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    Comparison of ELISA and RIA methods to quantify arginine vasopressin hormone levels in cell culture
    (Springer, 2023) Turkmen, Merve Ozcan; Karaduman, Tugce; Mergen, Hatice
    The radioimmunoassay (RIA) method is widely used to determine the levels of arginine vasopressin (AVP) in studies, especially in cell culture studies. However, there are many difficulties and disadvantages in performing this method. Therefore, this study aimed to assess the comparison between enzyme-linked immunosorbent assay (ELISA) and RIA methods by using the Bland-Altman statistical method and to investigate whether the commonly used RIA can be replaced by the ELISA method for measurement of AVP levels in cell culture medium. For this purpose, wild-type (WT) and three different mutant AVP-NPII vectors were transiently transfected to mouse neuroblastoma (Neuro2A) cells and AVP secretion into the cell culture medium by transfected Neuro2A cells was determined by both RIA and ELISA methods. Following the use of the normalization method, Bland-Altman method, currently the most commonly used statistical method assessing comparison between two methods, was performed to assess agreement between two methods. The order of normalized AVP values obtained using the RIA method was as follows: WT (0.921 +/- 0.08), 207_209delGGC mutant (0.801 +/- 0.09), G45C mutant (0.508 +/- 0.10), and G88V mutant (0.497 +/- 0.12). The normalized AVP values obtained using the ELISA method were as follows: WT (0.865 +/- 0.12), 207_209delGGC mutant (0.704 +/- 0.13), G88V mutant (0.255 +/- 0.16), and G45C mutant (0.250 +/- 0.15). The order of AVP levels measured from each transfected cell using both methods was quite similar. According to the Bland-Altman method, there is an agreement between RIA and ELISA for measuring the AVP levels in cell culture. It can be recommended to apply ELISA instead of the RIA method for determinating AVP levels.
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    Determination of Autophagy in Human Cervicovaginal Smears by Cytological and Immunocytochemical Methods
    (Wolters Kluwer Medknow Publications, 2023) Turkmen, Merve Ozcan; Demirezen, Sayeste; Beksac, Mehmet Sinan
    Background: Autophagy is a catabolic process whereby organelles and long-lived proteins are recycled through lysosomes to maintain cellular homeostasis. This process is being widely studied using culture techniques and animal models; however, cervicovaginal smears have not been used to detect autophagy. Aims: Our study aims to detect and evaluate autophagy in normal, malignant, infectious, and atypical cells in cervicovaginal smears by using cytological and immunocytochemical methods. Materials and Methods: Papanicolaou-stained 200 cervicovaginal smears were examined and 55 of 200 (27.5%) smears containing negative for intraepithelial lesion or malignancy (NILM) with identifiable infections and/or reactive/reparative changes (INF); briefly, NILM-INF (n = 31, 56.4%), atypical (n = 4, 7.3%), and malignant cells (n = 20, 36.3%) were evaluated as a study group. One hundred forty-five of 200 (72.5%) normal smears were accepted as the NILM without any identifiable infections (control group). The autophagy marker protein Microtubule-associated protein 1 light chain 3 A (MAP1LC3A) was used for immunocytochemical examination. Results: The staining intensity of the MAP1LC3A protein and autophagy positivity were lower in the malignant cells; however, they were higher in the NILM-INF and atypical cells. A statistically significant correlation between the malignant and normal cells was obtained for the autophagy positivity (P = 0.012). In view of the staining intensity of MAP1LC3A protein by the H-score method, a significant correlation was found between the NILM-INF and the normal cells (P = 0.015). Conclusions: Autophagy was detected in various cervicovaginal smears for the first time in this study. Our findings indicate that an autophagy process is essential in infectious cells as well as in the transformation of atypical cells into malignant cells in carcinogenesis.
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    Functional analyses of three different mutations in the AVP-NPII gene causing familial neurohypophyseal diabetes insipidus
    (Springer, 2021) Turkmen, Merve Ozcan; Karaduman, Tugce; Tuncdemir, Beril Erdem; Unal, Mehmet Altay; Mergen, Hatice
    Purpose Familial neurohypophyseal diabetes insipidus (FNDI), a rare disorder, which is clinically characterized by polyuria and polydipsia, results from mutations in the arginine vasopressin-neurophysin II (AVP-NPII) gene. The aim of this study was to perform functional analyses of three different mutations (p.G45C, 207_209delGGC, and p.G88V) defined in the AVP-NPII gene of patients diagnosed with FNDI, which are not included in the literature. Methods For functional analysis studies, the relevant mutations were created using PCR-based site-directed mutagenesis and restriction fragment replacement strategy and expressed in Neuro2A cells. AVP secretion into the cell culture medium was determined by radioimmunoassay (RIA) analysis. Fluorescence imaging studies were conducted to determine the differences in the intracellular trafficking of wild-type (WT) and mutant AVP-NPII precursors. Molecular dynamics (MD) simulations were performed to determine the changing of the conformational properties of domains for both WT and 207-209delGGC mutant structures and dynamics behavior of residues. Results Reduced levels of AVP in the supernatant culture medium of p.G45C and p.G88V transfected cells compared to 207_209delGGC and WT cells were found. Fluorescence imaging studies showed that a substantial portion of the mutant p.G45C and p.G88V AVP-NPII precursors appeared to be located in the endoplasmic reticulum (ER), whereas 207_209delGGC and WT AVP-NPII precursors were distributed throughout the cytoplasm. Conclusions The mutations p.G45C and p.G88V cause a failure in the intracellular trafficking of mutant AVP-NPII precursors. However, 207_209delGGC mutation does not result in impaired cellular trafficking, probably due to not having any significant effect in processes such as the proper folding, gain of three-dimensional structure, or processing. These results will provide valuable information for understanding the influence of mutations on the function of the AVP precursor hormone and cellular trafficking. Therefore, this study will contribute to elucidate the mechanisms of the molecular pathology of AVP-NPII mutations.