Moleküler Biyoloji ve Genetik Bölümü / Department of Molecular Biology and Genetics

Permanent URI for this collectionhttps://hdl.handle.net/11413/6788

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Open Access
Assessing the Efficacy of a Novel Sperm-Washing Medium Enriched With Serotonin, L-Carnitine, and Coenzyme Q10: An Observational Cohort Study
(Wolters Kluwer Medknow Publications, 2024) DOĞAN, SİNEM; Aydın, Turgut; Koroglu, Nadiye; Yılmazer, Yasemin; Albayrak, Nazlı; Çetin, Fadime; Moshfeghi, Elnaz; ÇELİK, ÖZGE
This observational cohort study investigated the potential of a novel sperm-washing medium (SWM) enriched with serotonin (5-HT), L-carnitine (L-C), and coenzyme Q10 (CoQ10) to enhance sperm motility and reduce DNA damage. It compared this innovative medium (5-HT/L-C/CoQ10 SWM) with two widely used commercial media (SWM 1 and SWM 2). Ninety-eight volunteers from an infertility clinic provided semen samples, which were divided into three aliquots for analysis in different SWMs: group 1, SWM was composed of hydroxyethyl piperazineethanesulfonic acid (HEPES), sodium bicarbonate, human serum albumin (HSA), taurine, and gentamicin sulfate (SWM 1); group 2, SWM was composed of HEPES, sodium bicarbonate, and HSA (SWM 2); and group 3, SWM was composed of HEPES-buffered human tubal fluid supplemented with 5-HT, L-C, and CoQ10 (5-HT/L-C/CoQ10 SWM). Sperm motility was categorized as progressive, nonprogressive, or immotile. Apoptosis, reactive oxygen species (ROS) production, and DNA fragmentation were also assessed. There were no significant differences in total or progressive sperm motility among the groups. Spermatozoa in group 3 exhibited reduced apoptosis, necrosis, and ROS levels and increased viability. No significant differences were observed in the DNA fragmentation index among groups. The 5-HT/L-C/CoQ10 SWM reduced sperm oxidative stress and apoptosis compared with those of the two commercially available SWMs, suggesting that 5-HT/L-C/CoQ10 SWM could be useful for enhancing in vitro fertilization success rates.
Open Access
Biochemical and Proteomic Analyses in Drought-Tolerant Wheat Mutants Obtained by Gamma Irradiation
(MDPI, 2024) Şen, Ayşe; GÜMÜŞ, TAMER; Temel, Aslıhan; Öztürk, İrfan; ÇELİK, ÖZGE
The bread wheat cultivar (Triticum aestivum L. cv. Sagittario) as a parental line and its mutant, drought-tolerant lines (Mutant lines 4 and 5) were subjected to polyethylene glycol (PEG)-induced drought. Drought stress resulted in decreased chlorophyll levels and the accumulation of proline and TBARS, despite increases in activities of catalase, peroxidase, and superoxide dismutase enzymes. Transcription of the genes encoding these enzymes and delta-1-pyrroline 5-carboxylase synthetase was induced by drought. 2-DE gel electrophoresis analysis identified differentially expressed proteins (DEPs) in the mutant lines, which are distinguished by "chloroplast", "mitochondrion", "pyruvate dehydrogenase complex", and "homeostatic process" terms. The drought tolerance of the mutant lines might be attributed to improved photosynthesis, efficient ATP synthesis, and modified antioxidant capacity. In addition to proteomics data, the drought tolerance of wheat genotypes might also be assessed by chlorophyll content and TaPOX gene expression. To our knowledge, this is the first proteomic analysis of gamma-induced mutants of bread wheat. These findings are expected to be utilized in plant breeding studies.
Open Access
Examining the Effect of Metformin on Cell Death Mechanisms in Relation to Hippo Signaling in MDA-MB-231 Breast Cancer Cells
(Si̇vas Cumhuri̇yet Üni̇versi̇tesi̇, Fen Fakültesi̇, 2024) RENCÜZOĞULLARI, ÖZGE; SONALP, ZEYNEP GÜLŞAH
Breast cancer is one of the most common cancer types in women in the world and our country. Antitumorigenic activity is achieved with various therapeutic drugs by directly suppressing the constantly active PI3K/Akt/mTOR signaling pathway or enabling AMPK activation. AMPK, a positive regulator of autophagy, ensures the induction of autophagy by suppressing the Akt/mTOR pathway. Metformin, an anti-diabetic drug, achieves its anti-tumorigenic effect by activating AMPK. Deregulation of the Hippo signaling pathway is a new therapeutic target because it causes cancer cells to become aggressive and evade cell death mechanisms. The study aims to reveal the effects of metformin treatment on Hippo signaling pathway activity on apoptosis and autophagy, depending on drug treatment in MDA-MB-231 breast cancer cells. Metformin decreased the cell viability through induction of mitochondria membrane potential loss in dose and time dependent manner in MDA-MB-231 cells. The colony forming potential of the MDA-MB-231 cells were suppressed by 10 mM metformin treatment which was induced apoptotic cell death and autophagy by increasing Bim, Bad, Bak and cleavage of caspase 3, 9, PARP and Beclin1, Atg5 and Atg7. Moreover, Hippo signaling related protein levels showed remarkable increase due to metformin treatment. It was shown that metformin treatment increased the activity of the hippo signaling pathway, resulting in the induction of apoptosis and autophagy.
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Lidocaine Inhibits Rat Prostate Cancer Cell Invasiveness and Voltage-Gated Sodium Channel Expression in Plasma Membrane
(Springer, 2024) Rizaner, Nahit; Fraser, Scott P.; Gül, İlknur Bugan; Purut, Esma; Djamgoz, Mustafa B. A.; ALTUN, SEYHAN
There is increasing evidence, mostly from breast cancer, that use of local anaesthetics during surgery can inhibit disease recurrence by suppressing the motility of the cancer cells dependent on inherent voltage-gated sodium channels (VGSCs). Here, the possibility that lidocaine could affect cellular behaviours associated with metastasis was tested using the Dunning cell model of rat prostate cancer. Mostly, the strongly metastatic (VGSC-expressing) Mat-LyLu cells were used under both normoxic and hypoxic conditions. The weakly metastatic AT-2 cells served for comparison in some experiments. Lidocaine (1-500 mu M) had no effect on cell viability or growth but suppressed Matrigel invasion dose dependently in both normoxia and hypoxia. Used as a control, tetrodotoxin produced similar effects. Exposure to hypoxia increased Nav1.7 mRNA expression but VGSC alpha protein level in plasma membrane was reduced. Lidocaine under both normoxia and hypoxia had no effect on Nav1.7 mRNA expression. VGSC alpha protein expression was suppressed by lidocaine under normoxia but no effect was seen in hypoxia. It is concluded that lidocaine can suppress prostate cancer invasiveness without effecting cellular growth or viability. Extended to the clinic, the results would suggest that use of lidocaine, and possibly other local anaesthetics, during surgery can suppress any tendency for post-operative progression of prostate cancer.
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Investigation of Tos17 LTR Retrotransposon Movements in Rice (Oryza sativa L.) Under Nickel and Boron Stress
(Springer Heidelberg, 2024) MERİÇ, SİNAN; AYAN, ALP; GÜNDÜZ, BURCU; ÖZPİRİNÇCİ, CAN; ÇELİK, ÖZGE; ATAK, ÇİMEN
Heavy metal and metalloid pollution caused by the industrialization became a leading stress factors for agricultural plants. The increase in the amount of nickel and boron in agricultural areas due to mining and increasing industrial activity is an important agricultural constraint. The difference between deficiency and toxicity levels of these heavy metal and metalloid is extremely critical. Nickel and boron are important micronutrients for plant growth, while they become toxic at critical densities. Plants exhibit different responses to these pollutants. It is essential to find specific biomarkers to discriminate the tolerant varieties to develop elite varieties. Transposable elements are known to have an efficient role against environmental stress factors. In this research, we evaluated the potential use of Tos17 retrotransposon movement as a molecular marker to identify the stress tolerances of two Oryza sativa L. varieties against nickel and boron pollutants.
Open Access
Mechanistic Approach on Melatonin-Induced Hormesis of Photosystem II Function in the Medicinal Plant Mentha spicata
(MDPI, 2023) Moustakas, Michael; Sperdouli, Ilektra; Adamakis, Ioannis-Dimosthenis S.; Sas, Begüm; İŞGÖREN, SUMRUNAZ; Moustaka, Julietta; Morales, Fermin
Melatonin (MT) is considered a new plant hormone having a universal distribution from prokaryotic bacteria to higher plants. It has been characterized as an antistress molecule playing a positive role in the acclimation of plants to stress conditions, but its impact on plants under non-stressed conditions is not well understood. In the current research, we evaluated the impact of MT application (10 and 100 mu M) on photosystem II (PSII) function, reactive oxygen species (ROS) generation, and chlorophyll content on mint (Mentha spicata L.) plants in order to elucidate the molecular mechanism of MT action on the photosynthetic electron transport process that under non-stressed conditions is still unclear. Seventy-two hours after the foliar spray of mint plants with 100 mu M MT, the improved chlorophyll content imported a higher amount of light energy capture, which caused a 6% increase in the quantum yield of PSII photochemistry (Phi(PSII)) and electron transport rate (ETR). Nevertheless, the spray with 100 mu M MT reduced the efficiency of the oxygen-evolving complex (OEC), causing donor-side photoinhibition, with a simultaneous slight increase in ROS. Even so, the application of 100 mu M MT decreased the excess excitation energy at PSII implying superior PSII efficiency. The decreased excitation pressure at PSII, after 100 mu M MT foliar spray, suggests that MT induced stomatal closure through ROS production. The response of Phi(PSII) to MT spray corresponds to a J-shaped hormetic curve, with Phi(PSII) enhancement by 100 mu M MT. It is suggested that the hormetic stimulation of PSII functionality was triggered by the non-photochemical quenching (NPQ) mechanism that stimulated ROS production, which enhanced the photosynthetic function. It is concluded that MT molecules can be used under both stress and non-stressed conditions as photosynthetic biostimulants for enhancing crop yields.
Open Access
Mechanistic Insights on Salicylic Acid Mediated Enhancement of Photosystem II Function in Oregano Seedlings Subjected to Moderate Drought Stress
(MDPI, 2023) Moustakas, Michael; Sperdouli, Ilektra; Moustaka, Julietta; Şaş, Begüm; İŞGÖREN, SUMRUNAZ; Morales, Fermin
Dramatic climate change has led to an increase in the intensity and frequency of drought episodes and, together with the high light conditions of the Mediterranean area, detrimentally influences crop production. Salicylic acid (SA) has been shown to supress phototoxicity, offering photosystem II (PSII) photoprotection. In the current study, we attempted to reveal the mechanism by which SA is improving PSII efficiency in oregano seedlings under moderate drought stress (MoDS). Foliar application of SA decreased chlorophyll content under normal growth conditions, but under MoDS increased chlorophyll content, compared to H2O-sprayed oregano seedlings. SA improved the PSII efficiency of oregano seedlings under normal growth conditions at high light (HL), and under MoDS, at both low light (LL) and HL. The mechanism by which, under normal growth conditions and HL, SA sprayed oregano seedlings compared to H2O-sprayed exhibited a more efficient PSII photochemistry, was the increased (17%) fraction of open PSII reaction centers (qp), and the increased (7%) efficiency of these open reaction centers (Fv '/Fm '), which resulted in an enhanced (24%) electron transport rate (ETR). SA application under MoDS, by modulating chlorophyll content, resulted in optimized antenna size and enhanced effective quantum yield of PSII photochemistry (phi(PSII)) under both LL (7%) and HL (25%), compared to non-SA-sprayed oregano seedlings. This increased effective quantum yield of PSII photochemistry (phi(PSII)) was due to the enhanced efficiency of the oxygen evolving complex (OEC), and the increased fraction of open PSII reaction centers (qp), which resulted in an increased electron transport rate (ETR) and a lower amount of singlet oxygen (O-1(2)) production with less excess excitation energy (EXC).
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In Situ Synthesis and Cell Line Studies of Nano-Hydroxyapatite/Graphene Oxide Composite Materials for Bone Support Applications
(Elsevier Science Ltd., 2023) Özder, Melike Nur; Çiftçi, Fatih; RENCÜZOĞULLARI, ÖZGE; Arısan, Elif Damla; Üstündağ, Cem Bülent
The current work presents and discusses the findings of on the structural, chemical and thermal properties of in situ synthesis of graphene oxide-hydroxyapatite (GO/HA) nanocomposite materials doped with graphene oxide (w/w) at different ratios of 0%, 0.1%, 0.5% and 1% for bone tissue support applications. Microstructure and crystallinity were investigated by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), pore structures by Brunauner-Emmett-Teller (BET) analysis, Dynamic light scattering (DLS). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were performed to characterize the morphology of the bone support scaffold materials. The attachment and cell viability of fibroblast NIH/3T3 cells were observed on the synthesized bone support composite materials. Excellent proliferation of bone support scaffolds containing 0.5% and 1% GO (w/w) was observed by fluorescence microscopy using 3,3 & PRIME;-Dihexyloxacarbocyanine iodide (Dio-6), 4 & PRIME;,6-diamidino-2-phenylindole (DAPI), MitoRed and Anti-Vinculin. The innovative materials revealed in this study demonstrated that GO and HA promoted cell proliferation and cell adhesion with their well-compatible properties and helped cell penetration and colonization by increasing the surface area of GO, making them promising materials for bone support applications.
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Prognostic Role of Long-Chain Acyl-Coenzyme A Synthetase Family Genes in Patients with Clear Cell Renal Cell Carcinoma: A Comprehensive Bioinformatics Analysis Confirmed with External Validation Cohorts
(CIG Media Group LP, 2023) TEMİZ, MUSTAFA ZAFER; Colakerol, Aykut; Sönmez, Salih Zeki; Gökçe, Adem; Canitez, İbrahim Oğulcan; Özsoy, Şule; Kandıralı, Engin; Semerciöz, Atilla; Müslümanoğlu, Ahmet Yaser
The prognostic role of long-chain acyl-CoA synthetases (ACSLs) was investigated using The Cancer Genome Atlas (TCGA) database for kidney clear cell carcinoma (KIRC) patients (n = 518). TCGA data were accessed via LinkedOmics database. We revealed lowered ACSL1 expression was associated with worse tumor histopathology and poor overall survival in ccRCC. It seems it may be used for prognostic marker for ccRCC. Introduction: We aimed to determine the prognostic role of long-chain acyl-CoA synthetases (ACSLs) as a disease marker for kidney clear cell carcinoma (KIRC). Patients and Methods: The Cancer Genome Atlas (TCGA) data were accessed via open access LinkedOmics database for KIRC. Provisional datasets were used for analysis as previously described and gene expression quantification data were downloaded. The corresponding clinical information of patients also were obtained from the database. Five ACSL family members, ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6, were investigated in the TCGA-KIRC cohort. Xena browser, cBioPortal and UALCAN, and Cancer Cell Line Encyclopedia (CCLE) databases were also used to confirm the results. Exter nal validation was perfor med using patient cohorts from the Gene Expression Omnibus (GEO-NCBI) database. Finally, the protein-protein interaction (PPI) was constructed based on the Search Tool for the Retrieval of Interacting Genes (STRING) database and visualized using Cytoscape software. Results: Pathological T3-T4 stage tumors had significantly lower ACSL1 mRNA expression ( P = .009). Patients with pathologically confirmed metastasis exhibited significantly lower expression, as well ( P = .02). ACSL1 mRNA expression was associated with overall survival (OS) and negatively correlated with OS time. Univariate and multivariate analyses showed that lower ACSL1 mRNA expression level was associated with mortality. Moreover, ACSL1 mRNA expression was exhibited significant difference in some VHL gene region mutations and PBRM1_p.R1010 mutation, and negatively correlated with HIF1-alpha mRNA expression ( P < .001). Confirmatory analyses and external validation also revealed similar findings. Conclusion: Lowered ACSL1 mRNA expression is associated with worse tumor histopathology and poor overall survival in KIRC. It may be used for prognostic marker for KIRC.
Open Access
The Protective Impact of Growth Hormone Against Rotenone-Induced Apoptotic Cell Death via Acting on Endoplasmic Reticulum Stress and Autophagy Axis
(Scientific and Technological Research Council Turkey, 2023) RENCÜZOĞULLARI, ÖZGE; TORNACI, SELAY; ÇELİK, YAĞMUR; TAŞ, NAYAT NAROT; YERLİKAYA, PINAR OBAKAN; Arısan, Elif Damla; Gürkan, Ajda Çoker
Human growth hormone (GH) is crucial modulator of cellular metabolisms, including cell proliferation and organ development, by stimulating insulin-like growth factor-1 (IGF-1), which has various functions such as cell proliferation, tissue growth, survival, or neuroprotection. Therefore, GH is implicated as a critical player in the cell and can enhance neurogenesis and provide neuroprotection during the treatment of neurological diseases such as Parkinson's disease (PD). In this study, the neuroprotective role of GH was investigated in rotenone-induced PD models for the first time. Both SH-SY5Y and SK-N-AS neuroblastoma cells were exposed to rotenone to mimic PD pathogenesis as stated in previous studies. Our data demonstrated that overexpression of GH led to the resistance of the SH-SY5Y and SK-N-AS cell lines to rotenone treatment. The levels of ER stress markers, CHOP, PERK, XBP-1, and ATF6, were higher in wt cells than GH+ SH-SY5Y cells. However, the level of autophagy markers LC3 increased and the levels of reactive oxygen species (ROS) decreased with the overexpression of GH. Furthermore, while rotenone significantly increased the SubG1 population in the cell cycle of SH-SY5Y wt cells, there was a minor alteration in GH+ cell population. Concomitantly, the levels of the proapoptotic marker, cleaved-PARP, and positive staining of Annexin V in SH-SY5Y wt cells were higher after rotenone treatment. Together, these results revealed that overexpression of GH enhanced the autophagy response by triggering the ER stress of SH-SY5Y cells to rotenone exposure and showed a neuroprotective effect in vitro PD models.
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Profile-Based Proteomic Investigation of Unintended Effects on Transgenic and Gamma Radiation Induced Mutant Soybean Plants
(Springer, 2023) MERİÇ, SİNAN; AYAN, ALP; ATAK, ÇİMEN; Arı, Şule
GM risk assessments are crucial for determination and prevention of potential adverse effects through early detection and proper evaluation of intended and potential unintended changes in molecular breeding. In this context, the concept of 'substantial equivalence' has been suggested for the safety tests of GM products for many years. This study evaluated differences between four types of Glycine max crops; transgenic soybean, its non-transgenic counterpart, gamma induced soybean mutant and its parental line, at proteomic level in context of substantial equivalence. The results revealed the ratio of differentially expressed protein spots to total protein spots in mutants compared to their parental line was 50.7%. This ratio was 41.2% in transgenic plants compared to their counterparts. Scatter plot analysis presented those mutant plants showed a wider spread than transgenic plants in terms of distribution of proteins. It was determined that up-regulated proteins in mutant plants have various biological roles in processes such as development, stress response, photorespiration and ribosomal subunit assembly. In transgenic plants, upregulated proteins were shown to have diverse biological roles in processes such as ribosomal subunit assembly, electron transport, amino acid and nucleic acid metabolism and cell wall biogenesis. Although debate on the potential unintended effects focuses on GM organisms, our results point out that the plants that gained new characteristics by various breeding methods should be characterized in all aspects case-by-case.
Open Access
The Protective Effects of Sodium Pentaborate Tetrahydrate Against UVB-induced Apoptosis in Human Keratinocytes
(Hitit Üniversitesi, 2022) ABDİK, EZGİ AVŞAR
Ultraviolet radiation (UV) is an environmental carcinogen causing human skin cancer. Exposure of the skin to UV produces apoptotic keratinocytes called sunburn cells within the epidermis. Boron, an essential element for plants, has several biological properties, such as anti-cancer, anti-microbial, and anti-oxidant. In the present study, the possible protective effects of sodium pentaborate tetrahydrate (SPT) against UVB-induced apoptosis in human keratinocyte cells, HaCaT, were investigated. They were treated with SPT at different concentrations (7.8-125 μg/mL) for 24h after UVB irradiation (20, 30 and 60mJ/cm2). Cell viability, annexin V assay, cell cycle analysis, and apoptosis-related gene levels were measured using RT-PCR. Treatment with SPT (15.6-31.25μg/mL) after 30 mJ/m2 UVB exposure significantly increased cell survival. Annexin V apoptosis analysis demonstrated a robust protective effect by treatment with SPT at concentrations of 15.6 and 31.25μg/mL after 30mJ/cm2 UVB irradiation. The cell cycle analysis revealed that UVB irradiation elevated the number of cells at the G0/G1 phase while SPT treatment after UVB irradiation increased the number of cells at G2/M phase, suggesting the changes were partially reversed. Furthermore, treatment with 15.6μg/mL SPT after 30 mJ/m2 UV irradiation blocked the activation of Caspase 3, Caspase 9, Bax, And P53. These results indicate that treatment with SPT exerts protective effects after UVB irradiation. Thus, treatment with SPT led to strong protection against UVB-induced apoptotic cell death in HaCaT cells.
Open Access
CRISPR/Cas9-Mediated Bag-1 Knockout Increased Mesenchymal Characteristics of MCF-7 Cells Via Akt Hyperactivation-Mediated Actin Cytoskeleton Remodeling
(Public Library of Science, 2022) KILBAŞ, PELİN ÖZFİLİZ; Can, Nisan Denizce; Kızılboğa, Tuğba; Ezberci, Fikret; Doğanay, Hamdi Levent; Doğanay, Gizem Dinler; ARISAN, ELİF DAMLA
Bag-1 protein is a crucial target in cancer to increase the survival and proliferation of cells. The Bag-1 expression is significantly upregulated in primary and metastatic cancer patients compared to normal breast tissue. Overexpression of Bag-1 decreases the efficiency of conventional chemotherapeutic drugs, whereas Bag-1 silencing enhances the apoptotic efficiency of therapeutics, mostly in hormone-positive breast cancer subtypes. In this study, we generated stable Bag-1 knockout (KO) MCF-7 breast cancer cells to monitor stress-mediated cellular alterations in comparison to wild type (wt) and Bag-1 overexpressing (Bag-1 OE) MCF-7 cells. Validation and characterization studies of Bag-1 KO cells showed different cellular morphology with hyperactive Akt signaling, which caused stress-mediated actin reorganization, focal adhesion decrease and led to mesenchymal characteristics in MCF-7 cells. A potent Akt inhibitor, MK-2206, suppressed mesenchymal transition in Bag-1 KO cells. Similar results were obtained following the recovery of Bag-1 isoforms (Bag-1S, M, or L) in Bag-1 KO cells. The findings of this study emphasized that Bag-1 is a mediator of actin-mediated cytoskeleton organization through regulating Akt activation. © 2022 Kilbas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Evaluation of the Spectrum of Proteolytic Activity of Micromycetes of the Genus Aspergillus in Relation to Proteins of the Hemostasis System
(Pleiades Publishing, 2022) Osmolovskiy, A.A.; ŞAŞ, B.; Aleksandrova, A.V.; Baranova, N.A.; Kreyer V.G.
Abstract: The activity of extracellular proteinases in seven strains of different species of micromycetes of the genus Aspergillus in relation to proteins of the hemostasis system was studied. Comparison of the values of enzymatic indices during the growth of strains on agar media with casein and fibrin, followed by their submerged fermentation fermentation and analysis of amidolytic activity with chromogenic peptide substrates of proteinases of the hemostasis system, made it possible to select the A. candidus A4 and A. crustosus A29 strains as promising producers of fibrinolytic proteinases. The proteinases formed by both strains were able to cleave the chromogenic peptide substrates of thrombin, plasmin, factor X, protein C, and urokinase, exhibiting high plasmin-like and thrombin-like activity and not having an activating effect on the proenzymes of the hemostasis system. © 2022, Allerton Press, Inc.
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Comparison of COVID-19 Laboratory Diagnosis by Commercial Kits: Effectivity of RT-PCR to the RT-LAMP
(Wiley, 2022) ARTIK, YAKUP; Coşgun, Alp B.; Cesur, Nevra P.; Hızel, Nedret; Uyar, Yavuz; Sur, Haydar; AYAN, ALP
Coronavirus disease 2019 or COVID-19 caused by novel coronavirus/severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or 2019-nCoV) is an ongoing pandemic that has emerging global effects and requires rapid and reliable diagnostic testing. Quantitative reverse transcription-polymerase chain reaction (q-RT-PCR) is the gold standard method for SARS-CoV-2 detections. On the other hand, new approaches remedy the diagnosis difficulties gradually. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) as one of these novel approaches may also contribute to faster and cheaper field-based testing. The present study was designed to evaluate this rapid screening diagnostic test that can give results in 30-45 min and to compare the effectiveness of LAMP to the q-RT-PCR. The 30 randomly chosen patient samples were generated by nasopharyngeal swabs with a portion of the SARS-CoV-2 nucleic sequence. The sample of quantification cycle (Cq) values was tested using RT-LAMP as well as by conventional q-RT-PCR. The patient samples were tested with four different kits (SENSObiz COVID-19 [SARS-CoV-2] LAMP Assay, the QIAseq DIRECT SARS-CoV-2 kit, Biospeedy SARS-CoV-2 Variant Plus kit, and CoVirion-CV19-2 SARS-CoV-2 OneStep RT-PCR kit) and two different PCR devices (GDS Rotor-Gene Q Thermocycler and Inovia Technologies GenX series). Based on 30 patient samples, the positive/negative ratio (P/N) was 30/0 as Biospeedy and Covirion (positivity 100%), 28/2 as Qiagen kit (positivity 93.3%) for the samples studied on the Inovia device while the same samples on the Rotor-Gene device were 30/0 as Biospeedy and Covirion (positivity 100%), 29/1 as Qiagen kit at the first day (96.7%). On the fifth day, the samples were studied in the Inovia device and the respective results were obtained: 27/3 as Biospeedy (positivity 90%), 16/14 as Qiagen (positivity 53.3%), 28/2 as Covirion kit (positivity 93.3%). When these samples were studied in the Rotor-Gene device, it was 29/1 in Biospeedy and Covirion (positivity 96.7%), 19/11 in the Qiagen kit (positivity 63.3%). When these samples were compared with the LAMP method it was found to be 19/11 (positivity 63.3%) on the first day and 18/12 (positivity 60%) on the fifth day. SARS-CoV-2 test studies will contribute to a proactive approach to the development of rapid diagnosis systems. The LAMP approach presents promising results to monitor exposed individuals and also improves screening efforts in potential ports of entry.
Open Access
Gemcitabine in Combination With Epibrassinolide Enhanced the Apoptotic Response in an ER Stress-Dependent Manner and Reduced the Epithelial-Mesenchymal Transition in Pancreatic Cancer Cells
(TUBITAK Scientific & Technical Research Council Turkey, 2022) Mehdizadehtapeh, Leila; RENCÜZOĞULLARI, ÖZGE; KURYAYEVA, FADINA; ÇEVİKLİ, SENA SEDEF; ÖZAGAR, SEVVAL; ODABAŞ, PINAR SİBEL; TUNÇKOL, SUDE; YETİM, HAKAN; Gürkan, Ajda Çoker; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN
Gemcitabine is a broad-spectrum antimetabolite and a deoxycytidine analog recognized as a standard therapy alone or in combination with other antineoplastic agents in the therapy of pancreas cancer. Drug resistance following gemcitabine treatment is a common phenomenon; therefore, combinational therapy models are usually preferred. Pancreatic ductal adenocarcinoma, or pancreas cancer, is the fourth leading cause of cancer-related deaths worldwide. With the increasing incidence of pancreatic cancer every year, the mortality rate is also rising significantly because of late diagnosis, and limited chemotherapy options. Adjuvant chemotherapy after surgical resection is the typical option for the treatment of early pancreatic cancer. Mostly, 5-fluorouracil/leucovorin with irinotecan and oxaliplatin (FOLFIRINOX) and gemcitabine/nab-paclitaxel is used for the prognosis of advanced pancreatic cancer; however, chemoresistance usually occurs limiting the effectiveness of the chemotherapy. Therefore, most of the studies are focused on gemcitabine combination with other drugs to overcome the situation.As an apoptotic agent and a member of brassinosteroids, epibrassinolide (EBR) induces endoplasmic reticulum (ER) stress-dependent cell death in different cancer cells, as shown by our group. In this study, we aimed to enhance the gemcitabine apoptotic effect by EBR combined treatment in pancreatic cancer cells. EBR treatment reduced cell viability and inhibited cell proliferation in PANC-1, MIA PaCa-2, and AsPC-1 cells. Each pancreatic cancer cell gave different responses to the EBR treatment because of different aggressiveness. However, EBR induced apoptosis through increasing ROS generation, which was associated with ER stress in PANC-1 and MIA PaCa-2 cells. Gemcitabine alone reduced the cell viability of each pancreatic cancer cell line; however, combination with EBR led to further induction of apoptotic cell death in each pancreatic cancer cell line. In addition, combined treatment of gemcitabine and EBR further decreased N-cadherin and vimentin expressions, suggesting that epithelial-mesenchymal transition of pancreatic cells is reduced. In conclusion, EBR had therapeutic potential to avoid the gemcitabine-induced side effects during the treatment of pancreatic cancer.
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Circulating MicroRNA Expression Profiles to Identify a Potential Link Between Prostate Cancer and Obesity
(Elsevier, 2022) Arışan, Serdar; KILBAŞ, PELİN ÖZFİLİZ; RENCÜZOĞULLARI, ÖZGE; Ünsal, Narcin Palavan; Çoker-Gürkan, Ajda; Uysal-Onganer, Pınar; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN
Effective diagnostic methods are needed to apply appropriate treatment strategies in patients with aggressive prostate cancer. From this point of view, risk factors that cause prostate cancer or its aggressiveness should be considered. Obesity is a critical risk factor for triggering prostate cancer's metastatic properties. microRNAs are used as biomarkers in diagnosing cancer and obesity depending on their tissue-specific expression patterns. This study investigates the role of obesity in the metastatic profile of prostate cancer depending on the differential expression signatures of selected miRNAs in prostate cancer and obese patients. The roles of miR-100, miR-141 and miR-145 in prostate cancer and obesity are partially known. However, their potential to become circular biomarkers in the blood is not elucidated. There is no previous data on miR-4463 and miR-653 on prostate cancer and obesity association. In this study, the blood samples were taken and obtained serum from 69 patients of 6 subgroups that consisted of one healthy group and five unhealthy groups based on their different prostate cancer or obesity levels. Five selected miRNA expression analyses (miR-100, miR-141, miR-145, miR-4463, and miR-653) were performed through total RNA isolation, which was confirmed via synthetic cel-miR-39 miRNA. Quantitative Real-Time PCR analyzed the expression levels of selected miRNAs. Data analysis was performed via normalising target miRNA expression levels with cel-miR-39. In this study, we found that the relationship between prostate cancer and obesity was investigated at the molecular level. It was suggested that target miR-100 could be a promising biomarker for non-obese and aggressive prostate cancer patients. miR-145 is a more potential biomarker than miR-141 for non-aggressive and non-obese patients. miR-4463 can be used to predict more prostate cancer patients than obese patients. Lastly, miR-653 can be a biomarker for non-aggressive prostate cancer cells.
Open Access
The Comparison of Differentially Expressed microRNAs in Bag-1 Deficient and Wild Type MCF-7 Breast Cancer Cells by Small RNA Sequencing
(TUBITAK Scientific & Technical Research Council Turkey, 2022) KILBAŞ, PELİN ÖZFİLİZ; Alkurt, Gizem; Çoker Gürkan, Ajda; DİNLER DOĞANAY, GİZEM; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN
The multifunctional BAG-1 (Bcl-2 athanogene-1) protein promotes breast cancer survival through direct or indirect interaction partners. The number of the interacting partners determines its cellular role in different conditions. As well as interaction partner variability, the amount of BAG-1 protein in the cells could cause dramatic alterations. According to previous studies, while the transient silencing of Bag-1 enhanced drug-induced apoptosis, deletion of BAG-1 could induce stemness properties and Akt-mediated actin remodeling in MCF-7 breast cancer cells. Considering the heterogeneity of breast cancer and the variability of BAG-1-mediated cell response, it has become essential to determine microRNA (miRNA) functions in breast cancer depending on Bag-1 expression level. This study aims to compare microRNA expression levels in wt and Bag-1 knockout (KO) MCF-7 breast cancer cells. hsa-miR-429 was selected as a potential miRNA in BAG-1KO MCF-7 cells because of the downregulation both in bioinformatics and validation qRT-PCR assay. According to predicted mRNA targets and functional enrichment analysis the ten hub proteins that are phosphatidylinositol4,5-biphosphate 3-kinase catalytic subunit alpha (PIK3CA), kinase insert domain receptor (KDR), GRB2 associated binding protein 1 (GAB1), Rac family small GTPase1 (RAC1), vascular endothelial growth factor A (VEGFA), Cbl proto-oncogene (CBL), syndecan 2 (SDC2), phospholipase C gamma 1 (PLCG1), E1A binding protein p300 (EP300), and CRK like proto-oncogene, adaptor protein (CRKL) were identified as targets of hsa-miR-429. The functional enrichment analysis showed that the most significant proteins were enriched in PI3K/Akt, focal adhesion, cytoskeleton regulation, proteoglycans in cancer, and Ras signaling pathways. It was determined that hsa-miR-429 targeted these pathways in Bag-1 deficient conditions and could be used as a potential therapeutic target in future studies.
Open Access
Synthesis and Characterization of Novel ssDNA X-Aptamers Targeting Growth Hormone Releasing Hormone (GHRH)
(Public Library Science, 2022) ŞAHİN, BURCU AYHAN; APAYDIN, ZEYNEP-ELİF; Çoker-Gürkan, Ajda; ARISAN, ELİF DAMLA; YERLİKAYA, PINAR OBAKAN
Background Growth Hormone Releasing Hormone (GHRH), 44 amino acids containing hypothalamic hormone, retains the biological activity by its first 29 amino acids. GHRH (NH2 1-29) peptide antagonists inhibit the growth of prostate, breast, ovarian, renal, gastric, pancreatic cancer in vitro and in vivo. Aptamers, single-strand RNA, or DNA oligonucleotides are capable of binding to target molecules with high affinity. Our aim in this study is to synthesize and select X-aptamers against both GHRH NH2 (1-29) and GHRH NH2 (1-44) and demonstrate synthesized aptamers' target binding activity as well as serum stability. Methods and results Aptamers against GHRH NH2 (1-44) and NH2 (1-29) peptides were synthesized, and binding affinity (K-d) of 24 putative X-aptamers was determined by the dot-blot method, co-immunofluorescence staining and, SPR analysis. The serum stability of TKY.T1.08, TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers was 90-120 h, respectively. The dose-dependent binding of TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers on GHRHR in MIA PaCa-2 was approved by co-IF assay results. Moreover, SPR analysis indicated the Kd (4.75, 1.21, and 4.0 nM) levels of TKY2.T1.13, TKY.T2.08, TKY.T2.09 putative X-aptamers, respectively. Conclusion Our results illustrate the synthesis of 24 putative X-aptamers against both GHRH NH2 (1-44) and NH2 (1-29) peptides and TKY1.T1.13, TKY.T2.08, TKY.T2.09 X-aptamers have high serum stability, high target binding potential with low K-d levels.
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Investigation of the Effect of STAT3 Inhibition on Apoptotic Process Associated with JAK/STAT Signaling Pathway in A-498 and ACHN Renal Carcinoma Cells
(Wiley, 2022) YILDIZHAN, K. Y.; AYDIN, Y. E.; KILBAŞ, PELİN ÖZFİLİZ; ŞAHİN, BURCU AYHAN; RENCÜZOĞULLARI, ÖZGE