Petrophysical Analyses of Rock Construction Materials from a Roman Rural Settlement in Podsilo Bay on Rab Island (North-East Adriatic, Croatia).

This article presents the results of petrophysical analyses of limestones and sandstones used for the construction of the wall structures of a Roman rural settlement located in Podsilo Bay on Rab Island (Croatia). An on-site analysis of the walls indicated the use of different lithotypes, which is an uncommon case in the area. So far, no petrophysical properties of the applied materials have been tested, and their provenance has not been specified. The aim of this research was to determine their usability as construction materials in an attempt to determine the possible reasons behind the usage of multiple lithotypes and their suitability as building materials. The following procedure was used to address these issues: (1) determination of the petrographic characteristics of the rocks, (2) performance of tests to characterise the mechanical properties in a complex stress state of uniaxial tension followed by uniaxial and triaxial compression, and, finally, (3) determination of the internal structure of the rocks using methods based on X-ray imaging. Multi-proxy characteristics of the materials including numerous observations and methods were performed: optical microscopy used to characterise rock petrography and mineralogy, scanning electron microscopy (SEM) coupled with EDS, as well as grinding tests; furthermore, mechanical properties were determined on cylindrical samples in accordance with European standards. X-ray microtomography using the XµCT method enabled microscopic observations and determination of the orientation of discontinuities and the rock structure. The performed analyses allowed us to distinguish three lithological types of sandstone and two types of limestone among the examined stone blocks. Stone blocks of fine- and medium-grained sandstone with carbonate binders, as well as sparitic limestone and mudstone with calcite veins, were used to build the studied structures. The analysed blocks showed traces of partial edge processing. Despite the defects in the material structure identified using XµCT, all the types of rock were characterised by high or very high strength. High values of longitudinal wave velocity confirmed the good quality of the material. These results contribute to a better understanding of the construction process and the related technological choices, and they provide the first dataset which can be used for the reconstruction of the building's original appearance in the future.

Salinomycin Modulates the Expression of mRNAs and miRNAs Related to Stemness in Endometrial Cancer.

BACKGROUND: Salinomycin, an ionophore antibiotic, has a strong anti-cancer effect, inducing the apoptosis of cancer cells and cancer stem cells. OBJECTIVE: The aim of the study was to assess the influence of salinomycin on the expression profile of genes related to stemness and miRNA regulating their expression in endometrial cancer cells. METHODS: Endometrial cancer cells of cell line Ishikawa were exposed to salinomycin at concentrations in the range of 0.1-100 µM, with the aim of determining its pro-apoptotic potential and the concentration which would cause the death of 50% of the cells (Sulforhodamine B test). In the following stages, the cells were incubated with the drug at a concentration of 1µM for 12,24 and 48 hour periods and compared to the control. Determining the changes in the expression of the genes related to stemness and regulating their miRNA was done using the microarray technique and RTqPCR. ELISA assay was performed in order to determine the level of TGFß2, COL14A1, CDH2, WNT5A in cell culture under salinomycin treatment in comparison to the control. RESULTS: Salinomycin caused the apoptosis of cells. For the concentration of 0.1 µM, a decrease in the population of living cells by 11.9% was determined. For 1 µM, it was 49.8%, for 10 µM -69.4%, and for a concentration of 100 µM - 87.9%. The most noticeable changes in the expression caused by the addition of salinomycin into the culture were noted for mRNA: TGFß2; WNT5A (up-regulated); COL14A1; CDH2 (down-regulated), as well as miRNA: hsa-miR-411 (up-regulated); hsa-miR-200a; hsa-miR-33a; hsa-miR-199a; hsa-miR-371-5p; hsa-miR-374; hsa-miR-374b (down-regulated). CONCLUSION: It was confirmed that salinomycin has an influence on the stemness process. The most noticeable changes in the expression were noted for mRNA: TGFß2; COL14A1; CDH2; WNT5A, as well as for miRNA: hsa-miR-200a; hsa-miR-33a; hsa-miR-199a; hsa-miR-371-5p; hsa-miR-411; hsa-miR- 374a; hsa-miR-374b.

Effect of Salinomycin on Expression Pattern of Genes Associated with Apoptosis in Endometrial Cancer Cell Line.

BACKGROUND: Salinomycin is part of a group of ionophore antibiotics characterized by an activity towards tumor cells. To this day, the mechanism through which salinomycin induces their apoptosis is not fully known yet. The goal of this study was to assess the expression pattern of genes and the proteins coded by them connected with the process of programmed cell death in an endometrial cancer cell Ishikawa culture exposed to salinomycin and compared to the control. MATERIALS AND METHODS: Analysis of the effect of salinomycin on Ishikawa endometrial cancer cells (ECACC 99040201) included a cytotoxicity MTT test (with a concentration range of 0.1-100 µM), assessment of the induction of apoptosis and necrosis by salinomycin at a concentration of 1 µM as well the assessment of the expression of the genes chosen in the microarray experiment (microarray HG-U 133A_2) and the proteins coded by them connected with apoptosis (RTqPCR, ELISA assay). The statistical significance level for all analyses carried out as part of this study was p<0.05. RESULTS: It was observed that salinomycin causes the death of about 50% of cells treated by it (50.74±0.80% of all cells) at a concentration of 1µM. The decrease in the number of living cells was determined directly after treatment of the cells with the drug (time 0). The average percent of late apoptotic cells was 1.65±0.24% and 0.57±0.01% for necrotic cells throughout the entire observation period. DISCUSSION: Microarray analysis indicated the following number of mRNA differentiating the culture depending on the time of incubation with the drug: H_12 vs C = 114 mRNA, H_8 vs C = 84 mRNA, H_48 vs. C = 27 mRNA, whereas 5 mRNAs were expressed differently at all times. During the whole incubation period of the cells with the drug, the following dependence of the expression profile of the analyzed transcripts was observed: Bax>p53>FASL>BIRC5>BCL2L. CONCLUSION: The analysis carried out indicated that salinomycin, at a concentration of 1 µM, stopped the proliferation of 50% of endometrial cancer cells, mainly by inducing the apoptotic process of the cells. The molecular exponent of the induction of programmed cell death was an observed increase in the transcriptional activity of pro-apoptotic genes: Bax;p53;FASL and a decrease in the expression of anti-apoptotic genes: BCL2L2; BIRC5.

Assessment of the Usefulness of the SEMA5A Concentration Profile Changes as a Molecular Marker in Endometrial Cancer.

BACKGROUND: Semaphorin 5A (SEMA5A) functions not only in the nervous system but also in cancer transformation where its role has not yet been sufficiently studied and described. OBJECTIVE: The aim of the study was to determine the changes in SEMA5A expression in endometrial cancer at various degrees of its differentiation (G1-G3) compared to control. MATERIALS AND METHODS: The study group consisted of 45 patients with endometrial cancer at various grades: G1, 17; G2, 15; G3, 13. The control consisted of 15 women without neoplastic changes in the routine gynecological examination. The statistical analysis of immunohistochemical assessment of SEMA5A level was carried out using the Statistica 12 program based on the Kruskal-Wallis test and Dunn's post-hoc test (p<0.05). RESULTS: The expression of SEMA5A (optical density) was observed in the control group (Me = 103.43) and in the study group (G1, Me = 140.72; G2, Me = 150.88; G3, Me = 173.77). Differences in expression between each grade and control and between individual grades turned out to be statistically significant (p<0.01). The protein level of SEMA5A expression increased with the decreasing degree of endometrial cancer differentiation. CONCLUSION: In our research, we indicated the overexpression of SEMA5A protein in endometrial cancer. It is a valuable starting point for further consideration of the role of SEMA5A as a new supplementary molecular marker in endometrial cancer.

Assessment of Expression of Homeobox A5 in Endometrial Cancer on the mRNA and Protein Level.

BACKGROUND: Endometrial cancer is one of the most common gynecological cancer in the developed countries and occurs mainly in postmenopausal women. Angiogenesis is important for cancer formation as it provides nutrients for growing tumor mass. Most tumors do not show detectable Homeobox A5 (HOXA5 level), suggesting its potential role as a cancer suppressor. It was demonstrated that HOXA5 is involved in the progression of various types of cancer and the loss of its expression correlates with higher pathological grade and poorer outcome. OBJECTIVE: The aim of the study was to evaluate HOXA5 expression at transcriptome and protein levels. MATERIALS AND METHODS: The study enrolled 45 women diagnosed with endometrial cancer and 15 without neoplastic changes. The histopathological examination allowed us to divide cancer tissue samples according to the degree of histological differentiation: G1, 17; G2, 15; G3, 13. The expression of the HOXA5 protein was determined by immunohistochemistry. Microarray and RT-qPCR techniques were used to assess HOXA5 expression at the mRNA level. RESULTS: The reaction to the HOXA5 protein was only visible in glandular cells in G1 endometrial cancer and was lower compared to the control. In grades 2 and 3, reactions were noted at the limit of the method's sensitivity. In addition, reduced HOXA5 expression was observed at the transcriptome level. CONCLUSION: HOXA5 may become a potential complementary molecular marker, allowing early detection of neoplastic changes in the endometrium. It also seems that detection of HOXA5 at the mRNA and protein levels may be helpful in improving the accuracy of diagnosis and planning effective oncological therapy.

Expression Profile of VEGF-C, VEGF-D, and VEGFR-3 in Different Grades of Endometrial Cancer.

BACKGROUND: Vascular endothelial growth factor (VEGF)-C, -D, and VEGF receptor-3 are proteins characterized as crucial for tumor lymphangiogenesis. It is accompanied by angiogenesis during wound healing, but also in the neoplastic process. The research studies have shown that the lymphatic system plays a key role in the progression of carcinogenesis. OBJECTIVE: The aim of this study was to evaluate changes in the expression of VEGF-C, VEGF-D and VEGFR-3 in different grades of endometrial cancer (G1-G3). METHODS: The study included 45 patients diagnosed with endometrial cancer (G1=17; G2=15; G3=13) and 15 patients without neoplastic changes. The expression of VEGF-C, VEGF-D, and VEGFR-3 was assessed using microarray technique and immunohistochemistry. Statistical analysis was performed using the one-way ANOVA and Tukey's post-hoc test. RESULTS: Statistically significant changes in the expression at the transcriptome level were found only in the case of VEGF-C (G1 vs. C, fold change - FC = -1.15; G2 vs. C, FC = -2.33; G3 vs. C, FC = - 1.68). However, VEGF-D and VEGFR-3 were expressed at the protein level. Analysis of VEGF-D expression showed that the optical density of the reaction product in G1 reached 101.7, while the values in G2 and G3 were 142.7 and 184.4, respectively. For VEGF-R3, the optical density of the reaction product reached the following levels: 72 in control, 118.77 in G1, 145.8 in G2, and 170.9 in G3. CONCLUSION: An increase in VEGF-D and VEGFR-3 levels may indicate that VEGF-D-dependent processes are intensified along with the dedifferentiation of tumor cells. The lack of VEGF-C expression in endometrial cancer samples may suggest that this tumor is characterized by a different mechanism of metastasis than EMT. Our study emphasizes that when analyzing the metastatic potential of cancer, the expression of more than one factor should be taken into account.

Changes in Expression Pattern of SEMA3F Depending on Endometrial Cancer Grade - Pilot Study.

BACKGROUND: In the course of neoplastic diseases, a reduction in SEMA3F expression is observed, which translates into an increase in the proliferative and proangiogenic potential of cells forming the tumor and the surrounding microenvironment. OBJECTIVE: The aim of this study was to determine the changes in SEMA3F level in endometrial cancer depending on its grade. METHODS: The study material consisted of tissue samples: 15 without neoplastic changes (control group) and 45 with endometrial cancer (G1, 17; G2, 15; G3, 13; study group). SEMA3F expression was assessed using the immune-histochemical method. RESULTS: The expression of SEMA3F was observed in the control group (Me = 159.38) and in the study group (G1, Me = 121.32; G2, Me = 0; G3, Me = 130.37). Differences between each grade and control and between individual grades were statistically significant. There were no significant correlations between SEMA3F expression and weight and Body Mass Index (BMI). The reduced SEMA3F expression in tumor tissue compared to healthy tissue indicates that this protein plays key roles in proliferation and angiogenesis. CONCLUSION: We found that depending on the severity of the disease, cancer adopts different survival strategies, where SEMA3F plays an important role. As a molecular marker, SEMA3F is not sensitive to weight and BMI.