Effects of iron oxide nanoparticles on the gene expression profiles of cerebral endotheliocytes and astrocytes.

Iron oxide nanoparticles (IONPs) are considered as the most biocompatible magnetic materials suitable for biomedical applications. Nevertheless, there are many evidences of their toxicity for living organisms and partially neurotoxicity. The central nervous system is protected from undesirable substances circulating in the bloodstream by the blood-brain barrier (BBB). And even if being small enough, some nanoparticles could be able to penetrate cell membranes in other cells but will often be delayed by the BBB cells. However, the neurotoxicity of iron oxide is described even in the cases when IONPs should not uptake to the nervous system by experimental design. The aim of this study was to investigate what molecular changes in the cells-components of BBB - endotheliocytes and underlying astrocytes - may be caused by IONPs in the blood vessels of the brain. For this, a two-layer in vitro BBB model was created, consisting of rat cerebral endothelial cells and astrocytes. It was revealed that 100 and 200 mg/L of the nanoparticles induce metabolism alteration in the cells under study. Using RNA-sequencing, the up-regulation of pro-inflammatory chemokines encoding genes and changes in the expression of genes associated with detoxification in the endotheliocytes were demonstrated under the influence of 100 mg/L IONPs.

Starch-Coated Magnetic Iron Oxide Nanoparticles for Affinity Purification of Recombinant Proteins.

Starch-coated magnetic iron oxide nanoparticles have been synthesized by a simple, fast, and cost-effective co-precipitation method with cornstarch as a stabilizing agent. The structural and magnetic characteristics of the synthesized material have been studied by transmission electron microscopy, Mössbauer spectroscopy, and vibrating sample magnetometry. The nature of bonds between ferrihydrite nanoparticles and a starch shell has been examined by Fourier transform infrared spectroscopy. The data on the magnetic response of the prepared composite particles have been obtained by magnetic measurements. The determined magnetic characteristics make the synthesized material a good candidate for use in magnetic separation. Starch-coated magnetic iron oxide nanoparticles have been tested as an affinity sorbent for one-step purification of several recombinant proteins (cardiac troponin I, survivin, and melanoma inhibitory activity protein) bearing the maltose-binding protein as an auxiliary fragment. It has been shown that, due to the highly specific binding of this fragment to the starch shell, the target fusion protein is selectively immobilized on magnetic nanoparticles and eluted with the maltose solution. The excellent efficiency of column-free purification, high binding capacity of the sorbent (100-500 µg of a recombinant protein per milligram of starch-coated magnetic iron oxide nanoparticles), and reusability of the obtained material have been demonstrated.

Silica-Coated Iron Oxide Nanoparticles for DNA Isolation for Molecular Genetic Studies in Hematology.

Aim: To develop magnetic nanoparticles (MNPs) based on iron oxide for DNA isolation from blood cells for quantitative molecular genetic analyses of the V617F mutation in the Januskinase 2 (JAK2) gene. Materials and Methods: MNPs were synthesized by the coprecipitation method and coated with tetraethyl orthosilicate (TEOS). The size and shape of the complexes were estimated using transmission electron microscopy. Twenty blood samples from patients with myeloproliferative disorders were used for DNA isolation with the MNPs. DNA quality and compatibility for molecular genetic studies of the JAK2 V617F mutation were investigated by gel electrophoresis and real-time polymerase chain reaction (RT-PCR). Results: The average amount of DNA isolated from 150 µL of whole blood was 75.2 ng when MNPs were used and 72.5 ng when standard silica sorbent was used. There was no DNA damage observed after interaction with MNPs. RT-PCR demonstrated similar values for the JAK2 V617F mutant DNA ratios in the samples after DNA isolation with MNPs and by standard sorption on silica. Conclusions: MNPs with silicate capsules of sufficient thickness were obtained and the undesirable damaging effect of iron oxides on nucleic acids during isolation from cells were eliminated. Designed MNPs allow obtaining intact DNA for molecular genetic studies using the example of the JAK2 V617F for study.

Interactions of Chemically Synthesized Ferrihydrite Nanoparticles with Human Serum Transferrin: Insights from Fluorescence Spectroscopic Studies.

Human serum transferrin (HST) is a glycoprotein involved in iron transport that may be a candidate for functionalized nanoparticles to bind and target cancer cells. In this study, the effects of the simple and doped with cobalt (Co) and copper (Cu) ferrihydrite nanoparticles (Fh-NPs, Cu-Fh-NPs, and Co-Fh-NPs) were studied by spectroscopic and molecular approaches. Fluorescence spectroscopy revealed a static quenching mechanism for all three types of Fh-NPs. All Fh-NPs interacted with HST with low affinity, and the binding was driven by hydrogen bonding and van der Waals forces for simple Fh-NPs and by hydrophobic interactions for Cu-Fh-NPs and Co-Fh-NPs binding, respectively. Of all samples, simple Fh-NPs bound the most to the HST binding site. Fluorescence resonance energy transfer (FRET) allowed the efficient determination of the energy transfer between HST and NPs and the distance at which the transfer takes place and confirmed the mechanism of quenching. The denaturation of the HST is an endothermic process, both in the case of apo HST and HST in the presence of the three types of Fh-NPs. Molecular docking studies revealed that Fh binds with a low affinity to HST (Ka = 9.17 × 103 M-1) in accord with the fluorescence results, where the interaction between simple Fh-NPs and HST was described by a binding constant of 9.54 × 103 M-1.

Response of Desulfovibrio vulgaris to alkaline stress.

The response of exponentially growing Desulfovibrio vulgaris Hildenborough to pH 10 stress was studied using oligonucleotide microarrays and a study set of mutants with genes suggested by microarray data to be involved in the alkaline stress response deleted. The data showed that the response of D. vulgaris to increased pH is generally similar to that of Escherichia coli but is apparently controlled by unique regulatory circuits since the alternative sigma factors (sigma S and sigma E) contributing to this stress response in E. coli appear to be absent in D. vulgaris. Genes previously reported to be up-regulated in E. coli were up-regulated in D. vulgaris; these genes included three ATPase genes and a tryptophan synthase gene. Transcription of chaperone and protease genes (encoding ATP-dependent Clp and La proteases and DnaK) was also elevated in D. vulgaris. As in E. coli, genes involved in flagellum synthesis were down-regulated. The transcriptional data also identified regulators, distinct from sigma S and sigma E, that are likely part of a D. vulgaris Hildenborough-specific stress response system. Characterization of a study set of mutants with genes implicated in alkaline stress response deleted confirmed that there was protective involvement of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative regulators/histidine kinases (DVU0331 and DVU2580).

Experimental protein mixture for validating tandem mass spectral analysis.

Several methods have been used to identify peptides that correspond to tandem mass spectra. In this work, we describe a data set of low energy tandem mass spectra generated from a control mixture of known protein components that can be used to evaluate the accuracy of these methods. As an example, these spectra were searched by the SEQUEST application against a human peptide sequence database. The numbers of resulting correct and incorrect peptide assignments were then determined. We show how the sensitivity and error rate are affected by the use of various filtering criteria based upon SEQUEST scores and the number of tryptic termini of assigned peptides.