Field-Based Planetary Protection Operations for Melt Probes: Validation of Clean Access into the Blood Falls, Antarctica, Englacial Ecosystem.

Subglacial environments on Earth offer important analogs to Ocean World targets in our solar system. These unique microbial ecosystems remain understudied due to the challenges of access through thick glacial ice (tens to hundreds of meters). Additionally, sub-ice collections must be conducted in a clean manner to ensure sample integrity for downstream microbiological and geochemical analyses. We describe the field-based cleaning of a melt probe that was used to collect brine samples from within a glacier conduit at Blood Falls, Antarctica, for geomicrobiological studies. We used a thermoelectric melting probe called the IceMole that was designed to be minimally invasive in that the logistical requirements in support of drilling operations were small and the probe could be cleaned, even in a remote field setting, so as to minimize potential contamination. In our study, the exterior bioburden on the IceMole was reduced to levels measured in most clean rooms, and below that of the ice surrounding our sampling target. Potential microbial contaminants were identified during the cleaning process; however, very few were detected in the final englacial sample collected with the IceMole and were present in extremely low abundances (∼0.063% of 16S rRNA gene amplicon sequences). This cleaning protocol can help minimize contamination when working in remote field locations, support microbiological sampling of terrestrial subglacial environments using melting probes, and help inform planetary protection challenges for Ocean World analog mission concepts.

Antimicrobial and wound healing properties of a bacterial cellulose based material containing B. subtilis cells.

A biocomposite composed of bacterial cellulose (BC) gel-film and Bacillus subtilis (BS) cells was obtained and characterized with a view to future biomedical applications. The inclusion of functional ingredient (1010/g viable BS cells) in the composite was carried out by their joint aggregation with the BC gel-film. Immobilized BS cells displayed high antagonistic activity towards causative agents of wound infections such as Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa. Application of the BC/BS-biocomposite for the treatment of excision wounds, performed on laboratory animals, stimulated reparative processes and shortened the healing time. Possible mechanisms of the wound-healing effect of BC/BS gel films are discussed. In this work we claim that the developed BC/BS-material can be positioned as a universal wound coating and sanitary-hygienic product.

Synthesis, characterization, in vitro biocompatibility and antibacterial properties study of nanocomposite materials based on hydroxyapatite-biphasic ZnO micro- and nanoparticles embedded in Alginate matrix.

The paper presents the results of studies of biocompatibility and antibacterial properties of multiphase nanocomposite materials based on HA-Alg-ZnO (hydroxyapatite­sodium alginate-biphasic zinc oxide) and HA-ZnO (hydroxyapatite­zinc oxide), which were synthesized from the analytically pure calcium nitrate tetrahydrate, ammonium hydrophosphate, hydrous ammonia, zinc nitrate hexahydrate and calcium chloride. The samples' antimicrobial activity assessment was carried out on Gram-negative (E. coli, P. aeruginosa) and Gram-positive bacteria (S. aureus and S. epidermidis) test cultures by the co-incubation and modified "agar diffusion" methods. The murine fibroblast cells were used for the biocompatibility tests and cytotoxicity evaluation. It was shown that synthesized nanocomposite material has a multiphase nanoscale architecture, where ZnO nanocrystals are represented by two lattices: cubic and hexagonal. The possible explanation of ZnO nanocrystals' phase transition is given. At the same time, a partial replacement of Ca2+ ions by Zn2+ ions in the HA lattice possibly occurs due to processing of composite by US radiation. The replacement was evidenced by the non-stoichiometric Ca/P ratio < 2.16, OPO lines' shifting on FTIR spectrum and TEM analysis. The studied composite demonstrate a pronounced antibacterial activity due to the incorporation of ZnO particles into sodium alginate and moistened powder of hydroxyapatite. Both forms of HA-ZnO (suspension) and HA-Alg-ZnO (beads) are biocompatible. An interpretation of the process of Zn ions' embedding into hydroxyapatite and alginate matrix is given, as well as their influence on the biomimetic composite properties is discussed in details. STATEMENT OF SIGNIFICANCE: A number of studies have shown that Zn effectively inhibits the growth and development of bacteria and yeast fungi. Zinc plays an important role in the creation of new antimicrobial agents, and zinc-doped hydroxyapatite will find further application in biomedicine. In this regard, the phase states of zinc oxide, as well as the processes of calcium replacement by zinc in calcium apatite and in alginate should be explored fully. Nowadays we have lack of information and the study's results about those interactions. The present study provides data of the multiphase morphology, antimicrobial activity, biocompatibility and cytotoxicity of the biomimetic nanostructured composite materials, such as sodium alginate/hydroxyapatite/ZnO based granules and hydroxyapatite/ZnO based hydrogel, and the establishing Zn ions' behavior patterns with another composite components.

Thermal fluctuations of haemoglobin from different species: adaptation to temperature via conformational dynamics.

Thermodynamic stability, configurational motions and internal forces of haemoglobin (Hb) of three endotherms (platypus, Ornithorhynchus anatinus; domestic chicken, Gallus gallus domesticus and human, Homo sapiens) and an ectotherm (salt water crocodile, Crocodylus porosus) were investigated using circular dichroism, incoherent elastic neutron scattering and coarse-grained Brownian dynamics simulations. The experimental results from Hb solutions revealed a direct correlation between protein resilience, melting temperature and average body temperature of the different species on the 0.1 ns time scale. Molecular forces appeared to be adapted to permit conformational fluctuations with a root mean square displacement close to 1.2 Å at the corresponding average body temperature of the endotherms. Strong forces within crocodile Hb maintain the amplitudes of motion within a narrow limit over the entire temperature range in which the animal lives. In fully hydrated powder samples of human and chicken, Hb mean square displacements and effective force constants on the 1 ns time scale showed no differences over the whole temperature range from 10 to 300 K, in contrast to the solution case. A complementary result of the study, therefore, is that one hydration layer is not sufficient to activate all conformational fluctuations of Hb in the pico- to nanosecond time scale which might be relevant for biological function. Coarse-grained Brownian dynamics simulations permitted to explore residue-specific effects. They indicated that temperature sensing of human and chicken Hb occurs mainly at residues lining internal cavities in the ß-subunits.

Contractile tension and beating rates of self-exciting monolayers and 3D-tissue constructs of neonatal rat cardiomyocytes.

The CellDrum technology (The term 'CellDrum technology' includes a couple of slightly different technological setups for measuring lateral mechanical tension in various types of cell monolayers or 3D-tissue constructs) was designed to quantify the contraction rate and mechanical tension of self-exciting cardiac myocytes. Cells were grown either within flexible, circular collagen gels or as monolayer on top of respective 1-mum thin silicone membranes. Membrane and cells were bulged outwards by air pressure. This biaxial strain distribution is rather similar the beating, blood-filled heart. The setup allowed presetting the mechanical residual stress level externally by adjusting the centre deflection, thus, mimicking hypertension in vitro. Tension was measured as oscillating differential pressure change between chamber and environment. A 0.5-mm thick collagen-cardiac myocyte tissue construct induced after 2 days of culturing (initial cell density 2 x 10(4) cells/ml), a mechanical tension of 1.62 +/- 0.17 microN/mm(2). Mechanical load is an important growth regulator in the developing heart, and the orientation and alignment of cardiomyocytes is stress sensitive. Therefore, it was necessary to develop the CellDrum technology with its biaxial stress-strain distribution and defined mechanical boundary conditions. Cells were exposed to strain in two directions, radially and circumferentially, which is similar to biaxial loading in real heart tissues. Thus, from a biomechanical point of view, the system is preferable to previous setups based on uniaxial stretching.

From powder to solution: hydration dependence of human hemoglobin dynamics correlated to body temperature.

A transition in hemoglobin (Hb), involving partial unfolding and aggregation, has been shown previously by various biophysical methods. The correlation between the transition temperature and body temperature for Hb from different species, suggested that it might be significant for biological function. To focus on such biologically relevant human Hb dynamics, we studied the protein internal picosecond motions as a response to hydration, by elastic and quasielastic neutron scattering. Rates of fast diffusive motions were found to be significantly enhanced with increasing hydration from fully hydrated powder to concentrated Hb solution. In concentrated protein solution, the data showed that amino acid side chains can explore larger volumes above body temperature than expected from normal temperature dependence. The body temperature transition in protein dynamics was absent in fully hydrated powder, indicating that picosecond protein dynamics responsible for the transition is activated only at a sufficient level of hydration. A collateral result from the study is that fully hydrated protein powder samples do not accurately describe all aspects of protein picosecond dynamics that might be necessary for biological function.

Hemoglobin dynamics in red blood cells: correlation to body temperature.

A transition in hemoglobin behavior at close to body temperature has been discovered recently by micropipette aspiration experiments on single red blood cells (RBCs) and circular dichroism spectroscopy on hemoglobin solutions. The transition temperature was directly correlated to the body temperatures of a variety of species. In an exploration of the molecular basis for the transition, we present neutron scattering measurements of the temperature dependence of hemoglobin dynamics in whole human RBCs in vivo. The data reveal a change in the geometry of internal protein motions at 36.9 degrees C, at human body temperature. Above that temperature, amino acid side-chain motions occupy larger volumes than expected from normal temperature dependence, indicating partial unfolding of the protein. Global protein diffusion in RBCs was also measured and the findings compared favorably with theoretical predictions for short-time self-diffusion of noncharged hard-sphere colloids. The results demonstrated that changes in molecular dynamics in the picosecond time range and angstrom length scale might well be connected to a macroscopic effect on whole RBCs that occurs at body temperature.

Molecular processes in biological thermosensation.

Since thermal gradients are almost everywhere, thermosensation could represent one of the oldest sensory transduction processes that evolved in organisms. There are many examples of temperature changes affecting the physiology of living cells. Almost all classes of biological macromolecules in a cell (nucleic acids, lipids, proteins) can present a target of the temperature-related stimuli. This review discusses some features of different classes of temperature-sensing molecules as well as molecular and biological processes that involve thermosensation. Biochemical, structural, and thermodynamic approaches are applied in the paper to organize the existing knowledge on molecular mechanisms of thermosensation. Special attention is paid to the fact that thermosensitive function cannot be assigned to any particular functional group or spatial structure but is rather of universal nature. For instance, the complex of thermodynamic, structural, and functional features of hemoglobin family proteins suggests their possible accessory role as "molecular thermometers".

Decrease in extracellular collagen crosslinking after NMR magnetic field application in skin fibroblasts.

Although biological effects of electromagnetic fields were investigated intensively, there is still no agreement on the significance of their effects. The underlying mechanisms and therapeutic importance are still mostly unknown too. In this study, primary cultures of human dermal fibroblasts were exposed to magnetic field at nuclear magnetic resonance (NMR) conditions for in total 5 days and 4 h/day. Among the investigated parameters were: cell proliferation rate, cell morphology, total protein concentration as well as content of skin-specific collagen types I, III, IV. NMR exposure induced distinct changes both in cellular and extracellular components. The extracellular matrix (ECM) of NMR-exposed cells had less cross-linked collagen. In particular, the increase of collagen of the soluble fraction was at 17.2 +/- 2.9% for type I, 27.0 +/- 1.86% for type III, 17.3 +/- 1.46% for type IV (N = 6). In the absence of resonance frequency, the effects of magnetic field on ECM were less profound.

Body temperature-related structural transitions of monotremal and human hemoglobin.

In this study, temperature-related structural changes were investigated in human, duck-billed platypus (Ornithorhynchus anatinus, body temperature T(b) = 31-33 degrees C), and echidna (Tachyglossus aculeatus, body temperature T(b) = 32-33 degrees C) hemoglobin using circular dichroism spectroscopy and dynamic light scattering. The average hydrodynamic radius (R(h)) and fractional (normalized) change in the ellipticity (F(obs)) at 222 +/- 2 nm of hemoglobin were measured. The temperature was varied stepwise from 25 degrees C to 45 degrees C. The existence of a structural transition of human hemoglobin at the critical temperature T(c) between 36-37 degrees C was previously shown by micropipette aspiration experiments, viscosimetry, and circular dichroism spectroscopy. Based on light-scattering measurements, this study proves the onset of molecular aggregation at T(c). In two different monotremal hemoglobins (echidna and platypus), the critical transition temperatures were found between 32-33 degrees C, which are close to the species' body temperature T(b). The data suggest that the correlation of the structural transition's critical temperature T(c) and the species' body temperature T(b) is not mere coincidence but, instead, is a more widespread structural phenomenon possibly including many other proteins.

NMR in vitro effects on proliferation, apoptosis, and viability of human chondrocytes and osteoblasts.

This study presents findings on the proliferation rate, cellular apoptosis, and viability of human chondrocyte and osteoblast cultures before and after treatment with NMR pulse sequences. A commercially available nuclear magnetic resonance machine (MBST(R)-Nuclear Magnetic Resonance Therapy) was used for treatment. The study was carried out for 19 days, including 9 days of NMR exposure in a controlled, double-blind, randomized manner, using commercially available human cell lines. The study revealed that NMR treatment did not induce apoptosis or inhibit cell viability, but revealed a tendency of an elevated cell proliferation rate as observed by cell count.

Bactericidal effects of plasma-generated cluster ions.

Air purification by plasma-generated cluster ions (PCIs) relies on a novel technology producing hydrated positive and negative ions. Phenomenological tests have shown strong evidence of lethal effects of the PCIs on various micro-organisms. However, the mechanisms of PCI action are still widely unknown. The aim was thus to test the bactericidal efficacy of PCI technology on common indoor micro-organisms and to explore possible PCI mechanisms of action. According to time/dose-dependent experiments with Staphylococcus, Enterococcus, Micrococcus and Bacillus, the inhibiting effects became apparent within the first few minutes of PCI exposure and led to an irreversible 99.9% destruction within the following 2-8 h of exposure. The destructive effect of the PCIs corresponded to membrane damage of the bacteria. Use of the techniques of both SDS PAGE and 2D PAGE revealed changes in the bacterial surface protein composition induced by the PCIs. In contrast, neither DNA nor cytoplasm protein damage was detected electrophoretically. The antimicrobial action of the PCIs seems to occur because of chemical modification of the surface proteins of bacteria. In situ hydroxyl radical formation on the surface of bacteria was proposed as the leading mechanism of the protein damage caused by the PCIs. At the same time, DNA damage seems not to be involved in the antibacterial action of the PCIs. The data obtained would broaden the knowledge concerning the antibacterial effects of air-born plasma-generated cluster ions and help to produce more efficient air-cleaning devices.

Evaluation of lateral mechanical tension in thin-film tissue constructs.

Fibroblast-populated collagen matrices provide a simplified tissue model for wound healing and development processes. A technology (CELLDRUM Technology) evaluating lateral mechanical tension in fibroblast-populated collagen matrices (tissue constructs) with a thickness of 1 mm was introduced. Defined mechanical boundary conditions together with the known number and orientation of the cells revealed precise data on the average tension exerted by a single cell. Circular cell-populated collagen gels were manufactured inside the CELLDRUM on top of a flexible membrane. The collagen matrix was then excited by a sound pulse. The resulting resonance oscillation was monitored by a laser-based deflection sensor and frequency and damping were analyzed giving information on mechanical properties of the tissue construct. Several evaluation experiments were performed. Calf serum enhanced contractile forces of fibroblasts dose dependently. After the gels were treated with cytochalasin D for 24 h, the cell forces were reduced by 42% of control. The remaining tension was attributed to the extracellular matrix remodeling occurring during cell growth and to other cytoskeletal structures like microtubules and intermediate filaments. We also found that only after a few hours of culture fibroblast-seeded collagen gels began developing significant mechanical tension. A mechanical tension profile of proliferating fibroblasts in collagen gels over culture time was obtained.

[Immunostimulating activity of a saponin-containing extract of Saponaria officinalis]. / Izuchenie immunostimuliruiushchei aktivnosti saponinsoderzhashchego ékstrakta Saponaria officinalis.

The immunostimulating activity of saponin-containing extract of Saponaria officinalis has been studied. Use of an S. officinalis extract in a concentration close to the critical concentration of saponin micella formation increased the immunogenicity of viral glycoproteins. The immunogenicity of glycoprotein complexes with S. officinalis was higher than the immunogenicity of intact virus and micellae of purified glycoproteins and was comparable to that of glycoprotein complexes with Quil A glycoside.

Assessment of dot-blot ELISA sensitivity on membrane sorbent using various peroxidase substrates.

The sensitivity of the peroxidase reaction in dot-blot ELISA significantly depends on the substrate. The highest sensitivity is observed using benzidine and diamine-phenol combinations as the substrates due to the reaction of the coupled oxidation (NADI).