In vitro assembly of semi-artificial molecular machine and its use for detection of DNA damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc). This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage. Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks), fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO(4) blunt-ended break (DNase I-type breaks), if T4 DNA ligase is present in the solution. When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO(4) blunt-ended breaks, the ligase reacts with 5'PO(4) DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO(4) blunt-ended DNA breaks. This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.     

Discrimination between invasive Ponto‐Caspian gobies using a PCR‐RFLP method

Accurate identification of invaders, and especially their juveniles and eggs, is a difficult task if several morphologically similar species co‐occur. The aim of the study was to develop and test a rapid and cost‐effective procedure for identification of five species of invasive gobies occurring in the middle Danube basin, namely round goby Neogobius melanostomus, bighead goby Ponticola kessleri, monkey goby Neogobius fluviatilis, racer goby Babka gymnotrachelus and tubenose goby Proterorhinus semilunaris. First, a 708 bp fragment of the cytochrome oxidase I gene was amplified and sequenced for representative samples of these five species. Appropriate sequences of the five species available in public databases were used for in silico analysis. A digestion of the amplified fragment with the BfaI enzyme was found to be suitable for the species identification, as it showed unique restriction patterns for each species. The technique was also successfully applied for fish remains from burbot Lota lota stomachs. Thus the technique could be a useful tool in monitoring biological invasions, especially by identifying specimens that could not be determined on the basis of morphological features. The results demonstrate that the PCR‐RFLP method may in some cases be more reliable for species identification than a standard DNA sequencing.     

Nanoblinker: Brownian Motion Powered Bio-Nanomachine for FRET Detection of Phagocytic Phase of Apoptosis

We describe a new type of bio-nanomachine which runs on thermal noise. The machine is solely powered by the random motion of water molecules in its environment and does not ever require re-fuelling. The construct, which is made of DNA and vaccinia virus topoisomerase protein, can detect DNA damage by employing fluorescence. It uses Brownian motion as a cyclic motor to continually separate and bring together two types of fluorescent hairpins participating in FRET. This bio-molecular oscillator is a fast and specific sensor of 5′OH double-strand DNA breaks present in phagocytic phase of apoptosis. The detection takes 30 s in solution and 3 min in cell suspensions. The phagocytic phase is critical for the effective execution of apoptosis as it ensures complete degradation of the dying cells’ DNA, preventing release of pathological, viral and tumor DNA and self-immunization. The construct can be used as a smart FRET probe in studies of cell death and phagocytosis.     

Small-size magnetically insulated plasma diodes for neutron generation

Results from experimental and theoretical studies of deuteron acceleration in small-size magnetically insulated plasma diodes are presented. The problems of creating accelerating tubes for neutron generation on the basis of magnetically insulated diodes are considered. The prospects of creating small-size neutron generators with neutron fluxes of 10¹⁰–10¹² neutrons/s into the full solid angle are estimated.     

Effects of adaptation to periodic and continuous hypoxia in disorders of electrical stability of the heart in postinfarction cardiosclerosis

The effects of adaptation to intermittent and continuous hypoxia on the electrical stability of the heart were compared in middle altitude conditions and in altitude chamber in Wistar rats with postinfarction cardiosclerosis. It has been shown that both forms of adaptation could restore the heart fibrillation threshold and restrict the ectopic activity in postinfarction cardiosclerosis. Beneficial effects of adaptation to intermittent hypoxia in conditions of the altitude chamber appeared to be more radical.     

Structural and functional characteristics of bacterial biofilms

Data on the structural and functional traits of bacterial biofilms are presented. The structures involved in different stages of biofilm formation are discussed in detail. Techniques for biofilm modeling and visualization are considered. Results of light and electron microscopic investigations are analyzed. Original data on microscopy of biofilms are presented. The possible formation of dormant and nonculturable forms in the biofilm is discussed.     

Formation of biofilms as an example of the social behavior of bacteria

This paper is a brief review of data on bacterial biofilms that occur inside and outside of host organisms. Such biofilms are of great ecological and clinical importance. The role of interspecies communications in the development of bacterial biofilms and infectious diseases is particularly emphasized. Considerable attention is given to the electron microscopic study of biofilms formed by Salmonella typhimurium cells incubated as a broth culture in microtubes without aeration. Bacterial samples taken from the biofilm and planktonic culture grown in the same microtube were comparatively investigated by transmission electron microscopy.