Low Level Phosphorylation of Histone H2AX on Serine 139 (gammaH2AX) Is Not Associated with DNA Double-Strand Breaks

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities

NIEINWAZYJNE METODY KONTROLNO-POMIAROWE W ZASTOSOWANIACH PRZEMYSŁOWYCH

Monitoring and control systems constitute an important group of diagnostic tools used in the industry. When selected properly, they allow for the early detection of adverse conditions of the process, making it possible to prevent catastrophes. While these systems are becoming more precise and effective it is essential to exploring the state of knowledge about the nature of the processes that are the domains of their proper execution. Based on a process of gravitational flow of solids proposed measuring and control system is described, which includes tools such as strain gauges, accelerometers and capacitance tomography sensors. Preliminary results of conducted tests confirmed the efficiency of the proposed system for the monitoring of an installation for storage and unloading of bulk solids. The flow rig is located in the Tom Dyakowski Process Tomography Lab at the Institute of Applied Computer Science.Systemy kontrolno-pomiarowe stanowią istotną grupę narzędzi diagnostycznych wykorzystywanych w przemyśle. Prawidłowo dobrane do charakteru procesu pozwalają na wczesne wykrywanie niepożądanych stanów procesu, dzięki czemu możliwe jest np. zapobieganie katastrofom przemysłowym. Nieustanny rozwój tych systemów powoduje, że są one coraz bardziej precyzyjne i efektywne, jednak od dokładności urządzeń często ważniejsze jest zgłębianie stanu wiedzy na temat natury badanych procesów. Na podstawie procesu grawitacyjnego przepływu substancji sypkich opisany został system pomiarowo-kontrolny, w skład którego wchodzą takie narzędzia jak: tensometry, akcelerometry oraz tomograficzne czujniki pojemnościowe. Wstępne wyniki przeprowadzonych eksperymentów potwierdzają skuteczność proponowanego systemu w odniesieniu do monitorowania przemysłowej instalacji przechowywania i rozładowywania materiałów sypkich, znajdującej się w Laboratorium Tomografii Procesowej im. T. Dyakowskiego Instytutu Informatyki Stosowanej

Biological properties of low-toxicity PLGA and PLGA/PHB fibrous nanocomposite implants for osseous tissue regeneration. Part I: evaluation of potential biotoxicity

In response to the demand for new implant materials characterized by high biocompatibility and bioresorption, two prototypes of fibrous nanocomposite implants for osseous tissue regeneration made of a newly developed blend of poly(L-lactide-co-glycolide) (PLGA) and syntheticpoly([R,S]-3-hydroxybutyrate), PLGA/PHB, have been developed and fabricated. Afibre-forming copolymer of glycolide and L-lactide (PLGA) was obtained by a unique method of synthesis carried out in blocksusing Zr(AcAc)4 as an initiator. The prototypes of the implants are composed of three layers of PLGA or PLGA/PHB, nonwoven fabrics with a pore structure designed to provide the best conditions for the cell proliferation. The bioactivity of the proposed implants has been imparted by introducing a hydroxyapatite material and IGF1, a growth factor. The developed prototypes of implants have been subjected to a set of in vitro and in vivobiocompatibility tests: in vitro cytotoxic effect, in vitro genotoxicity and systemic toxicity. Rabbitsshowed no signs of negative reactionafter implantation of the experimental implant prototypes

Evaluation of Terrestrial Laser Scanner Accuracy in the Control of Hydrotechnical Structures

In many cases of monitoring or load testing of hydrotechnical structures, the measurement results obtained from dial gauges may be affected by random or systematic errors resulting from the instability of the reference beam. For example, the measurement of wall displacement or pile settlement may be increased (or decreased) by displacements of the reference beam due to ground movement. The application of surveying methods such as high-precision levelling, motorized tacheometry or even terrestrial laser scanning makes it possible to provide an independent reference measurement free from systematic errors. It is very important in the case of walls and piles embedded in the rivers, where the construction of reference structure is even more difficult than usually. Construction of an independent reference system is also complicated when horizontal testing of sheet piles or diaphragm walls are considered. In this case, any underestimation of the horizontal displacement of an anchored or strutted construction leads to an understated value of the strut’s load. These measurements are even more important during modernization works and repairs of the hydrotechnical structures

Accuracy Assessment of Semi-Automatic Measuring Techniques Applied to Displacement Control in Self-Balanced Pile Capacity Testing Appliance

Static load tests of foundation piles are the basic method for the designing or verification of adopted design solutions which concern the foundation of a building structure. Preparation of a typical test station using the so-called inverted beam method is very expensive and labor-intensive. The settlement values of the loaded pile are usually recorded using accurate dial gauges. These gauges are attached to a reference beam located in close proximity to the pile under test, which may cause systematic errors (difficult to detect) caused by the displacement of the adopted reference beam. The application of geodetic methods makes it possible to maintain an independent, external reference system, and to verify the readouts from dial gauges. The article presents an innovative instrumentation for a self-balanced stand for the static load test made from a closed-end, double steel pipe. Instead of typical, precise geometric leveling, the semi-automatic measuring techniques were used: motorized total station measurement and terrestrial laser scanning controlled by a computer. The processing of the acquired data made it possible to determine the vertical displacements of both parts of the examined pile and compare displacements with the results from the dial gauges. On the basis of the excess of the collected observations, it was possible to assess the accuracy, which confirmed the usefulness of measuring techniques under study

New chitosan wound dressing – first step - the cytotoxicity evaluation

The cytotoxicity of chitosan with polyvinox (PCH) and crosslinked chitosan (CH) was studied and analyzed. Cell viability was determined by thiazolyl blue formazan (MTT) assay and cell morphology observations were carried out during cell culturing and MTT tests. Crosslinked chitosan was used as a protective foil (scaffold) for skin wounds. Studies in vitro and the other obtained in this work results prove that the studied materials CH and PCH do not cause cytotoxic activity to Balb 3T3 mouse fibroblasts. CH and PCH are promising biomaterials with prospective application as wound healing dressings

Influence of Porous Dressings Based on Butyric-Acetic Chitin Co-Polymer on Biological Processes In Vitro and In Vivo

In spite of intensively conducted research allowing for the development of more and more advanced wound dressing materials, there is still a need for dressings that stimulate not only reparative and regenerative processes, but also have a positive effect on infected and/or difficult-to-heal wounds. Porous dressing materials based on butyric-acetic chitin co-polyester containing 90% of butyryl and 10% of acetyl groups (BAC 90/10) can also be included in the group mentioned above. Two types of dressings were obtained by the salt leaching method, i.e. a porous sponge Medisorb R and Medisorb Ag with an antibacterial additive. The aim of the study was to evaluate biological effects of porous Medisorb R and Medisorb Ag dressings under in vitro and in vivo conditions. In an in vitro biodegradation test, no mass loss of Medisorb R dressing was observed within 14 days of incubation in physiological fluids at 37 °C. However, on the basis of the FTIR (Fourier Transform Infrared Spectroscopy) tests, surface degradation of Medisorb R dressing was observed. Additionally, the antibacterial activity of the porous Medisorb Ag dressing containing microsilver as an antibacterial additive was confirmed. The in vivo studies included inflammatory activity, skin irritation and sensitisation tests, as well an assessment of local effect after contact with subcutaneous tissue up to 6 months and skin wounds up to 21 days. In the in vivo tests, the dressings exhibited neither effects of skin irritation nor sensitisation. Under macroscopic examination, in full thickness defects of subcutaneous tissue and skin, the dressings caused wound healing with no inflammation, undergoing the most gradual biodegradation between weeks 4 and 8, and the observed differences were statistically significant. In the histological assessment, a weakened, limited inflammatory process associated with degradation of the material has been observed. The process of skin wound healing under Medisorb R dressing in the early period was accelerated compared to that observed in the control group with a gauze dressing

Relationship of DNA damage signaling to DNA replication following treatment with DNA topoisomerase inhibitors camptothecin/topotecan, mitoxantrone, or etoposide

DNA topoisomerase I (Top1) and topoisomerase II (Top2) inhibitors are widely used to treat a variety of cancers. Their mechanism of action involves stabilization of otherwise transient (“cleavable”) complexes between Top1 or Top2 and DNA; collisions of DNA replication forks with such stabilized complexes lead to formation of DNA double-strand breaks (DSBs). In this study, using 5-ethynyl-2′deoxyuridine (EdU) as a DNA precursor, we directly assessed the relationship between DNA replication and induction of DSBs revealed as γH2AX foci in A549 cells treated with Top1 inhibitors topotecan (Tpt) or camptothecin (Cpt) and Top2 inhibitors mitoxantrone (Mxt) and etoposide (Etp). Analysis of cells by multiparameter laser scanning cytometry following treatment with Tpt or Cpt revealed that only DNA replicating cells showed induction of γH2AX and a strong correlation between DNA replication and formation of DSBs (r = 0.86). In cells treated with Mxt or Etp, the correlation was weaker (r = 0.52 and 0.64). In addition, both Mtx and Etp caused induction of γH2AX in cells not replicating DNA. Confocal imaging of nuclei of cells treated with Tpt revealed the presence of γH2AX foci predominantly in DNA replicating cells and close association and co-localization of γH2AX foci with DNA replication sites. In cells treated with Mxt or Etp, the γH2AX foci were induced in DNA replicating as well as non-replicating cells but the close association between a large proportion of γH2AX foci and DNA replication sites was also apparent. The data are consistent with the view that collision of DNA replication forks with cleavable Top1–DNA complexes stabilized by Tpt/Cpt is the sole cause of induction of DSBs. Additional mechanisms such as involvement of transcription and/or generation of oxidative stress may contribute to DSBs induction by Mxt and Etp. The confocal analysis of the association between DNA replication sites and the sites of DSBs (γH2AX foci) opens a new approach for mechanistic studies of the involvement of DNA replication in induction of DNA damage