Optimization of PCR Conditions for Amplification of GC-Rich EGFR Promoter Sequence

BackgroundPolymerase chain reaction (PCR) is an extremely sensitive method that often demands optimization, especially when difficult templates need to be amplified. The aim of the present study was to optimize the PCR conditions for amplification of the epidermal growth factor receptor (EGFR) promoter sequence featuring an extremely high guanine-cytosine (GC) content in order to detect single nucleotide polymorphisms -216G gt T and -191C gt A. MethodsGenomic DNA used for amplification was extracted from formalin-fixed paraffin-embedded lung tumor tissue and PCR products were detected by agarose gel electrophoresis. ResultsResults showed that addition of 5% dimethyl sulfoxide (DMSO), as well as DNA concentration in PCR reaction of at least 2 g/ml, were necessary for successful amplification. Due to high GC content, optimal annealing temperature was 7 degrees C higher than calculated, while adequate MgCl2 concentration ranged from 1.5 to 2.0 mM. ConclusionIn conclusion, EGFR promoter region is a difficult PCR target, but it could be amplified after optimization of MgCl2 concentration and annealing temperature in the presence of DMSO and the DNA template of acceptable concentration

Frequencies of EGFR single nucleotide polymorphisms in non-small cell lung cancer patients and healthy individuals in the Republic of Serbia: a preliminary study

The purpose of this study was to determine the frequencies of EGFR -216G gt T, -191C gt A, and 181946C gt T in Serbian non-small cell lung cancer (NSCLC) patients, as well as to compare it with healthy individuals, in order to assess their potential importance for lung cancer in Serbia. The study involved 56 NSCLC patients and 53 unrelated healthy volunteers, and genotyping was performed on DNA samples obtained from formalin-fixed paraffin-embedded lung tumor tissue and blood, respectively. This was the first time to show genotype frequencies of those single nucleotide polymorphisms for this study group from the territory of the Republic of Serbia. There was very strong evidence of association between age and death due to lung cancer (Pearson chi-square = 43.439, df = 2, p lt 0,001), as well as between ever smoking and death due to lung cancer (Pearson chi-square = 31.727, df = 1, p lt 0.001). When dominant genetic model (GG vs. GT+TT) was used for -216G gt T, we have found significant association (p = 0.012) between -216GG genotype and NSCLC patients within smokers' subgroup. So, carriers of -216GG genotype had higher risk (OR = 4.33, 95 % CI = 1.324-14.179) than noncarriers (GT and TT) for developing non-small cell lung cancer in our patients

Spectroscopic and Computational Study of the Protonation Equilibria of Amino-Substituted benzo[b]thieno[2,3-b]pyrido[1,2-a]benzimidazoles as Novel pH-Sensing Materials

We present the synthesis and analytical, spectroscopic and computational characterization of three amino-substituted benzo[b]thieno[2, 3- b]pyrido[1, 2-a]benzimidazoles as novel pH probes with a potential application in pH-sensing materials. The designed systems differ in the number and position of the introduced isobutylamine groups on the pentacyclic aromatic core, which affects their photophysical and acid- base properties. The latter were investigated by UV-Vis absorption and fluorescence spectroscopies and interpreted by DFT calculations. An excellent agreement in experimentally measured and computationally determined pKa values and electronic excitations suggests that all systems are unionized at neutral pH, while their transition to monocationic forms occurs at pH values between 3 and 5, accompanied by substantial changes in spectroscopic responses that make them suitable for detecting acidic conditions in solutions. Computations identified imidazole imino nitrogen as the most favorable protonation site, further confirmed by analysis of perturbations in the chemical shifts of 1H and 13C NMR, and showed that the resulting basicity emerges as a compromise between the basicity-reducing effect of a nearby nitrile and a favorable contribution from the attached secondary amines. With this in mind, we designed a system with three amino substituents for which calculations predict pKa = 7.0 that we suggest as an excellent starting point for a potential pH sensor able to capture solution changes during the transition from neutral towards acidic media

Supplementary data for the article: Sović, I.; Cindrić, M.; Perin, N.; Boček, I.; Novaković, I.; Damjanović, A.; Stanojković, T.; Zlatović, M.; Hranjec, M.; Bertoša, B. Biological Potential of Novel Methoxy and Hydroxy Substituted Heteroaromatic Amides Designed as Promising Antioxidative Agents: Synthesis, 3D-QSAR Analysis, and Biological Activity. Chemical Research in Toxicology 2019, 32 (9), 1880–1892. https://doi.org/10.1021/acs.chemrestox.9b00256

Supplementary material for: [https://doi.org/10.1021/acs.chemrestox.9b00256]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3851]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3914

Supplementary data for the article: Sović, I.; Cindrić, M.; Perin, N.; Boček, I.; Novaković, I.; Damjanović, A.; Stanojković, T.; Zlatović, M.; Hranjec, M.; Bertoša, B. Biological Potential of Novel Methoxy and Hydroxy Substituted Heteroaromatic Amides Designed as Promising Antioxidative Agents: Synthesis, 3D-QSAR Analysis, and Biological Activity. Chemical Research in Toxicology 2019, 32 (9), 1880–1892. https://doi.org/10.1021/acs.chemrestox.9b00256

Supplementary material for: [https://doi.org/10.1021/acs.chemrestox.9b00256]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/3851]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3914

Chemical Composition, Antioxidant and Antibacterial Activity of Different Extracts of Poplar Type Propolis

Chemical composition, antioxidant activity and in vitro antibacterial activity of propolis from the central, continental part of Croatia were studied. Propolis hydro-ethanolic extracts (PHEE), prepared using three different methods and two solvent mixtures contained high amounts of flavonoids (20.95–28.11 % TIC), aromatic acids (8.17–15.91 % TIC) and their esters (9.27-11.91 % TIC). The PHEE obtained in this study showed high antioxidant activity (DPPH IC50 values from 9.96–19.95 µg/ml and FRAP 38.0–41.9 mM Fe2+/mg PHEE). Despite differences in composition, the PHEE samples exhibited significant antibacterial activities, affecting tested strains of Staphylococcus aures, Enterococcus faecalis, Escherichia coli and Moraxella catarrhalis. The use of different solvent ratio and extraction procedures selectively increases or decreases the content of specific propolis components in the extract which can have a beneficial effect on the application of propolis extracts. This work is licensed under a Creative Commons Attribution 4.0 International License