Denis Korneev

Monash University (Australia) · School of Biological Sciences, Clayton

Atomic force microscopy-based single virus particle force spectroscopy was developed using dielectrophoresis for fixing virions at the tip of an atomic force microscope (AFM) probe. Electron microscopic visualization was found to be necessary to prove the deposition of virus particles on the tip of the AFM probe, while fixation of single virions by incubating the tip with a virus suspension proved impossible. Force spectroscopy measurements were performed for the vaccinia virus, influenza virus, and bacteriophage AP22. ForceReader special software was designed for analyzing the force–distance curves.

The method of single-virus force spectroscopy (AFM)

The specific interactions of the pairs laminin binding protein (LBP)–purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus–cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus–cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30–70 pN) sharp globule–coil transition for LBP and LBP–fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus–cell membrane fusion process. Copyright © 2014 John Wiley & Sons, Ltd.

We developed a candidate vaccine CombiHIVvac, which combines the conserved polyepitope immunogens approaches in a novel self-adjuvanted microparticle concept. The experimental visualization of a theoretical predicted formation of microparticles was performed using the method of transmission electron microscopy (TEM) with negative staining. Obtained high resolution images of ComiHIVvac microparticles showed that vaccine structure mimic the size and organization of native viruses and yields insights into how this structure relates to high immunogenisity of vaccine.

Abstract Glycyrrhizin or glycyrrhizic acid (GA) - triterpene glycoside extracted from licorice root - has been intensively studied over the past decade and is considered to be a potential drug delivery system. Glycyrrhizin was found to enhance the therapeutic effect of various drugs; however the detailed mechanism of these effects is still unknown and attracts the attention of researchers. In this work, we have made an attempt to clarify the mechanism of Glycyrrhizin activity on molecular and cellular level. The influence of GA on the functional properties of biomembranes was investigated via NMR spectroscopy and atomic force microscopy (AFM) using human erythrocytes as a model system. GA was shown to increase the permeability (about 60%) and to decrease elasticity modulus of cell membranes (by an order of magnitude) even in micromolar concentrations. Changes on the erythrocyte surface were also detected by AFM. These results could provide a new insight on the mechanism of bioavailability enhancement of some drugs in the presence of glycyrrhizin, as well as the mechanism of its own biological activity. The role of cholesterol-glycyrrhizin binding in the observed effects is also discussed.

Background: The problem of bacterial colonization of implants used in medical practice continues to be relevant regardless of the material of the implant. Particular attention should be given to polymeric implants which are produced "ex tempore" from polymethyl methacrylate, for example, during orthopedic surgical interventions (so-called «bone cement»). The protection of such implants by antibiotic impregnation is subjected to multiple criticisms; therefore, as an alternative to antibiotics, lytic bacteriophages with a number of unique advantages can be used - however, no experimental studies have been published on the possibility of impregnating bacteriophages into polymethyl methacrylate and their antibacterial activity assessment under such conditions. Aims: To evaluate the possibility of physical placement of bacteriophages in polymethylmethacrylate and to characterize the lytic antibacterial effect of two different strains of bacteriophages when impregnated into polymer carrier ex tempore during the polymerization process in in vitro model. Materials and methods: First stage - Atomic force microscopy (AFM) of polymethyl methacrylate samples for medical purposes was used to determine the presence and size of caverns in polymethyl methacrylate after completion of its polymerization at various reaction temperatures (+6...+25°C and +18...+50°C). The second stage was performed in vitro and included an impregnation of two different bacteriophage strains (phage ph20 active against S. aureus and ph57 active against P. aeruginosa) into polymethyl methacrylate during the polymerization process, followed by determination of their antibacterial activity. Results: ACM showed the possibility of bacteriophages placement in the cavities of polymethyl methacrylate - the median of the section and the depth of cavities on the outer surface of the polymer sample polymerized at +18...+50°C were 100.0 and 40.0 nm, respectively, and on the surface of the transverse cleavage of the sample - 120.0 and 100.0 nm, respectively, which statistically did not differ from the geometric dimensions of the caverns of the sample polymerized at a temperature of +6...+25°C. The study of antibacterial activity showed that the ph20 bacteriophage impregnated in polymethyl methacrylate at +6...+25°C lost its effective titer within the first six days after the start of the experiment, while the phage ph57 retained an effective titer for at least 13 days. Conclusion: the study confirmed the possibility of bacteriophages impregnation into medical grade polymethyl methacrylate, maintaining the effective titer of the bacteriophage during phage emission into the external environment, which opens the way for the possible application of this method of bacteriophage delivery in clinical practice. It is also assumed that certain bacteriophages are susceptible to aggressive influences from the chemical components of «bone cement» and/or polymerization reaction products which requires strict selection of bacteriophage strains that could be suitable for this method of delivery.

The hierarchically structured carbon-carbon nanocomposites represent carbon microfibers covered with a layer of carbon nanomaterials. Having properties of both micro- and nano-level, such nanocomposites demonstrate perspectives in polymer reinforcement, membrane technologies and catalysis. The synthesis of the carbon-carbon nanocomposites with controlled properties is a challenging task. This paper describes effect of catalyst deposition technique on the properties of hierarchically structured carbon-carbon nanocomposites. The synthesized samples were analyzed by XRD, SEM, TEM and BET. It was shown how the way of catalyst deposition affects both the yield and structure of the carbon nanofibers grown on the microfiber surface.

The way to produce the nanostructured carbon filaments via H 2 -assisted catalytic decomposition of CF 2 Cl 2 over self-organizing Ni-based catalyst has been reported. The self-organizing 6%Ni/CNM catalyst, where CNM is a carbon nanomaterial, resulted from carbon erosion of bulk Ni-Cr alloy (nichrome) in C 2 H 4 Cl 2 vapors was also shown to be effective for catalytic chemical vapor deposition of CF 2 Cl 2 with formation of bimodal carbon structures. It was demonstrated that interaction of nichrome with CF 2 Cl 2 /H 2 reaction mixture at 600 °C leads to its rapid disintegration caused by carbon erosion to form disperse active Ni-particles catalyzing the growth of carbon filaments. The resulted filamentous carbon material is characterized with high textural parameters.

Catalytic chemical vapor deposition of 1,2-dichlorethane over Ni-based catalysts into carbon nanostructured materials was studied. The catalysts were prepared by mechanochemical activation and by metal dusting of bulk nickel-containing alloy precursors. Model Ni-M alloys, where M is Co, Cu, and Fe, were obtained by coprecipitation technique. Loading of M in the samples was varied in a range of 1–5 at.%. Pure nickel was used a reference. The kinetics of carbon deposition was investigated using flow reactor equipped with McBain balances. The samples of carbon product were characterized by nitrogen adsorption, scanning and transmission electron microscopies. The hydrogen addition into reaction mixture was shown to have opposite effect on both catalytic behavior and carbon yield depending on catalyst’s nature. Segmented structure of carbon filaments formed specifies its developed surface area. Both bulk chlorination of nickel particles and its blockage by dense carbon deposits in the case of mechanochemically prepared samples were suggested to be responsible for rapid deactivation of the catalyst.