion in the gas-phase. Bovine cytochrome c protein was studied in 8 different negative charge states ranging from 5- to 12-. Electron binding energies were extracted for all charge states and used as a direct probe of intramolecular Coulomb repulsion. Comparison of experimental results with simulations shows that the experimental outcome can be reproduced with a simple electrostatic model. Energetics are consistent with a structural transition from a folded to an unfolded conformational state of the protein as the number of charges increases. Furthermore, the additional ion-mobility data show that the onset of unfolding can be assigned to charge state 6- where three conformers can be distinguished.     

Recognition of beer brand based on multivariate analysis of volatile fingerprint

Automated head-space solid-phase microextraction (HS-SPME)-based sampling procedure, coupled to gas chromatography–time-of-flight mass spectrometry (GC–TOFMS), was developed and employed for obtaining of fingerprints (GC profiles) of beer volatiles. In total, 265 speciality beer samples were collected over a 1-year period with the aim to distinguish, based on analytical (profiling) data, (i) the beers labelled as Rochefort 8; (ii) a group consisting of Rochefort 6, 8, 10 beers; and (iii) Trappist beers. For the chemometric evaluation of the data, partial least squares discriminant analysis (PLS-DA), linear discriminant analysis (LDA), and artificial neural networks with multilayer perceptrons (ANN-MLP) were tested. The best prediction ability was obtained for the model that distinguished a group of Rochefort 6, 8, 10 beers from the rest of beers. In this case, all chemometric tools employed provided 100% correct classification. Slightly worse prediction abilities were achieved for the models “Trappist vs. non-Trappist beers” with the values of 93.9% (PLS-DA), 91.9% (LDA) and 97.0% (ANN-MLP) and “Rochefort 8 vs. the rest” with the values of 87.9% (PLS-DA) and 84.8% (LDA) and 93.9% (ANN-MLP). In addition to chromatographic profiling, also the potential of direct coupling of SPME (extraction/pre-concentration device) with high-resolution TOFMS employing a direct analysis in real time (DART) ion source has been demonstrated as a challenging profiling approach.     

Determination of insoluble avian eggshell matrix proteins

The organic components of bones and other mineralized tissues have a high impact on the organization and deposition of calcium, and consequently influence the mechanical properties of those tissues. The extractable proteins of avian eggshells have been studied extensively and many of them have been identified; insoluble (non-extractable) proteins have been sparsely studied, however. In the work discussed in this paper we studied EDTA-insoluble proteins by gradual decalcification of eggshell with EDTA. The insoluble proteinaceous films were chemically treated with cyanogen bromide and the mixtures of large fragments obtained were gradually precipitated with salt. The separated fractions were digested with trypsin and analyzed by HPLC–MS–MS (ion trap mass spectrometer). Analysis of the entire eggshell matrix (without precipitation steps) only enabled 6 proteins to be determined (ovocalyxins 32 and 36, ovocleidin 17 and 116, clusterin, and ovalbumin). Pretreatment of the individual eggshell layers and gradual precipitation with salt markedly increased the number of proteins identified – 28 proteins were determined. We identified for the first time collagens I (two chains) and III in the eggshell matrix, and Kunitz-like protease inhibitor as a major shell matrix protein. Besides the above mentioned proteins we can also mention EDIL3, fibronectin, sulfhydryl oxidase, tubulin alpha 1, lysozyme, Dickkopf-related protein 3, keratins, and ovotransferrin. The relative abundances of proteins in all eggshell layers were determined using the exponentially modified protein abundance index (emPAI). In the cuticle layer seven proteins were identified, whereas 16 proteins were described in the palisade layer and 23 in the mammillary layer.     

A rationale of the Baeyer-Villiger oxidation of cyclohexanone to ε-caprolactone with hydrogen peroxide: unprecedented evidence for a radical mechanism controlling reactivity

We demonstrate, for the first time, in the Baeyer-Villiger oxidation of cyclohexanone with aqueous hydrogen peroxide under conditions aimed at obtaining ε-caprolactone, that a thermally activated radical reaction leads to the concurrent formation of adipic acid, even when a stoichiometric amount of the oxidant is used. In fact, ε-caprolactone is the primary reaction product, but it is more reactive than cyclohexanone, and quickly undergoes consecutive transformations. When titanium silicalite-1 (TS-1) is used as a catalyst, the high concentration of hydroxy radicals within its pores accelerates the reaction rates, and the consecutive formation of adipic acid (and of lighter diacids as well) becomes largely kinetically preferred. The proper choice of the solvent, which also may act as a radical scavenger, both without catalyst and with TS-1, is a powerful tool for controlling the rates of the various reactions involved.     

Perturbation solutions of weakly compressible Newtonian Poiseuille flows with Navier slip at the wall

We consider both the planar and axisymmetric steady, laminar Poiseuille flows of a weakly compressible Newtonian fluid assuming that slip occurs along the wall following Navier’s slip equation and that the density obeys a linear equation of state. A perturbation analysis is performed in terms of the primary flow variables using the dimensionless isothermal compressibility as the perturbation parameter. Solutions up to the second order are derived and compared with available analytical results. The combined effects of slip, compressibility, and inertia are discussed with emphasis on the required pressure drop and the average Darcy friction factor.