Frequency and mutation type analysis of KRAS and BRAF genes in colorectal carcinomas in Serbian population

Uvod: Kolorektalni kancer (CRC) se po svojoj učestalosti i smrtnosti nalazi u samom vrhu svih kancera kod oba pola, kako u svetu, tako i u Srbiji. Za ranu kancerogenezu ovog maligniteta karakteristične su somatske genske promene KRAS i BRAF protoonkogena. Mutacije ovih gena predstavljaju molekularne biomarkere odgovora na ciljanu terapiju, dok su rezultati o njihovoj ulozi kao prognostičkih i prediktivnih faktora kontradiktorni. Podaci o učestalosti i tipu pojedinačnih KRAS i BRAF mutacija kod CRC-a u našoj zemlji su malobrojni, a njihova veza sa kliničko-histopatološkim karakteristikama tumora, karakteristikama bolesnika i tokom bolesti nije dovoljno ispitana. Cilj: Utvrđivanje prisustva, učestalosti i tipa KRAS mutacija i specifične BRAF p.V600E mutacije kod bolesnika sa CRC-om u populaciji Srbije i povezanost prisustva i tipa ovih mutacija sa kliničko-histopatološkim karakteristikama tumora i karakteristikama bolesnika. Takođe, ispitivanje navedenih mutacija kao prediktivnih biomarkera adjuvantne i EGFR-ciljane terapije. Materijal i metode: U radu je analizirano 188 parafinskih uzoraka karcinoma kolorektuma. DNK je izolovana komercijalnim kitom za izolaciju genomske DNK iz parafinskih tkiva (QIAamp® DNA FFPE Tissue kit, QIAGEN). Detekcija prisustva i tipa mutacija kodona 12 i 13 KRAS gena urađena je pomoću dva dijagnostički validirana testa bazirana na eseju lančane reakcije polimeraze (PCR) u realnom vremenu (DxS TheraScreen K-RAS PCR kit, QIAGEN) i PCR reakciji praćenoj reverznom hibridizacijom (KRAS StripAssayTM, ViennaLab Diagnostics). Detekcija p.V600E mutacije u BRAF genu urađena je analizom krive topljenja (HRM) PCR produkta i metodom direktnog sekvenciranja. Za statističku obradu podataka korišćeni su Fišerov egzaktni, χ2 i Log-Rank testovi...Introduction: Colorectal cancer (CRC) incidence and mortality rates are among the highest in both sexes worldwide, as well as in Serbia. The early carcinogenesis of this malignancy is characterized by somatic gene alterations of KRAS and BRAF protooncogenes. Mutations of these genes represent molecular biomarkers of response to targeted therapy, while their roles as prognostic and predictive factors are still contradictory. There are few data about the frequency and types of single KRAS and BRAF mutations in CRC in our country, and their correlation with clinicohistopathological characteristics of tumor, characteristics of patients and the course of disease is still to be established. Aim: Determination of presence, frequency and types of KRAS mutations and specific BRAF p.V600E mutation in CRC patients in Serbian population and correlation of presence and types of these mutations with tumor clinicohistopathological characteristics and patient characteristics. Also, the examination of specified mutations as potential predictive biomarkers of adjuvant and EGFR-targeted therapy. Material and methods: In this study 188 CRC paraffin samples were analyzed. DNA was extracted using the commercial kit for genomic DNA isolation from paraffin tissues (QIAamp® DNA FFPE Tissue kit, QIAGEN). Detection of presence and types of KRAS codon 12 and 13 mutations was performed with two validated diagnostic tests based on real-time polymerase chain reaction (PCR) assay (DxS TheraScreen K-RAS PCR kit, QIAGEN) and PCR reaction followed by reverse hybridization (KRAS StripAssayTM, ViennaLab Diagnostics). BRAF p.V600E mutation was assessed by high resolution melting (HRM) analysis of PCR products and automatic DNA sequencing. Fisher exact, χ2 and Log-Rank tests were used for statistical analysis..

Positive effects of naringenin on near-surface membrane fluidity in human erythrocytes

Purpose: Deformability/rheologic behavior of erythrocytes are related to near-surface membrane fluidity. Specific agents can increase erythrocyte membrane fluidity in order to adjust hemodynamics in cardiovascular diseases. Grapefruit flavanone naringenin has been proposed for potential use in an alternative therapy of cardiovascular conditions. In respect to this, we assessed here effects of two nutritionally relevant concentrations of naringenin (0.1 and 1 μg/ml) on near-surface membrane fluidity in human erythrocytes. Methods: We used electron paramagnetic resonance spectroscopy and fatty acid spin probes (5-DS and 7-DS), the spectra of which are dependent on membrane fluidity. Results: The results showed a significant (p = 0.029) increase of membrane fluidity near the hydrophilic surface in erythrocytes treated with higher concentration of naringenin. In the deeper layer, just below the erythrocyte membrane phospholipid heads, both lower and higher concentration of naringenin significantly increased membrane fluidity (p = 0.036 and p = 0.028, respectively). Conclusions: These data document the positive and dose dependent effect of naringenin on near-surface membrane fluidity in human erythrocytes, recommending its use in the cardiovascular conditions characterized by disturbed hemodynamics

Stipetum novakii ass. nova – a new association of serpentine rocky grassland vegetation (Halacsyetalia sendtneri) in Serbia

Phytosociological characteristics of grassland communities above serpentines (order Halacsyetalia sendtneri H. Ritter-Studni~ka 1970) in Serbia, are analyzed according to Braun-Blanquet methodology. In order to detect the basic floristic differentiation of analyzed communities ordinary correspondence analysiswas applied. Cluster analysis was also performed to see the structure and separation of the communities based on the floristic composition. In order to determine diagnostic species, fidelity indices with presence/ absence data and the size of all groups standardized to equal size were calculated. The new association Stipetum novakii is described in open rocky serpentine grasslands in Brdjani Gorge

Novel 1,3,4-thiadiazole-chalcone hybrids containing catechol moiety: synthesis, antioxidant activity, cytotoxicity and DNA interaction studies

Hybrid compounds that combine the 1,3,4-thiadiazole-containing catechol moiety with a chalcone motif were synthesized and examined for their antioxidant activity, cytotoxicity, and DNA-binding activity. A series of thirteen compounds showed strong antioxidant and cytotoxic effects on human acute promyelocytic leukemia HL-60 cells. Several compounds exerted good cytotoxic activities on cervical adenocarcinoma HeLa cells. The treatment of HeLa cells with IC50 and double IC50 concentrations of the compounds 5a, 5c, 5f, and 5m induced a statistically significant increase in the percentage of cells within a subG1 cell cycle phase. The examined compounds caused G2/M cell cycle arrest in HeLa cells. Each of these compounds triggered apoptosis in HeLa cells through activation of caspase-3, the main effector caspase, caspase-8, which is involved in the extrinsic apoptotic pathway, and caspase-9, which is involved in the intrinsic apoptotic pathway. All of the examined compounds decreased the expression levels of MMP2 in HeLa cells and levels of protumorigenic miR-133b. Compounds 5a and 5m lowered the expression level of oncogenic miR-21 in HeLa cells. In addition, compounds 5a, 5f, and 5m decreased the expression levels of oncogenic miR-155 while the treatment of HeLa cells with compounds 5a, 5c, and 5f increased expression of tumor-suppressive miR-206. Observed effects of these compounds on expression levels of four examined miRNAs suggest their prominent cancer-suppressive activity. An investigation by absorption and fluorescence spectroscopy showed more efficient calf thymus DNA binding activity of the compound 5m in comparison to other tested compounds. Results of a pUC19 plasmid cleavage study and comet assay showed DNA damaging activities of compounds 5a and 5c.Related to published version:[https://imagine.imgge.bg.ac.rs/handle/123456789/1168]This is the peer reviewed version of the paper: Jakovljević, K., Joksović, M. D., Matić, I., Petrović, N., Stanojković, T., Sladić, D., Vujčić, M., Janović, B., Joksović, L., Trifunović, S., & Marković, V. (2018). Novel 1,3,4-thiadiazole-chalcone hybrids containing catechol moiety: Synthesis, antioxidant activity, cytotoxicity and DNA interaction studies. Medchemcomm, 9(10), 1679–1697. [https://doi.org/10.1039/c8md00316e

The role of aldosterone inhibitors in cardiac ischemia–reperfusion injury

Myocardial ischaemia–reperfusion (I/R) injury is a well-known term for exacerbation of cellular destruction and dysfunction after the restoration of blood flow to a previously ischaemic heart. A vast number of studies that have demonstrated that the role of mineralocorticoids in cardiovascular diseases is based on the use of pharmacological mineralocorticoid receptor (MR) antagonists. This review paper aimed to summarize current knowledge on the effects of MR antagonists on myocardial I/R injury as well as postinfarction remodeling. Animal models, predominantly the Langendorff technique and left anterior descending coronary artery occlusion, have confirmed the potency of MR antagonists as preconditioning and postconditioning agents in limiting infarct size and postinfarction remodeling. Several preclinical studies in rodents have established and proved possible mechanisms of cardioprotection by MR antagonists, such as reduction of oxidative stress, reduction of inflammation, and apoptosis, therefore limiting the infarct zone. However, the results of some clinical trials are inconsistent, since they reported no benefit of MR antagonists in acute myocardial infarction. Due to this, further studies and the results of ongoing clinical trials regarding MR antagonist administration in patients with acute myocardial infarction are being awaited with great interest.This is the peer-reviewed version of the article: Dragasevic, N.; Jakovljevic, V.; Zivkovic, V.; Draginic, N.; Andjic, M.; Bolevich, S.; Jovic, S. The Role of Aldosterone Inhibitors in Cardiac Ischemia–Reperfusion Injury. Can. J. Physiol. Pharmacol. 2021, 99 (1), 18–29. [https://doi.org/10.1139/cjpp-2020-0276

Supplementary data for the article: Gojgic-Cvijovic, G. D.; Jakovljevic, D. M.; Loncarevic, B. D.; Todorovic, N. M.; Pergal, M. V.; Ciric, J.; Loos, K.; Beškoski, V.; Vrvić, M. M. Production of Levan by Bacillus Licheniformis NS032 in Sugar Beet Molasses-Based Medium. International Journal of Biological Macromolecules 2019, 121, 142–151. https://doi.org/10.1016/j.ijbiomac.2018.10.019

Supplementary material for: [https://www.sciencedirect.com/science/article/pii/S0141813018332847?via%3Dihub]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/350]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/2799

ANTIOXIDATIVE AND IMMUNOMODULATING POTENTIAL OF THE MUSHROOM Phellinus linteus

Phellinus linteus is a popular medicinal mushroom that is widely used in Asian countries. A number of studies have confirmed that P. linteus possesses exceptional biological potential useful for pharmacological applications, including anticancer and anti-inflammatory activities, as well as antidiabetic, hepatoprotective, and neuroprotective effects. The objective of the present study was to evaluate antioxidant and immunomodulatory activities of hot water polysaccharide extract obtained from the medicinal mushroom Phellinus linteus (Berk. et Curt.) Teng. FT-IR was used to study the polysaccharide profile of the extract. Its antioxidant potential was measured by the conjugated diene method in the linoleic acid model system. Immunomodulation was tested in vitro by measuring the synthesis of interferon-gamma (IFN-γ) in healthy human peripheral blood mononuclear cells (PBMCs) using enzyme linked immunosorbent assay (ELISA). The FT-IR spectrum of P. linteus hot water extracted polysaccharides showed a typical carbohydrate pattern. A small amount of proteins was also observed with characteristic absorptions at 1635, 1540 and 1412 cm-1 . Measurements of antioxidant properties in linoleic acid model system revealed relatively high antioxidant activity with EC50 value of 7.11 mg/mL. After 48 h of P. linteus polysaccharide extract incubation, the IFN-γ titer displayed immunosuppressive effect, 32.6 pg/mL. The IFN-γ titer for the suspension of PBMCs in PBS, which was used as a positive control, was found to be 135.2 pg/mL. Differences in IFN- γ contents in P. linteus extract vs. model control were strongly significant (p < 0.05). The results of this study suggest that the polysaccharide extract of P. linteus acts as a natural antioxidant and possesses immunomodulatory properties. Therefore, it can be a suitable raw material for the development of antioxidant food additives. In addition, due to the possible immunosuppressive effect P. linteus polysaccharide extract is particularly interesting and could find application in suppression of autoimmune diseases such as rheumatoid arthritis

Substrate type selection in diatom based lake water quality assessment

Various studies report contrasting results on the substrate-type effect on diatom community composition, but the particularly important question is whether or not it affects diatom-based assessments of water quality. We investigated whether the substrate type is a significant predictor of the diatom community composition and if it affects lake water quality assessment based on diatom indices. This study took place in Sava Lake (Serbia). We used glass, ceramic, willow and yew tree tiles as artificial substrates for periphyton development, and pebbles from the lake littoral as natural substrate. Results revealed differences in both the diatom community composition and diatom indices values related to the substrates. A distinction was recognized between natural, artificial wooden, and artificial inert substrates. However, the final lake quality assessment based on diatom indices was more or less similar in all substrate types in our study, and depended on value ranges associated with water quality classification and on diatom index choices. Artificial substrates in our study did show potential as an alternative for natural substrate, but further studies are required, particularly in various types of lentic ecosystems to confirm our findings and support artificial substrate employment in lake water quality assessment

Association of GSTT1 and GSTM1 gene polymorphisms with susceptibility to autoimmune diseases: A preliminary study

Autoimmune diseases (ADs) are chronic conditions initiated by the loss of immunological tolerance to self-antigens. The aim of this study was to detrmine how polymorphisms of glutathione S-transferases T1 and M1 (GSTT1 and GSTM1) genes influences on the occurrence of two autoimmune diseases: multiple sclerosis (MS) and Hashimoto's thyroditis (HT). A multiplex polymerase chain reaction (PCR) was used to detect the deletions in GSTT1 and GSTM1 genes. Our results showed that patients with ADs had significantly higher (p 0.05). This study suggests the potential role of GSTM1 deletion on ADs susceptibility, but on the other hand this study should be repeated in other patients with the same or similar diagnosis of ADs

Supplementary data for the article: Gojgic-Cvijovic, G. D.; Jakovljevic, D. M.; Loncarevic, B. D.; Todorovic, N. M.; Pergal, M. V.; Ciric, J.; Loos, K.; Beškoski, V.; Vrvić, M. M. Production of Levan by Bacillus Licheniformis NS032 in Sugar Beet Molasses-Based Medium. International Journal of Biological Macromolecules 2019, 121, 142–151. https://doi.org/10.1016/j.ijbiomac.2018.10.019

Supplementary material for: [https://www.sciencedirect.com/science/article/pii/S0141813018332847?via%3Dihub]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/350]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/2799