Plexin B3 promotes neurite outgrowth,interacts homophilically,and interacts with Rin

Background  

Plexins, known to date as receptors of semaphorins, are implicated in semaphorin-mediated axon repulsion and growth cone collapse. However, subtype-specific functions of the majority of the nine members of the mammalian plexin family are largely unknown. In order to investigate functional properties of B-plexins, we analyzed the expression of human and murine plexin B3 and expressed full-length human plexins B2 (B2) and B3 (B3) in NIH-3T3 cells.     

Paramagnetic Particles and PNA Probe for Automated Separation and Electrochemical Detection of Influenza

Considerable efforts have been devoted to the development of rapid and sensitive methods allowing the detection of viral nucleic acid. We herein describe an assay for identification of a specific influenza sequence. The suggested method was based on isolation using paramagnetic particles coupled with electrochemical detection of isolated product. Peptide nucleic acid (PNA) was used as a probe for hybridization and identification of the influenza-derived specific sequence. The use of PNA can show numerous benefits: PNA probe is not degradable by enzymes and the duplex of PNA with RNA/DNA is more thermostable and more resistant to pH changes than DNA/DNA or RNA/RNA duplexes. This PNA probe assay can be applied as a magnetically guidable tool for detection of DNA/RNA samples under different conditions.

     

A two‐step protocol for isolation of influenza A (H7N7) virions and their RNA for PCR diagnostics based on modified paramagnetic particles

Annual epidemics of influenza cause death of hundreds of thousands people and they also have a significant economic impact. Hence, a need for fast and cheap influenza diagnostic method is arising. The conventional methods for an isolation of the viruses are time‐consuming and require expensive instrumentation as well as trained personnel. In this study, we modified the surface of nanomaghemite (γ‐Fe₂O₃) paramagnetic core with tetraethyl orthosilicate and (3‐aminopropyl)triethoxysilane and the resulting particles were utilized for the isolation of H7N7 influenza virions. Consequently, we designed γ‐Fe₂O₃ paramagnetic core modified with calcium tripolyphosphate which was employed for the isolation of viral nucleic acid after virion's lysis. Both of these procedures can be performed rapidly in less than 10 min and, in combination with the RT‐PCR, the whole influenza detection can be shortened to few hours. Moreover, the whole protocol could be easily automated and/or miniaturized, and thus can serve as a basis for use in a lab‐on‐a‐chip device. We assume that magnetic isolation is an exceptional procedure which can significantly accelerate the diagnostic possibilities of a broad spectrum of diseases.     

High sensitivity biosensor measurement based on synchronous detection

The principle of synchronous detection (SD) has been applied to biosensor measurement. SD principle achieves significant increases in the signal to noise ratio, limit of detection and overall measurement robustness. Application of SD in biosensor measurement improves the analysis of the response and avoids the influence of interference/noise produced by stirring, electromagnetic effects and influence of parasitic currents. SD also enables the decomposition of signal to stimulation response and phenomena with long time of response. Second-order phenomena are identifiable in the signal. Linear statistical model was used to develop software for identification of the stimulation signal in the output current. SD was applied to the response signal of a Photosystem II complex (PSII) biosensor. PSII response to light stimulation follows first order kinetics. The inhibition kinetics of PSII has been studied. Kinetic constants of herbicide binding to PSII depend linearly on herbicide concentration and enable measurement of its concentration at low concentrations (linear range for diuron is 10?? to 10?? mM).     

Microfluidic tool based on the antibody-modified paramagnetic particles for detection of 8-hydroxy-2'-deoxyguanosine in urine of prostate cancer patients

Guanosine derivatives are important for diagnosis of oxidative DNA damage including 8-hydroxy-2'-deoxyguanosine (8-OHdG) as one of the most abundant products of DNA oxidation. This compound is commonly determined in urine, which makes 8-OHdG a good non-invasive marker of oxidation stress. In this study, we optimized and tested the isolation of 8-OHdG from biological matrix by using paramagnetic particles with an antibody-modified surface. 8-OHdG was determined using 1-naphthol generated by alkaline phosphatase conjugated with the secondary antibody. 1-Naphthol was determined by stopped flow injection analysis (SFIA) with electrochemical detector using a glassy carbon working electrode and by stationary electrochemical detection using linear sweep voltammetry. A special modular electrochemical SFIA system which needs only 10 μL of sample including working buffer for one analysis was completely designed and successfully verified. The recoveries in different matrices and analyte concentration were estimated. Detection limit (3 S/N) was estimated as 5 pg/mL of 8-OHdG. This method promises to be very easily modified to microfluidic systems as "lab on valve". The optimized method had sufficient selectivity and thus could be used for determination of 8-OHDG in human urine and therefore for estimation of oxidative DNA damage as a result of oxidation stress in prostate cancer patients.     

Paramagnetic Particles and PNA Probe for Automated Separation and Electrochemical Detection of Influenza

Considerable efforts have been devoted to the development of rapid and sensitive methods allowing the detection of viral nucleic acid. We herein describe an assay for identification of a specific influenza sequence. The suggested method was based on isolation using paramagnetic particles coupled with electrochemical detection of isolated product. Peptide nucleic acid (PNA) was used as a probe for hybridization and identification of the influenza-derived specific sequence. The use of PNA can show numerous benefits: PNA probe is not degradable by enzymes and the duplex of PNA with RNA/DNA is more thermostable and more resistant to pH changes than DNA/DNA or RNA/RNA duplexes. This PNA probe assay can be applied as a magnetically guidable tool for detection of DNA/RNA samples under different conditions.     

Fluorescence resonance energy transfer between green fluorescent protein and doxorubicin enabled by DNA nanotechnology

DNA nanotechnology is a rapidly growing research area, where DNA may be used for wide range of applications such as construction of nanodevices serving for large scale of diverse purposes. Likewise a panel of various purified fluorescent proteins is investigated for their ability to emit their typical fluorescence spectra under influence of particular excitation. Hence these proteins may form ideal donor molecules for assembly of fluorescence resonance emission transfer (FRET) constructions. To extend the application possibilities of fluorescent proteins, while using DNA nanotechnology, we developed nanoconstruction comprising green fluorescent protein (GFP) bound onto surface of surface active nanomaghemite and functionalized with gold nanoparticles. We took advantage of natural affinity between gold and thiol moieties, which were modified to bind DNA fragment. Finally we enclosed doxorubicin into fullerene cages. Doxorubicin intercalated in DNA fragment bound on the particles and thus we were able to connect these parts together. Because GFP behaved as a donor and doxorubicin as an acceptor using excitation wavelength for GFP (395 nm) in emission wavelength of doxorubicin (590 nm) FRET was observed. This nanoconstruction may serve as a double‐labeled transporter of doxorubicin guided by force of external magnetic force owing to the presence of nanomaghemite. Further nanomaghemite offers the possibility of using this technology for thermotherapy.     

Paramagnetic Particles Isolation of Influenza Oligonucleotide Labelled with CdS QDs

In this study, we describe hybridization design probes consisting of paramagnetic particles and quantum dots (QDs) with targeted DNA, and their application for detection of avian influenza virus (H5N1). Optical properties of QDs were beneficial, but the main attention was paid to the electroactivity of metal part of QDs and ODNs themselves. Differential pulse voltammetry was used for detection of cadmium(II) ions and square wave voltammetry for detection of cytosine–adenine peak in ODN-SH-Cd complex. It clearly follows from the obtained results that the optimized conditions were temperature of hybridization 25 °C, time of hybridization 35 min, and concentration of ODN-SH-Cd complex 20 μg mL^?1. The detection limit (3 signal/noise) was estimated as 15 ng mL^?1 of ODN-SH-Cd.     

Rapid superparamagnetic‐beads‐based automated immunoseparation of Zn‐proteins from Staphylococcus aureus with nanogram yield

Pathogenic bacteria have become a serious socio‐economic concern. Immunomagnetic separation‐based methods create new possibilities for rapidly recognizing many of these pathogens. The aim of this study was to use superparamagnetic particles‐based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II) containing proteins (Zn‐proteins). The isolated bacteria were immediately purified and disintegrated prior to immunoextraction of Zn‐proteins by superparamagnetic beads modified with chicken anti‐Zn(II) antibody. S. aureus culture was treated with ZnCl₂. Optimal pathogen isolation and subsequent disintegration assay steps were carried out with minimal handling. (i) Optimization of bacteria capturing: Superparamagnetic microparticles composed of human IgG were used as the binding surface for acquiring live S. aureus. The effect of antibodies concentration, ionic strength, and incubation time was concurrently investigated. (ii) Optimization of zinc proteins isolation: pure and intact bacteria isolated by the optimized method were sonicated. The extracts obtained were subsequently analyzed using superparamagnetic particles modified with chicken antibody against zinc(II) ions. (iii) Moreover, various types of bacterial zinc(II) proteins precipitations from particle–surface interactions were tested and associated protein profiles were identified using SDS‐PAGE. Use of a robotic pipetting system sped up sample preparation to less than 4 h. Cell lysis and Zn‐protein extractions were obtained from a minimum of 100 cells with sufficient yield for SDS‐PAGE (tens ng of proteins). Zn(II) content and cell count in the extracts increased exponentially. Furthermore, Zn(II) and proteins balances were determined in cell lysate, extract, and retentate.