Many faces of survey equipment failures during marine research at sea-Risk analysis.

Research of the marine environment is still a huge challenge for humanity. Each survey campaign is a complex project, where research vessels and relevant survey equipment is used. One of the problems that limit the success of working at sea are failures of survey equipment. The aim of this paper was to identify the most common survey equipment failures during marine research, find their causes and analyze identified risks. The authors employ risk assessment methodology in maritime research at sea and present its practical utility and contribution in social and organizational development. For this purpose we based the analysis on the review of relevant project documentation (Daily Progress Reports, Observation Cards) and the questionnaire addressed to specialists who carry out their survey work on board research vessels and also people involved in the implementation of offshore projects. The research reveals that 76.3% respondents participated in a project which had to be stopped due to a failure of the survey equipment that required return to the port which highlights that the problem which was analyzed is of particular importance. The questionnaire form was designed to obtain as much information as possible on the types of failures with examples and also their causes according to three groups: human factors, technical factors and forces of nature. Twelve risks were identified and analyzed. The authors also stress the relationship between the quality of research project management and its implementation in the context of the failure rate of measuring equipment.

On a risk of inhalation exposure during visits in Chernobyl exclusion zone.

In recent years Chernobyl exclusion zone has become a very popular tourist destination. Many people visiting power plant, Pripyat city or surrounding villages use different types of personal dosimeters to control external exposure, however very small group of tourist have opportunity to control internal contamination of respiratory tract using dedicated, high sensitive whole body counters. In this study 11 anti-dust masks collected from CEZ visitors and filters from one military MP-5 mask were analyzed using alpha, beta and gamma spectrometry to determine doses from actinides and fission products which can be inhaled without proper protective equipment. Results showed, that average effective dose from inhalation of contaminated aerosol in case of single-day trip (avoided due to use of mask) was 1.3 µSv per person, which is much smaller than potential effective dose after exploration of highly contaminated areas like Jupiter complex, where combined dose from all measured nuclides collected on MP-5 mask filter was 1.4 mSv.

Improved chemistry restraints for crystallographic refinement by integrating the Amber force field into Phenix.

The refinement of biomolecular crystallographic models relies on geometric restraints to help to address the paucity of experimental data typical in these experiments. Limitations in these restraints can degrade the quality of the resulting atomic models. Here, an integration of the full all-atom Amber molecular-dynamics force field into Phenix crystallographic refinement is presented, which enables more complete modeling of biomolecular chemistry. The advantages of the force field include a carefully derived set of torsion-angle potentials, an extensive and flexible set of atom types, Lennard-Jones treatment of nonbonded interactions and a full treatment of crystalline electrostatics. The new combined method was tested against conventional geometry restraints for over 22 000 protein structures. Structures refined with the new method show substantially improved model quality. On average, Ramachandran and rotamer scores are somewhat better, clashscores and MolProbity scores are significantly improved, and the modeling of electrostatics leads to structures that exhibit more, and more correct, hydrogen bonds than those refined using traditional geometry restraints. In general it is found that model improvements are greatest at lower resolutions, prompting plans to add the Amber target function to real-space refinement for use in electron cryo-microscopy. This work opens the door to the future development of more advanced applications such as Amber-based ensemble refinement, quantum-mechanical representation of active sites and improved geometric restraints for simulated annealing.

Gamma emitters in atmospheric precipitation in Krakow (Southern Poland) during the years 2005-2015.

The results of the sum of dry and wet activity deposition for naturally occurring 7Be, 210Pb, 40K, 22Na and anthropogenic 137Cs radionuclides in Krakow (Southern Poland) for the samples collected over 10 years (from August 2005 to July 2015) are presented and discussed. The radionuclides were determined using low background gamma spectrometry with HPGe detectors. Additionally, in this paper there are shown the results of activity concentrations in water from air precipitation for 7Be, 210Pb, 22Na, 40K and 137Cs radioisotopes from the period of 7 years (from August 2008 to July 2015). For all these series the statistical analysis including Spearman correlations, effects of seasonal variation and multiple regression models were conducted. After excluding two months from 2011 affected by the Fukushima accident, high Spearman correlation factors (R > 0.5) for activity deposition were noticed for the pair of the cosmogenic radionuclides 7Be and 22Na (R = 0.508) and between 210Pb and 7Be (R = 0.570). High correlation was noted between activity deposition and amount of precipitation for 7Be (R = 0.677). The seasonal correlations between 7Be-22Na, 40K-137Cs, 210Pb-137Cs and 7Be-210Pb were investigated and the highest correlation coefficient R = 0.731 for the 40K-137Cs pair was in the spring season. High correlations were observed also between 210Pb and 7Be for autumn (R = 0.594), 40K-137Cs in summer (R = 0.582), 7Be-22Na in spring (R = 0.635) and 210Pb-137Cs in autumn (R = 0.672). The multiple regression approach showed the interesting difference in scavenging mechanisms of cosmogenic and terrestrial radionuclides. According to that model, the deposition of cosmogenic nuclides was noticeably related to the amount of precipitation, while the deposition of terrestrial radionuclides did not show such dependence.

Improved ligand geometries in crystallographic refinement using AFITT in PHENIX.

Modern crystal structure refinement programs rely on geometry restraints to overcome the challenge of a low data-to-parameter ratio. While the classical Engh and Huber restraints work well for standard amino-acid residues, the chemical complexity of small-molecule ligands presents a particular challenge. Most current approaches either limit ligand restraints to those that can be readily described in the Crystallographic Information File (CIF) format, thus sacrificing chemical flexibility and energetic accuracy, or they employ protocols that substantially lengthen the refinement time, potentially hindering rapid automated refinement workflows. PHENIX-AFITT refinement uses a full molecular-mechanics force field for user-selected small-molecule ligands during refinement, eliminating the potentially difficult problem of finding or generating high-quality geometry restraints. It is fully integrated with a standard refinement protocol and requires practically no additional steps from the user, making it ideal for high-throughput workflows. PHENIX-AFITT refinements also handle multiple ligands in a single model, alternate conformations and covalently bound ligands. Here, the results of combining AFITT and the PHENIX software suite on a data set of 189 protein-ligand PDB structures are presented. Refinements using PHENIX-AFITT significantly reduce ligand conformational energy and lead to improved geometries without detriment to the fit to the experimental data. For the data presented, PHENIX-AFITT refinements result in more chemically accurate models for small-molecule ligands.

Lead shielding efficiency from the gamma background measurements in the salt cavern of the Polkowice-Sieroszowice copper mine.

The studies of lead shielding efficiency from the gamma background measurements were performed in the salt cavern of the copper mine - a site considered for an underground laboratory. Within the energy range of 50-2700 keV, the measured gamma-ray count rates normalized to the mass of the high-purity detectors germanium crystal are 5.93 and 6.32 s-1kg-1 for the used low-background and portable spectrometers, respectively. The gamma-ray flux of 0.124 (2) cm-2s-1 connected with the natural radioisotopes was observed by the portable HPGe, including 0.026 (1) cm-2s-1 contribution of radon decay products, whereas the photon flux at the spectrum continuum was 0.18 (5) cm-2s-1.

Molecular dynamics simulation of triclinic lysozyme in a crystal lattice.

Molecular dynamics simulations of crystals can enlighten interpretation of experimental X-ray crystallography data and elucidate structural dynamics and heterogeneity in biomolecular crystals. Furthermore, because of the direct comparison against experimental data, they can inform assessment of molecular dynamics methods and force fields. We present microsecond scale results for triclinic hen egg-white lysozyme in a supercell consisting of 12 independent unit cells using four contemporary force fields (Amber ff99SB, ff14ipq, ff14SB, and CHARMM 36) in crystalline and solvated states (for ff14SB only). We find the crystal simulations consistent across multiple runs of the same force field and robust to various solvent equilibration schemes. However, convergence is slow compared with solvent simulations. All the tested force fields reproduce experimental structural and dynamic properties well, but Amber ff14SB maintains structure and reproduces fluctuations closest to the experimental model: its average backbone structure differs from the deposited structure by 0.37Å; by contrast, the average backbone structure in solution differs from the deposited by 0.65Å. All the simulations are affected by a small progressive deterioration of the crystal lattice, presumably due to imperfect modeling of hydrogen bonding and other crystal contact interactions; this artifact is smallest in ff14SB, with average lattice positions deviating by 0.20Å from ideal. Side-chain disorder is surprisingly low with fewer than 30% of the nonglycine or alanine residues exhibiting significantly populated alternate rotamers. Our results provide helpful insight into the methodology of biomolecular crystal simulations and indicate directions for future work to obtain more accurate energy models for molecular dynamics.

All-atom crystal simulations of DNA and RNA duplexes.

BACKGROUND: Molecular dynamics simulations can complement experimental measures of structure and dynamics of biomolecules. The quality of such simulations can be tested by comparisons to models refined against experimental crystallographic data. METHODS: We report simulations of DNA and RNA duplexes in their crystalline environment. The calculations mimic the conditions for PDB entries 1D23 [d(CGATCGATCG)2] and 1RNA [(UUAUAUAUAUAUAA)2], and contain 8 unit cells, each with 4 copies of the Watson-Crick duplex; this yields in aggregate 64µs of duplex sampling for DNA and 16µs for RNA. RESULTS: The duplex structures conform much more closely to the average structure seen in the crystal than do structures extracted from a solution simulation with the same force field. Sequence-dependent variations in helical parameters, and in groove widths, are largely maintained in the crystal structure, but are smoothed out in solution. However, the integrity of the crystal lattice is slowly degraded in both simulations, with the result that the interfaces between chains become heterogeneous. This problem is more severe for the DNA crystal, which has fewer inter-chain hydrogen bond contacts than does the RNA crystal. CONCLUSIONS: Crystal simulations using current force fields reproduce many features of observed crystal structures, but suffer from a gradual degradation of the integrity of the crystal lattice. GENERAL SIGNIFICANCE: The results offer insights into force-field simulations that test their ability to preserve weak interactions between chains, which will be of importance also in non-crystalline applications that involve binding and recognition. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

Evaluation of acridine orange, LysoTracker Red, and quinacrine as fluorescent probes for long-term tracking of acidic vesicles.

Acidic vesicles can be imaged and tracked in live cells after staining with several low molecular weight fluorescent probes, or with fluorescently labeled proteins. Three fluorescent dyes, acridine orange, LysoTracker Red DND-99, and quinacrine, were evaluated as acidic vesicle tracers for confocal fluorescence imaging and quantitative analysis. The stability of fluorescent signals, achievable image contrast, and phototoxicity were taken into consideration. The three tested tracers exhibit different advantages and pose different problems in imaging experiments. Acridine orange makes it possible to distinguish acidic vesicles with different internal pH but is fairly phototoxic and can cause spectacular bursts of the dye-loaded vesicles. LysoTracker Red is less phototoxic but its rapid photobleaching limits the range of useful applications considerably. We demonstrate that quinacrine is most suitable for long-term imaging when a high number of frames is required. This capacity made it possible to trace acidic vesicles for several hours, during a process of drug-induced apoptosis. An ability to record the behavior of acidic vesicles over such long periods opens a possibility to study processes like autophagy or long-term effects of drugs on endocytosis and exocytosis.

Evidence for the role of active site residues in the hairpin ribozyme from molecular simulations along the reaction path.

The hairpin ribozyme accelerates a phosphoryl transfer reaction without catalytic participation of divalent metal ions. Residues A38 and G8 have been implicated as playing roles in general acid and base catalysis, respectively. Here we explore the structure and dynamics of key active site residues using more than 1 µs of molecular dynamics simulations of the hairpin ribozyme at different stages along the catalytic pathway. Analysis of results indicates hydrogen bond interactions between the nucleophile and proR nonbridging oxygen are correlated with active inline attack conformations. Further, the simulation results suggest a possible alternative role for G8 to promote inline fitness and facilitate activation of the nucleophile by hydrogen bonding, although this does not necessarily exclude an additional role as a general base. Finally, we suggest that substitution of G8 with N7- or N3-deazaguanosine which have elevated pKa values, both with and without thio modifications at the 5' leaving group position, would provide valuable insight into the specific role of G8 in catalysis.

Peptide crystal simulations reveal hidden dynamics.

Molecular dynamics simulations of biomolecular crystals at atomic resolution have the potential to recover information on dynamics and heterogeneity hidden in X-ray diffraction data. We present here 9.6 µs of dynamics in a small helical peptide crystal with 36 independent copies of the unit cell. The average simulation structure agrees with experiment to within 0.28 Å backbone and 0.42 Å all-atom RMSD; a model refined against the average simulation density agrees with the experimental structure to within 0.20 Å backbone and 0.33 Å all-atom RMSD. The R-factor between the experimental structure factors and those derived from this unrestrained simulation is 23% to 1.0 Å resolution. The B-factors for most heavy atoms agree well with experiment (Pearson correlation of 0.90), but B-factors obtained by refinement against the average simulation density underestimate the coordinate fluctuations in the underlying simulation where the simulation samples alternate conformations. A dynamic flow of water molecules through channels within the crystal lattice is observed, yet the average water density is in remarkable agreement with experiment. A minor population of unit cells is characterized by reduced water content, 310 helical propensity and a gauche(-) side-chain rotamer for one of the valine residues. Careful examination of the experimental data suggests that transitions of the helices are a simulation artifact, although there is indeed evidence for alternate valine conformers and variable water content. This study highlights the potential for crystal simulations to detect dynamics and heterogeneity in experimental diffraction data as well as to validate computational chemistry methods.

Rationale for the real-time and dynamic cell death assays using propidium iodide.

We have recently reported an innovative approach to use charged fluorochromes such as propidium iodide (PI) in the real-time, dynamic cell viability assays. This study was designed to provide a mechanistic rationale for the kinetic assays using cell permeability markers. Uptake of PI by live cells, effect on the cell cycle, long-term proliferation capacity, DNA damage response, and pharmacologic interactions with anticancer drugs were studied using both laser scanning microscopy and laser scanning cytometry. Exposure of human carcinomic alveolar basal epithelial A549 cells in cultures to 1.5 or 7.5 microM of PI for 24 h had minimal effect on cell cycle progression including DNA replication as measured by incorporation of 5'-ethynyl-2-deoxyuridine (EdU) detected by the "click chemistry" approach and measured by laser scanning cytometry. A modest reduction, from 44 to 40% or 33%, in frequency of DNA replicating cells was seen after 48 h at 1.5 or 7.5 microM concentration of PI. There was no evidence of increased phosphorylation of histone gammaH2AX in cells growing in the presence of 1.5 or 7.5 microM of PI for up to 48 h. Confocal image analysis of HeLa and NIH 3T3 mouse embryonic fibroblasts growing in the presence of PI showed granular distribution in cell cytoplasm suggesting PI accumulation in endosomes and progressive increase in fluorescence of nucleoli reflecting PI binding to nucleolar RNA. The overall responses of cells to cytotoxic agents were also not affected by the growth in the presence PI. Our data lend further support to the notion that PI can be effectively used in real-time, kinetic viability assays.