Fourier transform infrared microspectroscopy analysis of ovarian cancerous tissues in paraffin and deparaffinized tissue samples.

Ovarian cancer is one of the deadliest cancers occurring in women. This is typically due to late diagnosis of the disease and difficult treatment. Infrared microspectroscopy is a complementary research method that can be helpful in the diagnosis of this disease, because it allows for the analysis of the tissues biomolecular composition. In this study, archival paraffin-embedded preparations of ovarian tissues, tumours and control, were used. However, the paraffin present in such specimens is a strong absorber of infrared radiation, which makes it impossible to reliably analyse the biomolecular composition of the sample. The solution to this problem is to deparaffinize the tissue before the analysis. However, the extend to which the paraffinization and deparaffinization processes influence the biomolecular composition of the tissues is unclear. Analysed tissues in the form of cores were placed in a paraffin micromatrix and FTIR measurements were performed. Then the samples were deparaffinized and the measurements were taken again. For both sets of samples (embedded in paraffin and deparaffinized) ratios of integrated peaks and massifs within the obtained spectra were calculated. The obtained ratios were compared for different types of diseased and healthy, control tissues. The Kruskal-Wallis test revealed statistically significant differences of the calculated ratios between most of the types of tissues. Random Forest models clearly showed that both samples in paraffin and deparaffinized retain enough information to classify the tissues reliably. The feature analysis revealed the most important feature for distinguishing between different types of samples, i.e. 1080 cm-1/1240 cm-1 ratio and lipid saturation for the samples embedded in paraffin and deparaffinized respectively. The study showed that the deparaffinization process leads to changes in the biomolecular composition of the analysed tissues. Despite this, classification of the tissues based on FTIR measurements remains possible.

Combined spectroscopic, biochemical and chemometric approach toward finding of biochemical markers of obesity.

BACKGROUND: Early-stage detection of subclinical obesity-driven systemic changes is a challenging area of medical diagnostics, where the most popular existing measures - such as body mass index - BMI - often fall short of providing a realistic estimate of adiposity and, therefore, of ongoing pathologies at the systemic, tissue and cellular level. In the quest for identifying new more robust diagnostic markers, whole-organ analysis of chemical elements is a promising approach for identifying candidate proxies of obesity status in the system. METHODS: Total Reflection X-ray fluorescence (TXRF) coupled with biochemical assays, chemometrics and statistical validation was used as a new integrated pipeline for marker identification in external ear samples of obese animals. The specimens were taken from obese animals fed a high calorie diet as well as from lean intact animals fed a standard diet. RESULTS: The most significant differences in the content of K, Fe, Br, and Rb between the studied groups of the animals were identified. However, with the methodology applied Rb was found the most robust biochemical discriminator of early-stage obesity effects, as validated by the logistic regression model. We observed no relationship between the levels of the elements consumed by the animals and their apparent content in the earlobe tissue samples. CONCLUSIONS: Our preliminary study confirms that obesity alters tissue trace metal metabolism and shows the proposed new approach as an accurate and reliable methodology for detecting tissue elemental obesity-related alterations. GENERAL SIGNIFICANCE: This result can be of practical significance for designing new point-of-care systems for obesity screening tests, taking advantage of direct/indirect Rb measurements.

Elemental Composition of Skeletal Muscle Fibres Studied with Synchrotron Radiation X-ray Fluorescence (SR-XRF).

Diseases of the muscle tissue, particularly those disorders which result from the pathology of individual muscle cells, are often called myopathies. The diversity of the content of individual cells is of interest with regard to their role in both biochemical mechanisms and the structure of muscle tissue itself. These studies focus on the preliminary analysis of the differences that may occur between diseased tissues and tissues that have been recognised as a reference group. To do so, 13 samples of biopsied human muscle tissues were studied: 3 diagnosed as dystrophies, 6 as (non-dystrophic) myopathy and 4 regarded as references. From these sets of muscle biopsies, 135 completely measured muscle fibres were separated altogether, which were subjected to investigations using synchrotron radiation X-ray fluorescence (SR-XRF). Muscle fibres were analysed in terms of the composition of elements such as Br, Ca, Cl, Cr, Cu, Fe, K, Mn, P, S and Zn. The performed statistical tests indicate that all three groups (dystrophies-D; myopathies-M; references-R) show statistically significant differences in their elemental compositions, and the greatest impact, according to the multivariate discriminate analysis (MDA), comes from elements such as Ca, Cu, K, Cl and S.

Study of microvessel density and the expression of vascular endothelial growth factors in adrenal gland pheochromocytomas.

Angiogenesis (neoangiogenesis), a process of neovascularization, is an essential step for local tumor growth and distant metastasis formation. We have analysed angiogenesis status: vascular architecture, microvessel density, and vascular endothelial growth factors expression in 62 adrenal pheochromocytomas: 57 benign and 5 malignant. Immunohistochemical evaluation revealed that vascular architecture and vessel density are different in the central and subcapsular areas of the tumor. Furthermore, we have observed a strong correlation between number of macrophages and microvessel density in the central and subcapsular areas of the tumor and between the expression of VEGF-A in tumor cells and microvessel density in central and subcapsular areas of the tumor. Secondary changes in these tumors influence the results and both vascular architecture and microvessel density are markedly disturbed by hemorrhagic and cystic changes in pheochromocytomas. These changes are partially caused by laparoscopic operation technique. However, no differences in vascular parameters were found between pheochromocytomas with benign and malignant clinical behavior. Our observation showed that analysis of angiogenesis, as a single feature, does not help in differentiating malignant and benign pheochromocytomas and has no independent prognostic significance. On the other hand, high microvessel density in pheochromocytoma is a promising factor for antiangiogenic therapy in malignant cases.

Biological activity of pentachlorophenol on the digestive gland cells of the freshwater mussel Unio tumidus.

Many chlorinated phenols and their derivatives are used extensively as insecticides, fungicides and herbicides by industrial and agricultural users throughout the world. Among these substances, pentachlorophenol (PCP) is a broad-spectrum biocide, which is still used as a wood preservative. In this paper, the digestive gland cells were used to assess the effect of PCP in the range of concentrations 3.75-75 microM (0.01-0.2 ppm) on oxidative DNA damage, fluidity changes and peroxidation activity in the plasma membrane. The toxic property of PCP on DNA strand breakage was studied using the comet assay. The results showed that pentachlorophenol in the range of 37.5-75 microM contributed to these lesions. To demonstrate the changes in the fluidity of plasma membrane we used the spectrofluorimetric method using two fluorescence probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 12-(9-anthroyloxy) stearic acid (12-AS). It was shown that PC did not influence the surface of plasma membrane but contributed to the increase in the fluidity of the internal region of the lipid bilayer in the range of concentrations 18.75-75 microM (0.05-0.2 ppm). We also examined the effect of PCP on the lipid peroxidation. To imply its peroxidation properties the spectrophotometry method was used to measure the level of malondialdehyde (MDA), one of the endpoints of the peroxidation of polyunsaturated fatty acids. The obtained results showed that PCP in the used doses did not initiate the formation of lipid peroxides. Thus, our investigation indicates that PCP can behave as a prooxidant agent but its action depends on the used doses and parameters chosen for the research.