Molecular Biology and Genetics / Moleküler Biyoloji ve Genetik
Permanent URI for this collectionhttps://hdl.handle.net/11147/9
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Article Epithelial-Mesenchymal Transition as a Potential Route for Dapt Resistance in Breast Cancer Cells(Walter de Gruyter GmbH, 2023) Tellı, Kubra; Ozuysal, Ozden Yalcın; Telli, Kübra; Yalçın Özuysal, ÖzdenObjectives: Notch is a conserved pathway involved in cell- fate determination and homeostasis. Its dysregulation plays a role in poor prognosis and drug resistance in breast cancer. Targeting Notch signaling via inhibition of the gamma- secretase complex is in the spotlight of modern cancer treat- ments. Gamma-secretase inhibitors (GSI) have shown suc- cessful clinical activity in treating cancers, yet the possible resistance mechanism remains unstudied. Modeling the resistance and understanding culprit molecular mechanisms can improve GSI therapies. Accordingly, the aim of this study is to generate and analyze GSI-resistant breast cancer cells. Methods: Gradually increasing doses of DAPT, a well-known GSI, were applied to MCF-7 breast cancer cell lines to generate resistance. Cell viability, migration and gene expressions were assessed by MTT, wound healing and qRT-PCR analyses. Results: DAPT-resistant MCF-7 cells exhibited abnormal expression of Notch receptors, Notch targets (HES1, HES5, HEY1), and epithelial-mesenchymal transition (EMT) markers (E-cadherin, ZO-1, SNAIL2, N-cadherin) to overcome the continuous increase in DAPT toxicity by increased migration through mesenchymal transition. Conclusions: This study prospects into the role of EMT in the potential resistance mechanism against DAPT treatment for breast cancer cells. Complementary targeting of EMT should be investigated further for a possible effect to potentiate DAPT’s anti-cancer effects.Data Paper Knockdown of Death Receptor 5 Antisense Long Noncoding Rna and Cisplatin Treatment Modulate Similar Macromolecular and Metabolic Changes in Hela Cells(TÜBİTAK - Türkiye Bilimsel ve Teknolojik Araştırma Kurumu, 2022) Gürer, Dilek Cansu; Erdoğan Vatansever, İpek; Ceylan, Çağatay; Akgül, BünyaminBackground/aim: Despite great progress in complex gene regulatory mechanisms in the dynamic tumor microenvironment, the potential contribution of long noncoding RNAs (lncRNAs) to cancer cell metabolism is poorly understood. Death receptor 5 antisense (DR5-AS) is a cisplatin inducible lncRNA whose knockdown modulates cell morphology. However, its effect on cell metabolism is unknown. The aim of this study is to examine metabolic changes modulated by cisplatin and DR5-AS lncRNA in HeLa cells. Materials and methods: We used cisplatin as a universal cancer therapeutic drug to modulate metabolic changes in HeLa cervix cancer cells. We then examined the extent of metabolic changes by Fourier transform infrared spectroscopy (FTIR). We also performed transcriptomics analyses by generating new RNA-seq data with total RNAs isolated from cisplatin-treated HeLa cells. Then, we compared cisplatin-mediated transcriptomics and macromolecular changes with those mediated by DR5-AS knockdown. Results: Cisplatin treatment caused changes in the unsaturated fatty acid and lipid-to-protein ratios and the glycogen content. These observations in altered cellular metabolism were supported by transcriptomics analyses. FTIR spectroscopy analyses have revealed that DR5-AS knockdown causes a 20.9% elevation in the lipid/protein ratio and a 76.6% decrease in lipid peroxidation. Furthermore, we detected a 3.42% increase in the chain length of the aliphatic lipids, a higher content of RNA, and a lower amount of glycogen indicating relatively lower metabolic activity in the DR5-AS knockdown HeLa cells. Interestingly, we observed a similar gene expression pattern under cisplatin treatment and DR5-AS knockdown HeLa cells. Conclusion: These results suggest that DR5-AS lncRNA appears to account for a fraction of cisplatin-mediated macromolecular ametabolic changes in HeLa cervix cancer cells.Article Citation - WoS: 4Citation - Scopus: 4Protocol for Cell Surface Biotinylation of Magnetic Labeled and Captured Human Peripheral Blood Mononuclear Cells(Elsevier, 2022) Ayaz Güner, Şerife; Acar, Mustafa Burak; Boyvat, Dudu; Güner, Hüseyin; Bozalan, Habibe; Güzel, Melis; Yıldır, Selin Kübra; Altınsoy, Nilay; Fındık, Fatma; Karakükçü, Musa; Özcan, ServetAnalysis of the surfaceome of a blood cell subset requires cell sorting, followed by surface protein enrichment. Here, we present a protocol combining magnetically activated cell sorting (MACS) and surface biotinylation of the target cell subset from human peripheral blood mononuclear cells (PBMCs). We describe the steps for isolating target cells and their in-column surface biotinylation, followed by isolation and mass spectrometry analysis of biotinylated proteins. The protocol enables in-column surface biotinylation of specific cell subsets with minimal membrane disruption.Article Citation - WoS: 1Citation - Scopus: 1Cytoplasmically Localized Trna-Derived Fragments Inhibit Translation in Drosophila S2 Cells(TÜBİTAK - Türkiye Bilimsel ve Teknolojik Araştırma Kurumu, 2022) Hamid, Syed Muhammad; Akgül, BünyaminTransfer ribonucleic acids (tRNAs) serve not only as amino acid carriers during translation but also as a template for the biogenesis of short fragments that can regulate gene expression. Despite recent progress in the function of tRNA-derived fragments (tRFs), their intracellular localization, protein partners, and role in regulating translation are not well understood. We used synthetic tRFs to investigate their localization and function in Drosophila S2 cells. Under our experimental setting, all synthetic tRFs tested were localized at distinct sites within the cytoplasm in a similar manner in Drosophila S2 cells. Cytoplasmically-localized tRFs were positioned in close proximity to GW182 and XRN1 proteins. Functionally, tRFs, which slightly suppressed proliferation in S2 cells, inhibited translation without any major shift in the polysome profile. These results suggest that 5???-tRFs are cytoplasmically-localized and regulate gene expression through inhibition of translation in Drosophila.Article Citation - WoS: 3Citation - Scopus: 3Evaluation of Variation on Myostatin (mstn) Gene of Turkish Donkey Populations in Thrace Region of Turkey(Namık Kemal Üniversitesi, 2022) Işık, Raziye; Özdil, Fulya; Meral, SenaThe study aimed to determine the MSTN gene variation in 90 donkeys reared in the Thrace region of Turkey. Myostatin (MSTN), also named GDF-8 (growth differentiation factor 8) is a part of the transforming growth factor beta (TGF-beta) superfamily and it has a negative regulator role on muscle mass, growth and development in mammalian species. MSTN gene regulates the skeletal muscle growth in a negative way and has a significant role in homeostasis of skeletal muscles. Also, in muscle fibers balance of protein has been promoted by Myostatin factor. The total of 866 bp long partial intron 1 and 2, whole exon 2 regions of MSTN gene was amplified and PCR products analysed using DNA sequencing. In this study, a novel synonymous SNP was determined as g.4183919 G>A in the second exon region of the MSTN gene which does not cause an amino acid change in the protein. The G>A transition caused a silent mutation in leucine (leu) amino acid. Alterations in mRNA level and functionality of protein can occur due to synonymous mutations. Since leucine is an important amino acid that can avoid muscle mass loss and inhibits the expression of Myostatin, it can be said that silent mutation of Leu in donkeys may have altered the muscle mass and physical factor of donkeys in this study. Mutant leucine may have a lower efficient effect on preventing loss of muscles and causes more Myostatin protein expression. The identified SNP was firstly released and the DNA sequences of the MSTN gene in Turkish donkeys was revealed for the first time with recent study. Turkish donkeys lacked these mutations that were identified before in horses, which cause for the less might require for race ability of donkeys. The sequences of MSTN gene were submitted to the NCBI GenBank with the accession number: MW970078- MW970079. Further studies are needed to conduct, on protein and molecular levels, SNPs on the MSTN gene and their association with the morphological characters that may affect economic traits in donkey breeds.Article Citation - WoS: 14Citation - Scopus: 15Noncoding Rnas in Apoptosis: Identification and Function(TÜBİTAK, 2022) Tüncel, Özge; Kara, Merve; Yaylak, Bilge; Erdoğan, İpek; Akgül, BünyaminApoptosis is a vital cellular process that is critical for the maintenance of homeostasis in health and disease. The derailment of apoptotic mechanisms has severe consequences such as abnormal development, cancer, and neurodegenerative diseases. Thus, there exist complex regulatory mechanisms in eukaryotes to preserve the balance between cell growth and cell death. Initially, protein coding genes were prioritized in the search for such regulatory macromolecules involved in the regulation of apoptosis. However, recent genome annotations and transcriptomics studies have uncovered a plethora of regulatory noncoding RNAs that have the ability to modulate not only apoptosis but also many other biochemical processes in eukaryotes. In this review article, we will cover a brief summary of apoptosis and detection methods followed by an extensive discussion on microRNAs, circular RNAs, and long noncoding RNAs in apoptosis.Article Citation - WoS: 2Citation - Scopus: 2Antiviral Microrna Expression Signatures Are Altered in Subacute Sclerosing Panencephalitis(Wolters Kluwer Medknow Publications, 2021) Tüfekçi, Kemal Uğur; Allmer, Jens; Çarman, Kürşat Bora; Bayram, Erhan; Topçu, Yasemin; Hız, Semra; Genç, Şermin; Yiş, UluçBackground: Subacute sclerosing panencephalitis (SSPE) is a chronic, progressive disease caused by a persistent infection of the measles virus. Despite extensive efforts, the exact neurodegeneration mechanism in SSPE remains unknown. MicroRNAs (miRNAs) have emerged as an essential part of cellular antiviral defense mechanisms and can be modulated by antiviral cytokines Such as interferon-beta (IFN-beta). Aims and Objectives: In this study, we aimed to elucidate the role of antiviral miRNAs in the pathogenesis of SSPE and analyze the interaction between host antiviral miRNAs and virus genes. Materials and Methods: Thirty-seven patients who were followed with SSPE and age-matched healthy children were included in the study. Peripheral blood mononuclear cell levels of miR-196b, miR-296, miR-431, and miR-448 were analyzed using quantitative polymerase chain reaction. Target predictions and pathway constructions of deregulated miRNAs were assessed. Results: Here, we showed that IFN-beta-modulated miR-196b, miR-296, and miR-431 were significantly upregulated in patients with SSPE compared with healthy controls. Besides, sequence complementarity analysis showed that miR-296 and miR-196b predicted binding regions in measles virus genomic RNA. Conclusion: Our findings suggest that antiviral miRNAs are upregulated in patients with SSPE, which could be a part of the host antiviral defense mechanism. </p>Article Citation - WoS: 3Citation - Scopus: 3Chromatographic Analysis for Targeted Metabolomics of Antioxidant and Flavor-Related Metabolites in Tomato(Bio-Protocol, 2021) Gürbüz Çolak, Nergiz; Tek, Neslihan; Frary, Anne; Doğanlar, SamiTargeted metabolomics is a useful approach to evaluate crop breeding studies. Antioxidant and flavor-related traits are of increasing interest and are considered quality traits in tomato breeding. The present study presents chromatographic methods to study antioxidants (carotenoids, vitamin C, vitamin E, phenolic compounds, and glutathione) and flavor -related characters (sugars and organic acids) in tomato. Two different extraction methods (for polar and apolar entities) were applied to isolate the targeted compounds. The extraction methods developed in this work were time and cost-effective since no further purification was needed. Carotenoids, vitamin C, glutathione, and phenolic acids were analyzed by HPLC-PDA using a RP C18 column at an appropriate wavelength for each compound. Vitamin E and sugars were analyzed by HPLC with RP C18 and NH2 columns and detected by FLD and RI detectors, respectively. In addition, organic acids were analyzed with GC-FID using a Rtx 5DA column after derivatization with MSTFA. As a result, sensitive analytical methods to quantify important plant metabolites were developed and are described herein. These methods are not only applicable in tomato but are also useful to characterize other species for flavor-related and antioxidant compounds. Thus, these protocols can be used to guide selection in crop breeding.Article Citation - WoS: 3Citation - Scopus: 3Endogenous Heat Shock Protein Groel of A. Actinomycetemcomitans Preferentially Targets Primary Human Cd8+t Cells(TÜBİTAK, 2015) Kant, Melis; Akgül, Bünyamin; Nalbant Aldanmaz, AytenApoptosis can be used to manipulate host cells by bacterial products such as bacterial heat shock proteins (Hsp). One of the virulence factors of periodontal pathogen Aggregatibacter actinomycetemcomitans is heat shock protein GroEL (AaGroEL), which has been shown to interact with host cells. AaGroEL (Hsp64) also has the potential to modulate immune system cells. In this study we used endogenous AaGroEL protein as an antigen to study bacterial Hsp-induced apoptosis in different immune system cells. Human peripheral blood mononuclear cells and cell lines were cultured with different doses (50-1000 ng/mL) of endogenous AaGroEL at various time points. Apoptosis of the cells was measured by Annexin V and 7AAD labeling. Apoptotic cells were analyzed by flow cytometry. Our data suggested that AaGroEL-responding primary CD8+ T cells were more susceptible to apoptosis than CD4+ T cells. Furthermore, the magnitude of apoptosis in the Jurkat T cell line was higher than that in primary CD8+ T cells. There was no statistically significant level of apoptosis in the chronic myeloid leukemia (K562) cell line, which belongs to myeloid lineages. Thus, A. actinomycetemcomitans GroEL protein has more potent apoptotic effect on cells that are derived from a lymphoid progenitor.Article Citation - WoS: 8Citation - Scopus: 8Simple High-Performance Liquid Chromatographic Method for Determination of Donepezil Hcl in Pharmaceutical Formulations(ACG Publications, 2020) Bulduk, İbrahim; Aydın, Beyza SultanDonepezil HCl is a hydrochloride salt of a piperidine derivative acetylcholinesterase inhibitor and, it uses in treatment demantia of Alzheimer’s disease. In this study, a sensitive and rapid HPLC-UV method was developed and validated for determination of Donepezil HCl in API and tablet dosage forms. Chromatographic separation was performed using a Ace 5 C18 (5 ?m, 250 x 4.6 mm) by using isocratic phosphate buffer at pH:2.0 and acetonitrile (55:45, v/v) mobile phase was used at the rate of 1.2 mL/min. The column temperature was set at 30 ?C and the UV detection was recorded at 268 nm. The method was validated with respect to specificity, precision, accuracy, linearity, repeatability and reproducibility parameters in a concentration range of 25-125 µg/mL. The limit of detection (LOD) and limit of quantification (LOQ) were determined as 1.40 and4.20 µg/mL, respectively. The uncertainty budget of the measurement for Donepezil HCl was estiamted as 5.80 % at 95% confidence level (k = 2).