Influence of proton irradiation on the magnetic properties of two-dimensional Ni(II) molecular magnet.

The influence of 1.9 MeV proton irradiation on structural and magnetic properties has been explored in the two-dimensional (2D) NiSO4(1,3-phenylenediamine)2 coordination ferrimagnet. The X-ray powder diffraction and IR spectroscopy revealed that the octahedrons with Ni ion in the center remain unchanged regardless of the fluence a sample received. In contrast, proton irradiation greatly influences the hydrogen bonds in the flexible parts in which the 1,3-phenylenediamine is involved. Dc magnetic measurements revealed that several magnetic properties were modified with proton irradiation. The isothermal magnetization measured at T = 2.0 K varied with the proton dose, achieving a 50% increase in magnetization in the highest measured field µ0Hdc = 7 T or a 25% decrease in remanence. The most significant change was observed for the coercive field, which was reduced by 90% compared to the non-irradiated sample. The observed results are accounted for the increased freedom of magnetic moments rotation and the modification of intralayer exchange couplings.

Application of Spectroscopic Methods for the Identification of Superoxide Dismutases in Cyanobacteria.

Superoxide dismutases (SODs) belong to the group of metalloenzymes that remove superoxide anion radicals and they have been identified in three domains of life: Bacteria, Archaea and Eucarya. SODs in Synechocystis sp. PCC 6803, Gloeobacter violaceus CCALA 979, and Geitlerinema sp. ZHR1A were investigated. We hypothesized that iron (FeSOD) and/or manganese (MnSOD) dominate as active forms in these cyanobacteria. Activity staining and three different spectroscopic methods of SOD activity bands excised from the gels were used to identify a suitable metal in the separated samples. FeSODs or enzymes belonging to the Fe-MnSOD superfamily were detected. The spectroscopic analyses showed that only Fe is present in the SOD activity bands. We found FeSOD in Synechocystis sp. PCC 6803 while two forms in G. violaceus and Geitlerinema sp. ZHR1A: FeSOD1 and FeSOD2 were present. However, no active Cu/ZnSODs were identified in G. violaceus and Geitlerinema sp. ZHR1A. We have shown that selected spectroscopic techniques can be complementary to the commonly used method of staining for SOD activity in a gel. Furthermore, the occurrence of active SODs in the cyanobacteria studied is also discussed in the context of SOD evolution in oxyphotrophs.

Interdisciplinary Methods for Zoonotic Tissue Acellularization for Natural Heart Valve Substitute of Biomimetic Materials.

The goal of this work was to create a bioactive tissue-based scaffold using multi-disciplinary engineering materials and tissue engineering techniques. Materials & methods: Physical techniques such as direct laser interference lithography and proton radiation were selected as alternative methods of enzymatic and chemical decellularization to remove cells from a tissue without degradation of the extracellular matrix nor its protein structure. This study was an attempt to prepare a functional scaffold for cell culture from tissue of animal origin using new physical methods that have not been considered before. The work was carried out under full control of the histological and molecular analysis. Results & conclusions: The most important finding was that the physical methods used to obtain the decellularized tissue scaffold differed in the efficiency of cell removal from the tissue in favour of the laser method. Both the laser method and the proton method exhibited a destructive effect on tissue structure and the genetic material in cell nuclei. This effect was visible on histology images as blurred areas within the cell nucleus. The finite element 3D simulation of decellularization process of the three-layer tissue of animal origin sample reflected well the mechanical response of tissue described by hyperelastic material models and provided results comparable to the experimental ones.

Novel Approach to Tooth Chemistry. Quantification of the Dental-Enamel Junction.

The dentin-enamel junction (DEJ) is known for its special role in teeth. Several techniques were applied for the investigation of the DEJ in human sound molar teeth. The electron (EPMA) and proton (PIXE) microprobes gave consistent indications about the variability of elemental concentrations on this boundary. The locally increased and oscillating concentrations of Mg and Na were observed in the junction, in the layer adhering to the enamel and covering roughly half of the DEJ width. The chemical results were compared with the optical profiles of the junction. Our chemical and optical results were next compared with the micromechanical results (hardness, elastic modulus, friction coefficient) available in the world literature. A strong correlation of both result sets was proven, which testifies to the self-affinity of the junction structures for different locations and even for different kinds of teeth and techniques applied for studies. Energetic changes in tooth strictly connected with crystallographic transformations were calculated, and the minimum energetic status was discovered for DEJ zone. Modeling of both walls of the DEJ from optical data was demonstrated. Comparing the DEJ in human teeth with the same structure found in dinosaur, shark, and alligator teeth evidences the universality of dentin enamel junction in animal world. The paper makes a contribution to better understanding the joining of the different hard tissues.

Association of sulfur content in erythrocytes with cardiovascular parameters and blood pressure.

OBJECTIVE: The study aims at assessing the relationship between blood pressure, heart geometry parameters, and the erythrocyte content of sulfur, potassium, chlorine and phosphorus, in a group of patients with ambulatory systolic and diastolic blood pressure (SBP, DBP) below 140 or 90 mm Hg, respectively, who were otherwise healthy and untreated. METHODS: The study group consisted of 42 adults recruited in a primary care setting. The individuals were healthy, not undergoing any therapy and free from smoking. For each individual, data were obtained on: average 24-hour SBP and DBP, left ventricle geometry, complete blood count, lipids profile, fibrinogen, hs-CRP and the erythrocyte concentration of sulfur (S), potassium (K), chlorine (Cl) and phosphorus (P). RESULTS: Multivariate regression analysis showed statistically significant relationships of diastolic posterior wall thickness (PWTd) and relative wall thickness (RWT) with the concentration ratio of sulfur and potassium (S/K) in erythrocytes: PWTd and RWT increase as the S/K ratio increases. Also, SBP was found to be positively correlated with the S/K ratio. CONCLUSIONS: The increase in sulfur content in RBCs could be an indicator of the downregulation of nitric oxide (NO) erythrocyte bioavailability exerted by endogenously produced hydrogen sulfide (H2S), and, in consequence, a marker of the development of hypertension and/or adverse changes in heart geometry.

Nitrogen-Vacancy Color Centers Created by Proton Implantation in a Diamond.

We present an experimental study of the longitudinal and transverse relaxation of ensembles of negatively charged nitrogen-vacancy (NV-) centers in a diamond monocrystal prepared by 1.8 MeV proton implantation. The focused proton beam was used to introduce vacancies at a 20 µµm depth layer. Applied doses were in the range of 1.5×1013 to 1.5×1017 ions/cm2. The samples were subsequently annealed in vacuum which resulted in a migration of vacancies and their association with the nitrogen present in the diamond matrix. The proton implantation technique proved versatile to control production of nitrogen-vacancy color centers in thin films.

Biophysical and Biochemical Characteristics as Complementary Indicators of Melanoma Progression.

The multistep character of cancer progression makes it difficult to define a unique biomarker of the disease. Interdisciplinary approaches, combining various complementary techniques, especially those operating at a nanoscale level, potentially accelerate characterization of cancer cells or tissue properties. Here, we study a relation between the surface and biomechanical properties of melanoma cells, measured by mass spectrometry (ToF-SIMS) and atomic force microscopy (AFM). In total, seven cell lines have been studied. Six of them were melanoma cells derived from various stages of tumor progression: (1) WM115 cells derived from a 55 year old female skin melanoma at a vertical growth phase (VGP) in the primary melanoma site, (2) WM793 cells established from the vertical growth phase (VGP) of a primary skin melanoma lesion, (3) WM266-4 cells established from a cutaneous skin metastasis detected in the same patient as WM115 cells, (4) WM239 cells derived from a cutaneous skin metastasis, (5) 1205Lu cells originated from a lung metastasis diagnosed in the same patient as WM793 cells, and (6) A375P-cells were derived from a solid malignant tumor located in the lung. As a reference cell line, human epidermal melanocytes from adult skin (primary cell line HEMa-LP) were used. Results reveal low, medium, and large deformability of melanoma cells originating from vertical growth phase (VGP), and skin and lung metastasis, respectively. These changes were accompanied by distinct outcome from principal component analysis (PCA). In relation to VGP melanoma cells, cells from skin and lung metastasis reveal similar or significantly different surface properties. The largest deformability difference observed for cells from VGP and lung metastasis was accompanied by the largest separation of unspecific changes in their surface properties. In this way, we show the evidence that biomechanical and surface biochemical properties of cells change in parallel, indicating a potential of being used as nanobiophysical fingerprints of melanoma progression.

Biological effect of hydrothermally synthesized silica nanoparticles within crystalline hydroxyapatite coatings for titanium implants.

Development of functional coatings for artificial bone implants that strengthen the osseointegration and accelerate bone healing processes is urgently needed in the biomedical field. In this study we present biological effect of novel composite coatings with different concentration of silica nanoparticles within crystalline hydroxyapatite matrix (HAp-SiO2) synthesized on titanium under hydrothermal conditions. Samples were analyzed for their elemental composition, structure, bioactivity and in vitro cytotoxicity. The results indicate the formation and homogeneous distribution of silica nanoparticles on the surface of hexagonal hydroxyapatite (HAp) crystals. The coatings show improved bioactivity in comparison with pure HAp after 4 days of immersion in simulated body fluid (SBF). The responses of human osteoblast-like cells (MG-63) cultured onto the synthesized materials provide evidence that HAp-SiO2 composites exhibit good biocompatibility. We propose that this is because HAp-SiO2 composites favor biomineralization process with cell proliferation remaining unaffected, regardless of the amount of silica. Furthermore, SEM and fluorescence measurements demonstrate that HAp-SiO2 had positive effect on cell morphology, favoring cell adhesion.

Comparison of PIXE and XRF in the analysis of silver denarii of the early Piast.

The collection of denarii from the time of development of a Polish medieval state was studied using the proton induced X-ray emission spectroscopy. The major elements detected for these denarii are Ag and Cu, while minor elements such as Pb, Fe, Au, Bi, and Zn may also be present. The aim of the study was to cross-compare the results with a previous micro-X-ray fluorescence data and to perform a better quantification of the denarii elemental composition, especially for trace elements, providing suggestions of the origin of alloy compounds.

Changes in cellular response to the damage induced in PC-3 prostate cancer cells by proton microbeam irradiation.

The aim of this research was to find out whether the passage number effect may influence on the PC-3 cells (the human prostate cancer line derived from bone metastases) response to proton radiation. 2 MeV horizontally focused proton microbeam was used as a radiation source. The cells were treated with a counted number of H(+) ions (50-8000) corresponding to doses of 1.3-209 Gy/cell. For comparison, cell death was also induced by UVC radiation. All cells were stained with Hoechst 33342 and propidium iodide and visualized under a fluorescence microscope. Necrosis was observed at: a) 8000 protons per cell (corresponding to ∼209 Gy/cell) after 2-4 passages, b) 3200 protons per cell (corresponding to ∼84 Gy/cell) for cells after 11-14 passages and c) only 800 protons per cell (corresponding to ∼2 Gy/cell ) after 47-50 passages. Apoptosis was efficiently induced, by protons, only in cells after 50 passages. The results showed that the laboratory conditions affected cellular response of PC-3 cell line to the proton irradiation. The cellular response to the radiation treatment strongly depends on number of passages.

Cancer cell recognition--mechanical phenotype.

The major characteristics of cancer metastasis is the ability of the primary tumor cells to migrate by way of the blood or lymph vessels and to form tumors at multiple, distant sites. There are evidences that cancer progression is characterized by disruption and/or reorganization of cytoskeleton (i.e. cellular scaffold). This is accompanied by various molecular alterations influencing the overall mechanical resistance of cells. Current approach in diagnosis focuses mainly on microbiological, immunological, and pathological aspects rather than on the biomechanics of diseases. The determination of mechanical properties of an individual living cell has became possible with the development of local measurement techniques, such as atomic force microscopy, magnetic or optical tweezers. The advantage of them lies in the capability to measure living cells at a single cell level and in liquid conditions, close to natural environment. Here, we present the studies on mechanical properties of single cells originating from various cancers. The results show that, independently of the cancer type (bladder, melanoma, prostate, breast and colon), single cells are characterized by the lower Young's modulus, denoting higher deformability of cancerous cells. However, the obtained Young's modulus values were dependent on various factors, like the properties of substrates used for cell growth, force loading rate, or indentation depth. Their influence on elastic properties of cells was considered. Based on these findings, the identification of cancerous cells based on their elastic properties was performed. These results proved the AFM capability in recognition of a single, mechanically altered cell, also in cases when morphological changes are not visible. The quantitative analysis of cell deformability carried out using normal (reference) and cancerous cells and, more precisely, their characterization (qualitative and quantitative) can have a significant impact on the development of methodological approaches toward precise identification of pathological cells and would allow for more effective detection of cancer-related changes.

Characterization of N-cadherin unbinding properties in non-malignant (HCV29) and malignant (T24) bladder cells.

The expression of N-cadherin, characteristic of various cancers, very often leads to changes in the cells' adhesive properties. Thus, we sought to find out if N-cadherin expressed in various, but cancer-related cells, differs in its functional properties that could contribute to variations in cells' phenotypes. In our work, measurements of an unbinding force of a single N-cadherin molecule, probed with the same antibody both on a surface of living non-malignant (HCV29) and malignant cells (T24) of bladder cancer, were carried out with the use of an atomic force microscopy. The results show the enhanced N-cadherin level in T24 malignant cells (8.7% vs. 3.6% obtained for non-malignant one), confirmed by the Western blot and the immunohistochemical staining. The effect was accompanied by changes in unbinding properties of an individual N-cadherin molecule. Lower unbinding force values (26.1 ± 7.1 pN) in non-malignant cells reveal less stable N-cadherin complexes, as compared to malignant cells (61.7 ± 14.6 pN). This suggests the cancer-related changes in a structure of the binding site of the antibody, located at the extracellular domain of N-cadherin.

Dose response and kinetics of foci disappearance following exposure to high- and low-LET ionizing radiation.

PURPOSE: The effect of different radiation qualities on (i) 53BP1 (p53 Binding Protein 1) and p-ATM (phosphorylated ataxia telangiectasia mutated) foci induction, and (ii) on the kinetics of foci disappearance was analysed. MATERIAL AND METHODS: Normal human skin fibroblasts were exposed to 240 kV broad-field X-rays or targeted with individually counted helium ((3)He) particles or protons ((1)H) from a Charged Particle Microbeam. Anti-p-ATM and anti-53BP1 antibodies were used for foci visualisation via immunocytochemistry. RESULTS: 1 Gy of X-rays yielded approximately 33 53BP1-positive foci/cell. The ratio between the number of delivered particles and yielded tracks was found to be 1:1 and 3:1 after targeted (3)He and (1)H irradiation, respectively. It was determined that approximately 50% of radiation-induced damage was repaired as measured by loss of foci during the first 2, 6, and 10 hours following X-ray, protons, and (3)He irradiation, respectively. CONCLUSIONS: There was significant radiation quality dependence for 53BP1- and p-ATM-positive foci induction observed. Foci disappearance was radiation dose-independent in the samples irradiated with X-rays. Our results confirm that kinetics of foci disappearance depends on radiation quality, even when individual ions are targeted to cells.

Stiffness alterations of single cells induced by UV in the presence of nanoTiO2.

Nanocrystalline titanium dioxide (nanoTiO2) has been reported to generate reactive oxygen species (ROS) under UV illumination. In our studies, changes in mechanical properties of human skin fibroblasts, exposed to the oxidative stress induced in the presence of nanoTiO2 and UV light, were studied using atomic force microscopy (AFM). The exposure of cells to the action of ROS was performed at low TiO2 concentration (4 microg/mL) and under illumination with low-intensity UVA (8 and 20 mW/cm2) or UVC (0.1 mW/ cm2). AFM measurements of the cell stiffness were carried out immediately after exposure of cells to the oxidative stress. The data suggest that under illumination with low-intensity UVA nanoTiO2 generates ROS, which, in turn, damage cellular and subcellular structures. This process was detected by AFM as a marked drop in the cellular stiffness of ca. 30-75%, which occurred rapidly, in the time frame of 1 min. The photo-oxidative stress inducing the decrease of cell stiffness was cancelled in the presence of a well-established antioxidant, beta-carotene. The results highlight the sensitivity of AFM to detect early changes in mechanical properties of cells exposed to oxidative stress.

Specific detection of glycans on a plasma membrane of living cells with atomic force microscopy.

Among the many alterations of cancer cells is the expression of different surface oligosaccharides. In this work, oligosaccharide expression in living cells (cancer and reference ones) was studied with atomic force microscopy by using lectins as probes. The unbinding force obtained for the same lectin type (concanavalin A or Sambucus nigra) suggested slightly dissimilar structures of binding sites of the same ligand type. For the lectin from Phaseolus vulgaris, a much larger unbinding force indicated a distinct structure of the binding site in cancer cells. The unbinding probability confirmed a higher content of both sialic acid and mannose-containing ligands in cancer and reference cells, respectively. These results demonstrate the potential of atomic force microscopy to directly probe the presence of molecules on a living cell surface, together with the quantitative description of their expression.

Friction force microscopy as an alternative method to probe molecular interactions.

Friction force microscopy was applied to study protein-carbohydrate interactions that are important in many cellular recognition processes. The expression and structure of carbohydrates can be investigated using lectins as molecular probes since they recognize different types of sugar molecules. Lectins (concanavalin A and lentil lectin, recognizing mannose-type carbohydrates) were attached to the probing tip and carboxypeptidase Y (possessing complementary carbohydrates) was immobilized on a modified glass surface using microcontact printing. The results obtained from friction force maps and dependencies on the loading rate (measured in a physiological buffer) were divided in two distinct groups. The first group of results obtained for lectin-protein complexes was assigned to molecular recognition events, whereas the other including all control measurements was attributed to nonspecific interaction. All results presented here indicate that friction force microscopy can be successfully employed to study recognition processes.

Expression of prostate specific membrane antigen in androgen-independent prostate cancer cell line PC-3.

During the progression of prostate cancer from androgen-dependence or sensitivity to androgen-independence, the overall expression of prostate specific membrane antigen (PSMA) increases with its appearance in plasma membrane. However, surprisingly some androgen-independent metastatic prostate cancer cell lines do not express this protein. Estradiol (E2) and basic fibroblast growth factor (bFGF) due to their recognized and strong involvement in prostate growth, development, and pathology were selected with the aim of restoring the expression of PSMA in markedly dedifferentiated prostate cancer PC-3 cells and in Du 145. E2 (10(-7)-10(-11)M) and bFGF (10ng/ml) stimulated the expression of mRNAs for PSMA (2- to 4-fold increase) that apparently were further translated and processed to its membrane form in LNCaP, PC-3, and Du 145 cells. The values of interaction force between the same anti-PSMA antibodies and all studied cells were almost identical (45-64pN), indicating antigenic similarity of the membrane form of PSMA expressed in LNCaP, PC-3, and Du 145 cells.