Научно-методические основы подготовки студентов профессионально-педагогического вуза в условиях проблемно-модельного обучения

В статье рассматриваются научно-методические основы подготовки студентов профессионально-педагогического вуза в условиях проблемно-модельного обучени

Implementation of at-line capillary zone electrophoresis for fast and reliable determination of adenovirus concentrations in vaccine manufacturing

A CZE method was validated and implemented for fast and accurate in-process determination of adenovirus concentrations of downstream process samples obtained during manufacturing of adenovirus vector-based vaccines. An analytical-quality-by-design approach was embraced for method development, method implementation, and method maintenance. CZE provided separation of adenovirus particles from sample matrix components, such as cell debris, residual DNA and proteins. The intermediate precision of the virus particle concentration was 6.9% RSD and the relative bias was 2.3%. In comparison, the CZE method is intended to replace a quantitative polymerase chain reaction method which requires three replicates in three analytical runs to achieve an intermediate precision of 8.1% RSD. Given that, in addition, the time from sampling till reporting results of the CZE method was less than 2 h, whereas quantitative polymerase chain reaction requires 3 days, it follows that the CZE method enables faster processing times in downstream processing

Control of film morphology by folding hydrogen-bonded oligo(p-phenylenevinylen) polymers in solution

The film morphology of -conjugated oligomers has been controlled by self-assembly in solution. To this end supramolecular hydrogen-bonded systems of oligo(p-phenylenevinylene) (OPV) carrying ureido-s-triazine hydrogen-bonding groups are used. Neutron scattering experiments in dodecane solutions show that columnar stacks are formed. Films with thicknesses on the order of 100 nm are made that have a supramolecular organization resembling the organization present in solution. Uniform rodlike morphological domains range over several hundreds of nanometers as shown by atomic force microscopy. The rodlike morphology of the OPVs was also preserved when blended with a C60 derivative, producing stable photovoltaic devices

Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor

Mouse fibroblast cells overexpressing phosphatidylinositol transfer protein alpha [PI-TPalpha; sense PI-TPalpha (SPIalpha) cells] show a significantly increased rate of proliferation and an extreme resistance toward ultraviolet- or tumor necrosis factor-alpha-induced apoptosis. The conditioned medium (CM) from SPIalpha cells or the neutral lipid extract from CM stimulated the proliferation of quiescent wild-type NIH3T3 cells. CM was also highly effective in increasing resistance toward induced apoptosis in both wild-type cells and the highly apoptosis-sensitive SPIbeta cells (i.e., wild-type cells overexpressing PI-TPbeta). CM from SPIalpha cells grown in the presence of NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, expressed a diminished mitogenic and antiapoptotic activity. This strongly suggests that at least one of the bioactive factor(s) is an eicosanoid. In accordance, SPIalpha cells express enhanced levels of COX-1 and COX-2. The antiapoptotic activity of CM from SPIalpha cells tested on SPIbeta cells was inhibited by approximately 50% by pertussis toxin and suramin as well as by SR141716A, a specific antagonist of the cannabinoid 1 receptor. These inhibitors had virtually no effect on the COX-2-independent antiapoptotic activity of CM from SPIalpha cells. The latter results imply that PI-TPalpha mediates the production of a COX-2-dependent eicosanoid that activates a G-protein-coupled receptor, most probably a cannabinoid 1-like recepto

Fast, selective and quantitative protein profiling of adenovirus-vector based vaccines by ultra-performance liquid chromatography

A method for the quantitative determination of the protein composition of adenovirus-vector based vaccines was developed. The final method used RP-UPLC with UV absorbance detection, a C4 column (300 Å, 1.7 μm, 2.1 × 150 mm), and a water- acetonitrile (ACN) gradient containing trifluoroacetic acid (TFA) as ion-pairing agent. The chromatographic resolution between the various adenovirus proteins was optimized by studying the effect of the TFA concentration and the column temperature, applying a full factorial design of experiments. A reproducible baseline separation of all relevant adenovirus proteins could be achieved within 17 min run time. Samples containing adenovirus particles could be directly injected into the UPLC system without sample pretreatment. The viruses reproducibly dissociate into proteins in the UPLC system upon contact with the mobile phase containing ACN. The new RP-UPLC method was successfully validated for protein profiling and relative quantification of proteins in vaccine products based on adenovirus vector types 26 and 35. The intermediate precision of the relative peak areas of all proteins was between 1% and 14% RSD, except for the peak assigned to protein V (26% RSD). The method proved to be stability indicating with respect to thermal and oxidation stress of the adenovirus-vector based vaccine and was successfully implemented for the characterization of adenovirus-based products

Pre-existing subclones determine radioresistance in rectal cancer organoids

Summary: More than half of all patients with cancer receive radiation therapy, but resistance is commonly observed. Currently, it is unknown whether resistance to radiation therapy is acquired or inherently present. Here, we employed organoids derived from rectal cancer and single-cell whole-genome sequencing to investigate the long-term evolution of subclones in response to radiation. Comparing single-cell whole-genome karyotypes between in-vitro-unirradiated and -irradiated organoids revealed three patterns of subclonal evolution: (1) subclonal persistence, (2) subclonal extinction, and (3) subclonal expansion. Organoids in which subclonal shifts occurred (i.e., expansion or extinction) became more resistant to radiation. Although radioresistant subclones did not share recurrent copy-number alterations that could explain their radioresistance, resistance was associated with reduced chromosomal instability, an association that was also observed in 529 human cancer cell lines. These data suggest that resistance to radiation is inherently present and associated with reduced chromosomal instability

Side Chains Control Dynamics and Self-Sorting in Fluorescent Organic Nanoparticles

To develop fluorescent organic nanoparticles with tailored properties for imaging and sensing, full control over the size, fluorescence, stability, dynamics, and supramolecular organization of these particles is crucial. We have designed, synthesized, and fully characterized 12 nonionic fluorene co-oligomers that formed self-assembled fluorescent nanoparticles in water. In these series of molecules, the ratio of hydrophilic ethylene glycol and hydrophobic alkyl side chains was systematically altered to investigate its role on the above-mentioned properties. The nanoparticles consisting of π-conjugated oligomers containing polar ethylene glycol side chains were less stable and larger in size, while nanoparticles self-assembled from oligomers containing nonpolar pendant chains were more stable, smaller, and generally had a higher fluorescence quantum yield. Furthermore, the dynamics of the molecules between the nanoparticles was enhanced if the number of hydrophilic side chains increased. Energy transfer studies between naphthalene and benzothiadiazole fluorene co-oligomers with the same side chains showed no exchange of molecules between the particles for the apolar molecules. For the more polar systems, the exchange of molecules between nanoparticles took place at room temperature or after annealing. Self-assembled nanoparticles consisting of π-conjugated oligomers having different side chains caused self-sorting, resulting either in the formation of domains within particles or the formation of separate nanoparticles. Our results show that we can control the stability, fluorescence, dynamics, and self-sorting properties of the nanoparticles by simply changing the nature of the side chains of the π-conjugated oligomers. These findings are not only important for the field of self-assembled nanoparticles but also for the construction of well-defined multicomponent supramolecular materials in general

IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105–107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)). The binding affinity for PtdInsP(3), together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP(3) effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules