Molecular Biology and Genetics / Moleküler Biyoloji ve Genetik
Permanent URI for this collectionhttps://hdl.handle.net/11147/9
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Article Citation - WoS: 4Citation - Scopus: 4Sensitive and Specific Detection of Ligands Using Engineered Riboswitches(Elsevier Ltd., 2018) Morse, Daniel P.; Nevins, Colin E.; Aggrey-Fynn, Joana Efua; Bravo, Rick J.; Pfaeffle, Herman O.I.; Laney, Jess E.Riboswitches are RNA elements found in non-coding regions of messenger RNAs that regulate gene expression through a ligand-triggered conformational change. Riboswitches typically bind tightly and specifically to their ligands, so they have the potential to serve as highly effective sensors in vitro. In B. subtilis and other gram-positive bacteria, purine nucleotide synthesis is regulated by riboswitches that bind to guanine. We modified the xpt-pbuX guanine riboswitch for use in a fluorescence quenching assay that allowed us to specifically detect and quantify guanine in vitro. Using this assay, we reproducibly detected as little as 5 nM guanine. We then produced sensors for 2′-deoxyguanosine and cyclic diguanylate (c-diGMP) by appending the P1 stem of the guanine riboswitch to the ligand-binding domains of a 2′-deoxyguanosine riboswitch and a c-diGMP riboswitch. These hybrid sensors could detect 15 nM 2′-deoxyguanosine and 3 nM c-diGMP, respectively. Each sensor retained the ligand specificity of its corresponding natural riboswitch. In order to extend the utility of our approach, we developed a strategy for the in vitro selection of sensors with novel ligand specificity. Here we report a proof-of-principle experiment that demonstrated the feasibility of our selection strategy.Article Citation - WoS: 1Cellular Distribution of Invadopodia Is Regulated by Nanometer Scale Surface Protein Patterns(Elsevier Ltd., 2017) Batı Ayaz, Gizem; Can, Ali; Pesen Okvur, DevrimInvadopodia are proteolytic structures formed by cancer cells. It is not known whether their cellular distribution can be regulated by the organization of the extracellular matrix or the organization of the golgi complex or whether they have an adhesion requirement. Here, we used electron beam lithography to fabricate fibronectin (FN) nanodots with isotropic and gradient micrometer scale spacings on K-casein and laminin backgrounds. Investigating cancer cells cultured on protein nanopatterns, we showed that (i) presence of FN nanodots on a K-casein background decreased percent of cells with neutral invadopodia polarization compared to FN control surfaces; (ii) presence of a gradient of FN nanodots on a K-casein background increased percent of cells with negative invadopodia polarization compared to FN control surfaces; (iii) polarization of the golgi complex was similar to that of invadopodia in agreement with a spatial link; (iv) local adhesion did not necessarily appear to be a prerequisite for invadopodia formation.Article Citation - WoS: 51Citation - Scopus: 58Synthesis, Cytotoxic and Antimicrobial Activities of Novel Cobalt and Zinc Complexes of Benzimidazole Derivatives(Elsevier Ltd., 2017) Apohan, Elif; Yılmaz, Ülkü; Yılmaz, Özgür; Serindağ, Ayfer; Küçükbay, Hasan; Yeşilada, Özfer; Baran, YusufIn this study fourteen novel cobalt (II) or zinc (II) complexes of benzimidazoles were synthesized from the 1-(4-substitutedbenzyl)-1H-benzimidazoles and CoCl2·6H2O or ZnCl2. Cytotoxic activities of novel complexes were investigated against lung cancer cells (A549) and BEAS-2B. Three of the examined compounds (1, 4 and 5) showed high cytotoxic activity against A549. While the IC50 of the cisplatin was 2.56 μg/mL for A549 cells at 72 h, the IC50 values of compounds 1, 4 and 5 were 1.97, 1.87 and 1.9 μg/mL, respectively. IC50 values of these compounds for BEAS-2B cells were higher than the IC50 values for A549. While the IC50 values for BEAS-2B cells were 59.8, 24.5 and 32.67 μg/mL, respectively, the IC50 of the cisplatin was determined as 2.53 μg/mL in the present work. Three of the compounds have also high antimicrobial activity against all the microorganisms used.Article Citation - WoS: 56Citation - Scopus: 66Molecular Mechanisms of Quercitrin-Induced Apoptosis in Non-Small Cell Lung Cancer(Elsevier Ltd., 2014) Çinçin, Zeynep Birsu; Ünlü, Miray; Kıran, Bayram; Bireller, Elif Sinem; Baran, Yusuf; Çakmakoğlu, BediaBackground and Aims: Quercitrin (QR; quercetin-3-O-rhamnoside) has been used previously as an antibacterial agent and has been shown to inhibit the oxidation of low-density lipoproteins and prevent an allergic reaction. Furthermore, it was demonstrated that quercitrin exerts protective effects against H2O2-induced dysfunction in lung fibroblast cells. However, the mechanisms of quercitrin effects on cancer cell proliferation and apoptosis is not well understood. The aim of this study is to investigate the cytotoxic and apoptotic effects of quercitrin and the molecular mechanisms of quercitrin-induced apoptosis in non-small cell lung cancer (NSCLC) cell lines. Methods: Time- and dose-dependent antiproliferative and apoptotic effects of quercitrin determined by WST-1cell proliferation assay, lactate dehydrogenase (LDH) cytotoxicity assay, determination of nucleosome enrichment factor, changes in caspase-3 activity, loss of mitochondrial membrane potential (MMP) and also the localization of phosphatidylserine in the plasma membrane. Changes in whole genome gene expression levels were examined by Illumina Human HT-12v4 beadchip microarrays. Results: There were significant increases in caspase-3 activity, loss of MMP, and increases in apoptotic cell population in response to quercitrin in A549 and NCI-H358 NSCLC cells in a time- and dose-dependent manner. Conclusion: Our results demonstrated that genes involved in leukocyte transendothelial migration, cell adhesion and phosphatidylinositol signaling system pathways were the most statistically significant pathways in NCI-H358 and A549cells. These results revealed that quercitrin has antiproliferative and apoptotic effects on lung cancer cells by modulating the immune response. After confirming its anticarcinogenic effects invivo, quercitrin could be a novel and strong anticancer agent against NSCLC.Article Citation - WoS: 246Citation - Scopus: 245Step-By Quantitative Analysis of Focal Adhesions(Elsevier Ltd., 2014) Horzum, Utku; Özdil, Berrin; Pesen Okvur, DevrimFocal adhesions (FAs) are specialized adhesive structures which serve as cellular communication units between cells and the surrounding extracellular matrix. FAs are involved in signal transduction and actin cytoskeleton organization. FAs mediate cell adhesion, which is a critical phenomenon in cancer research. Since cells can form many and micrometer scale FAs, their quantitative analysis demands well-optimized image analysis approaches [1-3]. Here, we have optimized the analysis of FAs of MDA-MB-231 breast cancer cells. The optimization is based on proper processing of immunofluorescence images of vinculin, which is one of the markers of FAs. All image processing steps are carried out using the ImageJ software, which is freely available and in the public domain. The advantages of our method are:The analysis steps are simplified by combining different plugins of the ImageJ program.FAs are better detected with minimal false negatives due to optimized processing of fluorescent images.This approach can be applied to quantify a variety of fluorescent images comprising focal and/or localized signals within a high background such as FAs, one of the many complex signaling structures in a cell.Article Citation - WoS: 6Citation - Scopus: 6Fabrication of 3d Controlled in Vitro Microenvironments(Elsevier Ltd., 2014) Özdil, Berrin; Önal, Sevgi; Oruç, Tuğçe; Pesen Okvur, DevrimMicrofluidics-based lab-on-a-chips have many advantages, one of which is to provide physiologically relevant settings for cell biology experiments. Thus there is an ever increasing interest in their fabrication. Our goal is to construct three dimensional (3D) Controlled in vitro Microenvironments (CivMs) that mimic the in vivo microenvironments. Here, we present our optimized fabrication method that works for various lab-on-a-chip designs with a wide range of dimensions. The most crucial points are:While using one type of SU-8 photoresist (SU-2075), fine tuning of ramp, dwell time, spin speed, durations of soft bake, UV exposure and development allows fabrication of SU-8 masters with various heights from 40 to 600 μm.Molding PDMS (polydimethylsiloxane) at room temperature for at least two days instead of baking at higher temperatures prevents not only tears and bubbles in PDMS stamps but also cracks in the SU-8 master.3D nature of the CivMs is ensured by keeping the devices inverted during gel polymerization.Article Citation - WoS: 34Citation - Scopus: 41Autologous Rabbit Adipose Tissue-Derived Mesenchymal Stromal Cells for the Treatment of Bone Injuries With Distraction Osteogenesis(Elsevier Ltd., 2013) Sunay, Özgür; Can, Geylani; Çakır, Zeynep; Denek, Ziya; Kozanoglu, İlknur; Erbil, Güven; Yılmaz, Mustafa; Baran, YusufBackground aims: Adipose tissue-derived mesenchymal stromal cells (MSCs) have a higher capacity for proliferation and differentiation compared with other cell lineages. Although distraction osteogenesis is the most important therapy for treating bone defects, this treatment is restricted in many situations. The aim of this study was to examine the therapeutic potential of adipose tissue-derived MSCs and osteoblasts differentiated from adipose tissue-derived MSCs in the treatment of bone defects. Methods: Bone defects were produced in the tibias of New Zealand rabbits that had previously undergone adipose tissue extraction. Tibial osteotomy was performed, and a distractor was placed on the right leg of the rabbits. The rabbits were placed in control (group I), stem cell (group II) and osteoblast-differentiated stem cell (group III) treatment groups. The rabbits were sacrificed, and the defect area was evaluated by radiologic, biomechanical and histopathologic tests to examine the therapeutic effects of adipose tissue-derived MSCs. Results: Radiologic analyses revealed that callus density and the ossification rate increased in group III compared with group I and group II. In biomechanical tests, the highest ossification rate was observed in group III. Histopathologic studies showed that the quality of newly formed bone and the number of cells active in bone formation were significantly higher in group III rabbits compared with group I and group II rabbits. Conclusions: These data reveal that osteoblasts differentiated from adipose tissue-derived MSCs shorten the consolidation period of distraction osteogenesis. Stem cells could be used as an effective treatment for bone defects.Article Citation - WoS: 20Citation - Scopus: 20Genome-Wide Identification of Genes That Play a Role in Boron Stress Response in Yeast(Elsevier Ltd., 2011) Uluışık, İrem; Kaya, Alaattin; Ünlü, Ercan Selçuk; Avşar, Kadir; Karakaya, Hüseyin Çağlar; Yalçın, Talat; Koç, AhmetBoron is an essential micronutrient for plants and it is either necessary or beneficial for animals. Studies identified only few genes related to boron metabolism thus far and details of how boron is imported into cells and used in cell metabolism are largely unknown. In order to identify genes that play roles in boron metabolism, we screened the entire set of yeast haploid deletion mutants and identified 6 mutants that were resistant to toxic levels of boron, and 21 mutants that were highly sensitive to boron treatment. Furthermore, we performed a proteomic approach to identify additional proteins that are significantly up-regulated by boron treatment. Our results revealed many genes and pathways related to boron stress response and suggest a possible link between boron toxicity and translational control.Article Citation - WoS: 23Citation - Scopus: 24Proteasome Inhibitor Bortezomib Increases Radiation Sensitivity in Androgen Independent Human Prostate Cancer Cells(Elsevier Ltd., 2010) Göktaş, Serdar; Baran, Yusuf; Ural, Ali Uğur; Yazıcı, Sertaç; Aydur, Emin; Başal, Şeref; Avcu, Ferit; Pekel, Aysel; Dirican, Bahar; Beyzadeoğlu, MuratObjectives: To investigate the effects of a strong proteasome inhibitor, bortezomib alone or in combination with radiotherapy on androgen-independent DU145 human prostate cancer cells. Proteasomes play important roles in cell cycle, proliferation, apoptosis, angiogenesis, and cellular resistance to chemotherapy and radiotherapy. Methods: Increasing concentrations of bortezomib alone or in combination with radiation were applied to DU145 cells and IC50 values that inhibited cell growth by 50% were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium-bromide assay. Apoptosis was determined using annexin V staining by flow cytometry. mRNA levels of proapoptotic caspase-3 and antiapoptotic Bcl-2 genes were examined by reverse transcriptase polymerase chain reaction. Results: The IC50 value of bortezomib was found to be 28 μm although 400- and 800-cGy radiation decreased the cell proliferation by 14% and 28%, respectively. In 400- and 800-cGy radiation applied DU145 cells, IC50 value of bortezomib decreased to 23- and 12 μm, respectively. Exposure to 5 μm bortezomib for 48 hours caused apoptosis in 35% of the population whereas 800-cGy radiation resulted apoptosis in 14% of cells. However, 42% of DU145 cells that were exposed to 800 cGy and 5 μm bortezomib underwent apoptosis. Reverse transcriptase polymerase chain reaction results showed a significant decrease in mRNA levels of antiapoptotic Bcl-2 gene and an increase in proapoptotic caspase-3 gene expression in the combination group compared to control group. Conclusions: Bortezomib increases radiation sensitivity in androgen-independent human DU145 prostate cancer cells through inhibition of Bcl-2 and induction of caspase-3 genes. © 2010 Elsevier Inc. All rights reserved.Article Citation - WoS: 3Citation - Scopus: 3Regulation of Mrna Stability Through a Pentobarbital-Responsive Element(Elsevier Ltd., 2007) Akgül, Bünyamin; Tu, Chen-Pei D.Pentobarbital, a general anesthetic and non-genotoxic carcinogen, can induce gene expression by activating transcription. In the Drosophila glutathione S-transferase D21 (gstD21) gene, pentobarbital's regulatory influence extends to the level of mRNA turnover. Transcribed from an intronless gene, gstD21 mRNA is intrinsically very labile. But exposure to pentobarbital renders it stabilized beyond what can be attributed to transcriptional activation. We aim here to identify cis-acting element(s) of gstD21 mRNA as contributors to the molecule's pentobarbital-mediated stabilization. In the context of hsp70 5′UTR and the 3′UTR of act5C, gstD21 mRNA, minus its native UTRs, is stable. Maintaining the same context of heterologous UTRs, we can reconstitute using the full-length gstD21 sequence the inherent instability of gstD21 mRNA and its stabilization by pentobarbital. Transgenic flies that express these chimeric gstD21 mRNA exhibit decay intermediates lacking 3′UTR, which are not stabilized by PB treatment. The 3′UTR sequence, when inserted downstream from a reporter transcript, stabilizes it 1.6-fold under PB treatment. The analysis of the decay intermediates suggests a polysome-associated decay pattern. We propose a regulatory model that features a 59-nucleotide pentobarbital-responsive element (PBRE) in the 3′UTR of gstD21 mRNA.