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Publication Metadata only Antioxidant activity of CAPE (caffeic acid phenethyl ester) in vitro can protect human sperm deoxyribonucleic acid from oxidative damage(Elsevier, 2018) Ayla, Sule; Tunali, Gulden; Bilgic, Bulent E.; Sofuoglu, Kenan; Ozdemir, A. Arman; Tanriverdi, Gamze; Ozdemir, Semra; Soner, B. Cem; Ozturk, Bahar; Aslan, Esra Guler; Seckin, Ismail; N/A; Karahüseyinoğlu, Serçin; Faculty Member; School of Medicine; 110772Purpose: Sperm processing (e.g., centrifugation) used in preparation for assisted reproduction can result in excessive generation of reactive oxygen species (ROS) and potential sperm damage. The use of antioxidants during sperm processing has been shown to prevent iatrogenic sperm damage, including DNA damage. In this study, we evaluated the effect of caffeic acid phenethyl ester (CAPE) on oxidative stress mediated sperm dysfunction and DNA damage. Methods: Semen samples were obtained to liquefy at room temperature. After centrifugation and washing protocols, spermatozoa were incubated in a single step supplemented medium with either of 10, 50 or 100 mu mol/L CAPE for 2 hours at 36 degrees C. After incubation period, MDA levels of seminal plasma were measured. The fragmentation in sperm DNA was detected by light microscopy via use of an aniline blue assay, while ultrastructural morphology was analyzed by transmission electron microscopy. Results: Significant increase has been observed in percent chromatin condensation (assessed by aniline blue staining) and Malondialdehyde (Mmol/L) in oligoasthenoteratozoospermia group before the centrifugation (0.57 +/- 0.15). Incubation of samples with 100 mu mol/L CAPE after centrifugation resulted in a significantly lower percent chromatin condensation compared to samples incubated without CAPE (0.42 +/- 0.12) (P < 0.0033). Incubation of all samples with CAPE (10 mu mol/L, 50 mu mol/L, 100 mu mol/L.) after centrifugation resulted in a significantly lower percentage of Malondialdehyde levels. Conclusions: The data suggests that preincubation of spermatozoa with the antioxidant CAPE offers protection against oxidative DNA damage in vitro.Publication Metadata only Generation of induced pluripotent stem cell line (UCSFi001-A-77) carrying a biallelic frameshift variant in exon 4 of SGIP1 through CRISPR/Cas9(Elsevier, 2024) Fatima, Neelam; Dillen, Lieke; Hommersom, Marina P.; Fatima, Fareeha; van Beusekom, Ellen; Albert, Silvia; Khan, Asma Ali; de Brouwer, Arjan P. M.; van Bokhoven, Hans; Çepni, Ece; Graduate School of Health SciencesSGIP1 encodes a protein Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1. It is involved in the regulation of clathrinmediated endocytosis along with having a role in energy homeostasis in neuronal systems. We generated an isogenic human induced pluripotent stem cell (iPSC) line with a biallelic frameshift variant in SGIP1. . This exon has been shown to be subject to alternative splicing, leading to an isoform lacking 24 amino acids that are present in the longest SGIP isoform. The newly generated iPSC line will be helpful to dissect the differential properties of the two SGIP isoforms.Publication Metadata only Generation of induced pluripotent stem cell lines from two unrelated patients affected by intellectual disability carrying homozygous variants in SGIP1(Elsevier, 2024) Dillen, Lieke; Fatima, Neelam; Hommersom, Marina P.; Fatima, Fareeha; van Beusekom, Ellen; Albert, Silvia; Hagen, Johanna M. van; Vries, Bert B. A. de; Khan, Asma Ali; Brouwer, Arjan P. M. de; van Bokhoven, Hans; Çepni, Ece; Graduate School of Health SciencesIntellectual disability (ID) is a diverse neurodevelopmental condition and almost half of the cases have a genetic etiology. SGIP1 acts as an endocytic protein that influences the signaling of receptors in neuronal systems related to energy homeostasis through its interaction with endophilins. This study focuses on the generation and characterization of induced pluripotent stem cells (iPSC) from two unrelated patients due to a frameshift variant (c.764dupA, NM_032291.4) and a splice donor site variant (c.74 + 1G > A, NM_032291.4) in the SGIP1 gene.Publication Metadata only Increased expression of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 is required for growth of mouse embryonic stem cells that are undergoing differentiation(Springer, 2023) Güzel, Saime; Altunok, Tuğba Hazal; Yalçın, Abdullah; N/A; Gürpınar, Yunus; Researcher; Koç University Research Center for Translational Medicine (KUTTAM) / Koç Üniversitesi Translasyonel Tıp Araştırma Merkezi (KUTTAM); N/A; N/A; N/AThe unlimited proliferation capacity of embryonic stem cells (ESCs) coupled with their capability to differentiate into several cell types makes them an attractive candidate for studying the molecular mechanisms regulating self-renewal and transition from pluripotent state. Although the roles of 6-phosphofructo-2-kinase/fructose-2,6-bisphos-phatase family (PFKFB1-4) in cell survival, proliferation, and differentiation in tumor cells have been studied, their role in mouse ESC (mESC) biology is currently unkown. In the current study, Pfkfb isoenzyme expressions were analyzed in R1 and J1 mESCs that were cultured in the presence and absence of leukemia inhibitory factor (LIF). We report that expression of the Pfkfb3 isoenzyme was markedly increased when mESCs were promoted to differentiate upon LIF removal. We then demonstrated that Pfkfb3 silencing induced the differentiation marker Brachyury suggesting that Pfkfb3 may be required for the regulation of mesodermal differentiation of mESCs. Furthermore, we show that the increase in Pfkfb3 expression is required for the growth of early differentiated mESCs. Although these results provide important insights into the early differentiation of mESCs with regard to Pfkfb expressions, further mechanistic studies will be needed for understanding the pathways and mechanisms involved in regulation of proliferation and early differentiation of mESCs through Pfkfb3.Publication Metadata only Inhibiting effect of oleocanthal on neuroblastoma cancer cell proliferation in culture(Taylor & Francis, 2020) Mete, Mesut; Aydemir, Işıl; Duransoy, Yusuf Kurtuluş; Umur, Ahmet Şükrü; Tuğlu, Mehmet İbrahim; Ünsal, Ülkün Ünlü; Doctor; School of Medicine; Koç University Hospital; N/AWe investigated the potential anticancer effects of oleocanthal (OC) on neuroblastoma cells. Cells were divided into four groups: group 1, neuroblastoma cells were treated with OC; group 2, neurons that differentiated from neuroblastoma cells were treated with phosphate-buffered saline(PBS); group 3, bone marrow derived neuronal (BMDN) cells that were differentiated from bone marrow derived mesenchymal stem cells (BMSCs) were treated with OC; group 4, BMDN cells that were differentiated from BMSCs were treated with PBS. Groups 2 and 4 were control groups. The effects of OC on cell viability, oxidative stress, neurite inhibition and apoptosis at IC50 dose were investigated using MTT analysis, i-NOS and e-NOS measurement, neurotoxicity screening test (NST) and TUNEL staining, respectively. MTT analysis demonstrated that cells were significantly less viable in group 1 than in group 3. i-NOS and e-NOS staining intensity was significantly greater in group 1 than in group 3. NST revealed that OC inhibited neurite growth in both neuroblastoma and BMND cells; inhibition was significantly less in group 3 than in group 1. Significantly more TUNEL labeled cells were found in group 1 than in group 3. We found that OC prevented growth and proliferation of neuroblastoma cells in culture by increasing oxidative stress and apoptosis. We also found that the cytotoxicity of OC is negligible in BMDN cells.Publication Metadata only Management of cytological material, pre-analytical procedures and bio-banking in effusion cytopathology(Wiley, 2019) Engels, Marianne; Michael, Claire; Dobra, Katalin; Hjerpe, Anders; Fassina, Ambrogio; N/A; Fırat, Pınar Arıkan; Faculty Member; School of Medicine; 207545Serous effusion fluid is one of the most commonly encountered specimens in routine cytopathology practice. It provides invaluable information about the patient and the clinical status; but to get the most of it, specimen handling and processing must be carried out properly. Cytomorphology is the basis of a successful analysis which should complemented by ancillary tests when needed. A wide spectrum of ancillary techniques - ranging from immunocytochemistry and flow cytometry to different assays of molecular pathology - can be applied to serous effusions. This article describes the acquisition and management of serous effusion fluids, methods for preservation and transportation, different techniques of cytopreparation, application of immunocytochemistry, flow cytometry, and fluorescence in-situ hybridization (FISH), as well as DNA extraction for polymerase chain reaction (PCR) and next generation sequencing (NGS). Principles of bio-banking of effusion samples are also discussed which is getting more important in correlation with the developments in personalized medicine.Publication Metadata only Probing mammalian centrosome structure using BioID proximity-dependent biotinylation(Elsevier, 2015) Stearns, Tim; Department of Molecular Biology and Genetics; Karalar, Elif Nur Fırat; Faculty Member; Department of Molecular Biology and Genetics; College of Sciences; 206349Understanding the structure and function of the centrosome will require identification of its constituent components and a detailed characterization of the interactions among these components. Here, we describe the application of proximity-dependent biotin identification (BioID) to identify spatial and temporal relationships among centrosome proteins. The BioID method relies on protein fusions to a promiscuous mutant of the Escherichia coli biotin ligase BirA, which biotinylates proteins that are in a similar to 10 nm labeling radius of the enzyme. The biotinylated proteins are captured by affinity and are identified by mass spectrometry. Proteins identified in this way are referred to as "proximity interactors." Application of BioID to a set of centrosome proteins demonstrated the utility of this approach in overcoming inherent limitations in probing centrosome structure. These studies also demonstrated the potential of BioID for building large-scale proximity interaction maps among centrosome proteins. In this chapter, we describe the work flow for identification of proximity interactions of centrosome proteins, including materials and methods for the generation and characterization of a BirA*-fusion protein expression plasmid, expression of BirA*-fusion proteins in cells, and purification and identification of proximity partners by mass spectrometry.Publication Metadata only Quantitative analysis of immunogold labeling for basic fibroblast growth factor according to the activational stages of mast cells(Sci Printers & Publ Inc, 2013) Kayton, Robert J.; N/A; Department of Mathematics; N/A; Aktaş, Ranan Gülhan; Çağlar, Mine; Oktayer, Adviye Gözde; Faculty Member; Faculty Member; Master Student; Department of Mathematics; School of Medicine; College of Sciences; Graduate School of Health Sciences; 137519; 105131; N/AOBJECTIVE: To analyze whether immunogold labeling density for basic fibroblastic growth factor in granules is compatible with the activation stage of mast cells. STUDY DESIGN: Cytoplasmic features and granules of 46 mast cells were examined at the ultrastructural level. The cells were classified according to their activation stage, namely, whether resting, initially activated, fully degranulated or piecemeal degranulated. Granules were classified as electron lucent, moderate or dense granules. Gold particles per secretory granules in the cells were counted. Recently described quantitative analysis techniques were used for evaluation. RESULTS: There is a statistically meaningful relationship between the activation stage of mast cells and their immunogold labeling density. The number of different types of granules encountered in a cell depends on the type of the cell. The distribution of gold particles among the secretory granules depends upon the cell. The type of granule does not have an individual effect on the number of particles, as indicated by an overall statistical analysis of granules, cells and their interaction effects. CONCLUSION: A count of gold particles in the cells can be used as a strong biological indicator. Therefore the number of gold particles might be very useful for comparative studies related to the secretion of this growth factor under different conditions.Publication Metadata only The effects of magnesium sulfate on cyclophosphamide-induced ovarian damage: Folliculogenesis(Elsevier, 2020) Yılmaz, Tuğba Ekiz; Taşdemir, Müge; Arıcan, Nadir; N/A; Kaya, Mehmet; Ahıshalı, Bülent; Faculty Member; Faculty Member; School of Medicine; School of Medicine; 10486; 9509Cyclophosphamide (CYP) is one of the alkylating chemotherapeutic agents and its adverse effects on folliculogenesis in the ovary are well-known due to the previous scientific research on this topic. Magnesium has various effects in organisms, including catalytic functions on the activation and inhibition of many enzymes, and regulatory functions on cell proliferation, cell cycle, and differentiation. In this study, the effects of magnesium sulfate (MgSO4) on CYP induced ovarian damage were investigated. Immature Wistar-Albino female rats of 28-days were treated with pregnant mare serum gonadotrophin (PMSG) to develop the first generation of preovulatory follicles. Rats of the experimental groups were then treated with either CYP (100 mg/kg, i.p) and MgSO4 (270 mg/kg loading dose; 27 mg/kg maintenance doseX12, i.p) solely or in combination. Following in-vivo 5-bromo-2-deoxyuridine (BrdU) labeling, animals were sacrificed and ovaries were embedded in paraffin and Epon. In the ovaries, added to the evaluation of general morphology and follicle count; BrdU and TUNEL-labeling, cleaved caspase-3 and p27 (cyclin-dependent kinase inhibitor) staining was also performed immunohistochemically and an ultrastructural evaluation was performed by transmission electron microscopy (TEM). The number of primordial follicles were decreased and multilaminar primary and atretic follicles were increased in CYP group. After MgSO4 treatment, while primordial follicle pool were elevated, the number of atretic follicles were decreased. Additionally, decreased BrdU-labeling, increased cleaved caspase 3 immunoreactivity and increased TUNEL labeling were observed in CYP group. In CYP treated animals, observations showed that while MgSO4 administration caused no alterations in BrdU proliferation index and caspase-3 immunoreactivity, it significantly reduced the TUNEL labeling. It was also observed that, while p27 immunoreactivity significantly increased in the nuclei of granulosa and theca cells in the CYP group; MgSO4 treatment significantly reduced these immunoreactivities. The ultrastructural observations showed frequent apoptotic profiles in granulosa and theca cells in both early and advanced stages of follicles in the CYP group and the MgSO4 treatment before the CYP application led to ultrastructural alleviation of the apoptotic process. In conclusion, our data suggest that MgSO4 may provide an option of pharmacologic treatment for fertility preservation owing to the beneficial effects of on chemotherapy-induced accelerated follicular apoptotic process, and the protection of the primordial follicle pool.Publication Open Access Turkish ectodermal dysplasia cohort: from phenotype to genotype in 17 families(Karger Publishers, 2019) Güven, Yeliz; Bal, Elodie; Altunoğlu, Umut; Hadj-Rabia, Smail; Koruyucu, Mine; Tuna, Elif Bahar; Çıldır, Şule; Aktören, Oya; Bodemer, Christine; Uyguner, Zehra Oya; Smahi, Asma; N/A; Börklü Yücel, Esra; Kayserili, Hülya; PhD Student; Faculty Member; School of Medicine; N/A; 7945Hypohidrotic or anhidrotic ectodermal dysplasia (HED/EDA) is characterized by impaired development of the hair, teeth, or sweat glands. HED/EDA is inherited in an X-linked, autosomal dominant, or autosomal recessive pattern and caused by the pathogenic variants in 4 genes: EDA, EDAR, EDARADD, and WNT10A. The aim of the present study was to perform molecular screening of these 4 genes in a cohort of Turkish individuals diagnosed with HED/EDA. We screened for pathogenic variants of WNT10A, EDA, EDAR, and EDARADD through Sanger sequencing. We further assessed the clinical profiles of the affected individuals in order to establish phenotype-genotype correlation. In 17 (63%) out of 27 families, 17 pathogenic variants, 8 being novel, were detected in the 4 well-known ectodermal dysplasia genes. EDAR and EDA variants were identified in 6 families each, WNT10A variants in 4, and an EDARADD variant in 1, accounting for 35.3, 35.3, 23.5, and 5.9% of mutation-positive families, respectively. The low mutation detection rate of the cohort and the number of the EDAR pathogenic variants being as high as the EDA ones were the most noteworthy findings which could be attributed to the high consanguinity rate.