Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach

In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a “double-edged sword”—it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.     

Valence-induced effects on the electrical properties of NiMn2O4 ceramics with different Ni sources

Spinel-structured NiMn₂O₄ ceramics, with different valence Ni sources, were originally prepared using Ni₂O₃ and NiO as raw materials, and the effects of different valence Ni sources on their electrical properties were first investigated. XRD patterns show that both Ni₂O₃-based and NiO-based NiMn₂O₄ ceramics are single cubic spinel structures. SEM/EDS images indicate that the NiMn₂O₄ ceramics exhibited high density at the experiment-determined sintering temperatures. XPS results and Raman drifts prove that the Ni valence-induced changes in Mn ions at B sites played a significant role in the electrical properties and thermal stability of NiMn₂O₄ ceramics. Compared with NiO-based NiMn₂O₄, the resistivity at 25°C (ρ_25°C) of Ni₂O₃-based NiMn₂O₄ increased dramatically from 3109 to 106958 Ω cm, the thermal constant (B_25/50) increased from 3264 to 4473 K, and the resistance shifts after annealing for 1000 h at 150°C decreased from 0.80% to 0.74%. The investigation of the relationship between the material properties and valence of Ni sources has provided a new and effective way for designing the spinel-structured negative temperature coefficient (NTC) materials by modulating the valence of ions at A sites in the raw materials.     

Determination of Fexofenadine in Tablets by Capillary Electrophoresis in Free Solution and in Solution with Cyclodextrins as Analyte Carriers

ABSTRACT

Capillary electrophoresis (CE) methods for the determination of fexofenadine (FEX) in commercial pharmaceuticals were developed. It was demonstrated that FEX could be effectively analyzed in free solution cationic CE at low pH. Another analytical approach studied was based on cyclodextrin (CD) modified CE where highly charged CD derivatives served as analyte carriers. In this way, the separation range was spread to physiological pH region and a CE analysis of FEX, present actually in its zwitterionic form, could be accomplished. Several parameters affecting the separations were studied, including the type and concentration of carrier ion, counterion, analyte carrier, and pH of the buffer. The methods based on the free solution CE and CD-modified CE were compared each other, validated, and applied for the determination of FEX in tablets.     

A Quinacrine Analogue Selective Against Gastric Cancer Cells: Insight from Biochemical and Biophysical Studies

One of the earliest synthetic antimalarial drugs, quinacrine, was recently reported as interesting for the treatment of acute myeloid leukemia. Inspired by this and similar findings, we evaluated a set of quinacrine analogues against gastric (MKN‐28), colon (Caco‐2), and breast (MFC‐7) cancer cell lines and one normal human fibroblast cell line (HFF‐1). All the compounds, previously developed by us as dual‐stage antimalarial leads, displayed antiproliferative activity, and one of the set stood out as selective toward the gastric cancer cell line, MKN‐28. Interestingly, this compound was transported across an in vitro MKN‐28 model cell line in low amounts, and approximately 80 % was trapped inside those cells. Nuclear targeting of the same compound and its interactions with calf thymus DNA were assessed through combined fluorescence microscopy, spectroscopy, and calorimetry studies, which provided evidence for the compound's ability to reach the nucleus and to interact with DNA.     

Apoferritin Modified Magnetic Particles as Doxorubicin Carriers for Anticancer Drug Delivery

Magnetic particle mediated transport in combination with nanomaterial based drug carrier has a great potential for targeted cancer therapy. In this study, doxorubicin encapsulation into the apoferritin and its conjugation with magnetic particles was investigated by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The quantification of encapsulated doxorubicin was performed by fluorescence spectroscopy and compared to CE-LIF. Moreover, the significant enhancement of the doxorubicin signal was observed by addition of methanol into the sample solution.     

A Novel Insight into the Cardiotoxicity of Antineoplastic Drug Doxorubicin

Doxorubicin is a commonly used antineoplastic agent in the treatment of many types of cancer. Little is known about the interactions of doxorubicin with cardiac biomolecules. Serious cardiotoxicity including dilated cardiomyopathy often resulting in a fatal congestive heart failure may occur as a consequence of chemotherapy with doxorubicin. The purpose of this study was to determine the effect of exposure to doxorubicin on the changes in major amino acids in tissue of cardiac muscle (proline, taurine, glutamic acid, arginine, aspartic acid, leucine, glycine, valine, alanine, isoleucine, threonine, lysine and serine). An in vitro interaction study was performed as a comparison of amino acid profiles in heart tissue before and after application of doxorubicin. We found that doxorubicin directly influences myocardial amino acid representation even at low concentrations. In addition, we performed an interaction study that resulted in the determination of breaking points for each of analyzed amino acids. Lysine, arginine, β-alanine, valine and serine were determined as the most sensitive amino acids. Additionally we compared amino acid profiles of myocardium before and after exposure to doxorubicin. The amount of amino acids after interaction with doxorubicin was significantly reduced (p = 0.05). This fact points at an ability of doxorubicin to induce changes in quantitative composition of amino acids in myocardium. Moreover, this confirms that the interactions between doxorubicin and amino acids may act as another factor most likely responsible for adverse effects of doxorubicin on myocardium.     

MatLab program for precision calibration of optical tweezers

Optical tweezers are used as force transducers in many types of experiments. The force they exert in a given experiment is known only after a calibration. Computer codes that calibrate optical tweezers with high precision and reliability in the (x,y)-plane orthogonal to the laser beam axis were written in MatLab (MathWorks Inc.) and are presented here. The calibration is based on the power spectrum of the Brownian motion of a dielectric bead trapped in the tweezers. Precision is achieved by accounting for a number of factors that affect this power spectrum. First, cross-talk between channels in 2D position measurements is tested for, and eliminated if detected. Then, the Lorentzian power spectrum that results from the Einstein-Ornstein-Uhlenbeck theory, is fitted to the low-frequency part of the experimental spectrum in order to obtain an initial guess for parameters to be fitted. Finally, a more complete theory is fitted, a theory that optionally accounts for the frequency dependence of the hydrodynamic drag force and hydrodynamic interaction with a nearby cover slip, for effects of finite sampling frequency (aliasing), for effects of anti-aliasing filters in the data acquisition electronics, and for unintended “virtual” filtering caused by the position detection system. Each of these effects can be left out or included as the user prefers, with user-defined parameters. Several tests are applied to the experimental data during calibration to ensure that the data comply with the theory used for their interpretation: Independence of x- and y-coordinates, Hooke's law, exponential distribution of power spectral values, uncorrelated Gaussian scatter of residual values. Results are given with statistical errors and covariance matrix.

Program summary

Title of program: tweezercalibCatalogue identifier: ADTVProgram obtainable from: CPC Program Library, Queen's University of Belfast, N. Ireland.Program Summary URL:http://cpc.cs.qub.ac.uk/summaries/ADTVComputer for which the program is designed and others on which it has been tested: General computer running MatLab (MathWorks Inc.).Programming language used: MatLab (MathWorks Inc.). Uses “Optimization Toolbox” and “Statistics Toolbox”.Memory required to execute with typical data: Of order 4 times the size of the data file.High speed storage required: NoneNo. of lines in distributed program, including test data, etc.: 133 183No. of bytes in distributed program, including test data, etc.: 1 043 674Distribution format: tar gzip fileNature of physical problem: Calibrate optical tweezers with precision by fitting theory to experimental power spectrum of position of bead doing Brownian motion in incompressible fluid, possibly near microscope cover slip, while trapped in optical tweezers. Thereby determine spring constant of optical trap and conversion factor for arbitrary-units-to-nanometers for detection system.Method of solution: Elimination of cross-talk between quadrant photo-diode's output channels for positions (optional). Check that distribution of recorded positions agrees with Boltzmann distribution of bead in harmonic trap. Data compression and noise reduction by blocking method applied to power spectrum. Full accounting for hydrodynamic effects: Frequency-dependent drag force and interaction with nearby cover slip (optional). Full accounting for electronic filters (optional), for “virtual filtering” caused by detection system (optional). Full accounting for aliasing caused by finite sampling rate (optional). Standard non-linear least-squares fitting. Statistical support for fit is given, with several plots suitable for inspection of consistency and quality of data and fit.Restrictions on the complexity of the problem: Data should be positions of bead doing Brownian motion while held by optical tweezers. For high precision in final results, data should be time series measured over a long time, with sufficiently high experimental sampling rate: The sampling rate should be well above the characteristic frequency of the trap, the so-called corner frequency. Thus, the sampling frequency should typically be larger than 10 kHz. The Fast Fourier Transform applied requires the time series to contain 2ⁿ data points, and long measurement time is obtained with n>12-15. Finally, the optics should be set to ensure a harmonic trapping potential in the range of positions visited by the bead. The fitting procedure checks for harmonic potential.Typical running time: (Tens of) minutesUnusual features of the program: NoneReferences: The theoretical underpinnings for the procedure are found in [K. Berg-Sørensen, H. Flyvbjerg, Rev. Sci. Instrum. 75 (3) (2004) 594].     

DRYING RESEARCH AND DEVELOPMENT IN CZECHOSLOVAKIA

This paper provides general information on drying R 6D activities in Czechoslovakia. This survey report lists the main topics in drying according to individual industrial sectors in which drying plays an important role. Drying R 6D carried out in Czechoslovak Universities is also cited. The paper includes an extensive list of references, mainly proceedings of national drying conferences, symposia, and meetings     

Influence of Zn on the oxide layer on AISI 316L(NG) stainless steel in simulated pressurised water reactor coolant

The oxidation of AISI 316L(NG) stainless steel in simulated pressurised water reactor (PWR) coolant with or without addition of 1 ppm Zn at 280 °C for up to 96 h has been characterised in situ by electrochemical impedance spectroscopy (EIS), both at the corrosion potential and under anodic polarisation up to 0.5 V vs. the reversible hydrogen electrode (RHE). Additional tests were performed in simulated PWR coolant with the addition of 0.01 M Na₂B₄O₇ to exclude the effect of pH excursions probably due to Zn hydrolysis reactions. The thickness and in-depth composition of the oxide films formed at open circuit and at 0.5 V vs. RHE in the investigated electrolytes have been estimated from X-ray photoelectron spectroscopy (XPS) depth profiles. The kinetic and transport parameters characterising the oxide layer growth have been estimated using a calculational procedure based on the mixed conduction model for oxide films. Successful simulations of both the EIS and XPS data have been obtained. The parameter estimates are discussed in terms of the effect of Zn on the oxide layers on stainless steel in PWR conditions.