Molecular Biology and Genetics / Moleküler Biyoloji ve Genetik
Permanent URI for this collectionhttps://hdl.handle.net/11147/9
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Article Citation - Scopus: 11Μdacs Platform: a Hybrid Microfluidic Platform Using Magnetic Levitation Technique and Integrating Magnetic, Gravitational, and Drag Forces for Density-Based Rare Cancer Cell Sorting(Elsevier, 2023) Keçili, Seren; Yılmaz, Esra; Özçelik, Özge Solmaz; Anıl İnevi, Müge; Günyüz, Zehra Elif; Yalçın Özuysal, Özden; Özçivici, Engin; Tekin, Hüseyin CumhurCirculating tumor cells (CTCs) are crucial indicators of cancer metastasis. However, their rarity in the bloodstream and the heterogeneity of their surface biomarkers present challenges for their isolation. Here, we developed a hybrid microfluidic platform (microfluidic-based density-associated cell sorting (µDACS) platform) that utilizes density as a biophysical marker to sort cancer cells from the population of white blood cells (WBCs). The platform utilizes the magnetic levitation technique on a microfluidic chip to sort cells based on their specific density ranges, operating under a continuous flow condition. By harnessing magnetic, gravitational, and drag forces, the platform efficiently separates cells. This approach involves a microfluidic chip equipped with a microseparator, which directs cells into top and bottom outlets depending on their levitation heights, which are inversely proportional to their densities. Hence, low-density cancer cells are collected from the top outlet, while high-density WBCs are collected from the bottom outlet. We optimized the sorting efficiency by varying the flow rates, and concentrations of the sorting medium's paramagnetic properties using standard densities of polymeric microspheres. To demonstrate the platform's applicability, we performed hybrid microfluidic sorting on MDA-MB-231 human breast cancer cells and U-937 human monocytes. The results showed efficient sorting of rare cancer cells (≥100 cells/mL) from serum samples, achieving a sorting efficiency of ∼70% at a fast-processing speed of 1 mL h−1. This label-free approach holds promise for rapid and cost-effective CTC sorting, facilitating in-vitro diagnosis and prognosis of cancer. © 2023 The Author(s)Article Citation - WoS: 4Citation - Scopus: 4Mitigation Potential of Zingerone and Rutin on Toxicity Mechanisms of Nickel To Zebrafish Based on Morphological, Dna Damage and Apoptosis Outcome Analysis(Elsevier, 2023) Köktürk, Mine; Yıldırım, Serkan; Atamanalp, Muhammed; Kılıçoğlu, Metin; Uçar, Arzu; Özhan, Güneş; Alak, GoncaAlthough nickel (Ni) is an important cofactor for various enzymes in biological systems, it can cause serious problems when insufficient or excessive in an organism. Therefore, it is very important to investigate Ni in biological systems, especially in cells with its related pathogenic mechanism. This study was carried out to demonstrate the effects of zingerone (ZO) and rutin (RN) administration against nickel chloride (NiCl2) toxicity on neurobehavioral performance and brain oxidative status in zebrafish (Danio rerio) embryos/larvae on histological perspective. The experimental design of the study, which included twenty groups of fish, each containing 10 embryos, was prepared as semi-static and the trial continued for 96 hpf. In the obtained findings, it was determined that ZO and RN had a mitigating effect in this toxicity table where Ni caused oxidative stress in zebrafish larvae, induced DNA damage and apoptosis. A similar picture is valid for malformation processes as well as survival and hatching rates. These results showed that nickel is toxic to developing embryos via acting different mechanisms. In conclusion, we observed that ZO and RN have a greater effect on physiology, DNA damage and apoptosis than gross morphology, with a significant ameliorative effect.Conference Object Elimination of the B4galnt1 Gene Normalizes Lifespan and Prevents Pathology in Tay-Sachs Disease Mice(Elsevier, 2023) Seyrantepe, VolkanTay-Sachs disease is a neurodegenerative lysosomal storage disorder caused by mutations in the Hexa gene, which encodes the alpha subunit of lysosomal ß-hexaminidase A (HEXA). HEXA is responsible for the conversion of GM2 to GM3, therefore the deficiency leads to the accumulation of GM2 in the lysosomes, neurodegeneration, and eventual death. Currently, there is no efficient therapy for the disease yet.Article Citation - WoS: 4Citation - Scopus: 3Boron Stress Signal Is Transmitted Through the Tor Pathway(Elsevier, 2023) Uluışık, İrem; Koç, AhmetAlthough boron is an essential element for many organisms, an excess amount of it can cause toxicity, and the mechanism behind this toxicity is not yet fully understood. The Gcn4 transcription factor plays a crucial role in the boron stress response by directly activating the expression of the boron efflux pump Atr1. More than a dozen transcription factors and multiple cell signaling pathways have roles in regulating the Gcn4 transcription factor under various circumstances. However, it is unknown which pathways or factors mediate boron signaling to Gcn4. Using the yeast Saccharomyces cerevisiae as a model, we analyzed the factors that converge on the Gcn4 transcription factor to assess their possible roles in boron stress signaling. Our findings show that the GCN system is activated by uncharged tRNA stress in response to boron treatment and that GCN1, which plays a role in transferring uncharged tRNAs to Gcn2, is necessary for the kinase activity of Gcn2. The SNF and PKA pathways were not involved in mediating boron stress, even though they interact with Gcn4. Mutations in TOR pathway genes, such as GLN3 and TOR1, abolished Gcn4 and ATR1 activation in response to boric acid treatment. Therefore, our study suggests that the TOR pathway must be functional to form a proper response against boric acid stress.Article Citation - WoS: 17Citation - Scopus: 22Protein Corona Formation on Silver Nanoparticles Under Different Conditions(Elsevier, 2022) Tomak, Aysel; Yılancıoğlu, Buket; Winkler, David; Öksel Karakuş, CeydaThe surfaces of nanoparticles become covered by biomolecules in biological fluids. This protein ‘corona’ modifies materials’ characteristics and biological activity. The composition of the protein corona is dynamic, abundant biomolecules that bind first are subsequently replaced by less abundant but more tightly bound ones. Here, we explore the formation of the silver nanoparticle protein corona on exposure to cell culture media containing 10 % fetal bovine serum supplemented Dulbecco's Modified Eagle's medium. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry analysis were used to monitor how different parameters such as incubation time, heating duration, cell culture medium, incubation temperature, and the number of washes affect the nanoparticle–protein corona complex. silver nanoparticles with and without bound proteins were characterized by electron microscopy, dynamic light scattering, and ultraviolet-visible-near-IR spectroscopy. The tetrazolium-based MTT assay was used to determine viability of A549 human lung adenocarcinoma cells treated with silver nanoparticles. Characterization of the nanoparticles before and after protein binding provided insights into their changing morphology on corona formation. Our results confirmed that the physiological environment directly affects protein corona formation on nanoparticle surfaces. In particular, incubation condition-dependent differences in the amount of bound proteins were observed. This work highlights the importance of environmental drivers of protein adsorption, which should be considered when predicting and/or controlling protein targets of silver nanoparticles.Article Citation - WoS: 9Citation - Scopus: 10Characterization of Long Living Yeast Deletion Mutants That Lack Mitochondrial Metabolism Genes Dss1, Ppa2 and Afg3(Elsevier, 2019) Muid, Khandaker Ashfaqul; Kimyon, Önder; Reza, Shahadat Hasan; Karakaya, Hüseyin Çağlar; Koç, AhmetMolecular mechanisms of aging and longevity are still mostly unknown. Mitochondria play central roles in cellular metabolism and aging. In this study, we identified three deletion mutants of mitochondrial metabolism genes (ppa2 Delta, dss1 Delta, and afg3 Delta) that live longer than wild-type cells. These long-lived cells harbored significantly decreased amount of mitochondria] DNA (mtDNA) and reactive oxygen species (ROS). Compared to the serpentine nature of wild-type mitochondria, a different dynamics and distribution pattern of mitochondria were observed in the mutants. Both young and old long-lived cells produced relatively low but adequate levels of ATP for cellular activities. The status of the retrograde signaling was checked by expression of CIT2 gene and found activated in long-lived mutants. The mutant cells were also profiled for their gene expression patterns, and genes that were differentially regulated were determined. All long-lived cells comprised similar pleiotropic phenotype regarding mitochondrial dynamics and functions. Thus, this study suggests that DSS1, PPA2, and AFG3 genes modulate the lifespan by altering the mitochondrial morphology and functions.Correction Citation - WoS: 1Citation - Scopus: 2Corrigendum To “the Importance of Boron in Biological Systems” [j. Trace Elem. Med. Biol. 45 (2018) 156–162](Elsevier, 2019) Uluışık, İrem; Karakaya, Hüseyin Çağlar; Koç, Ahmet[No abstract available]Article Citation - WoS: 17Citation - Scopus: 19Pro-Metastatic Functions of Notch Signaling Is Mediated by Cyr61 in Breast Cells(Elsevier, 2020) Küçükköse, Cansu; Efe, Eda; Günyüz, Zehra Elif; Fıratlıgil, Burcu; Doğan, Hülya; Yalçın Özuysal, Özden; İlhan, MustafaMetastasis is the main cause of cancer related deaths, and unfolding the molecular mechanisms underlying metastatic progression is critical for the development of novel therapeutic approaches. Notch is one of the key signaling pathways involved in breast tumorigenesis and metastasis. Notch activation induces pro-metastatic processes such as migration, invasion and epithelial to mesenchymal transition (EMT). However, molecular mediators working downstream of Notch in these processes are not fully elucidated. CYR61 is a secreted protein implicated in metastasis, and its inhibition by a monoclonal antibody suppresses metastasis in xenograft breast tumors, indicating the clinical importance of CYR61 targeting. Here, we aimed to investigate whether CYR61 works downstream of Notch in inducing pro-metastatic phenotypes in breast cells. We showed that CYR61 expression is positively regulated by Notch activity in breast cells. Notch1-induced migration, invasion and anchorage independent growth of a normal breast cell line, MCF10A, were abrogated by CYR61 silencing. Furthermore, upregulation of core EMT markers upon Notch1-activation was impaired in the absence of CYR61. However, reduced migration and invasion of highly metastatic cell line, MDA MB 231, cells upon Notch inhibition was not dependent on CYR61 downregulation. In conclusion, we showed that in normal breast cell line MCF10A, CYR61 is a mediator of Notch1-induced pro-metastatic phenotypes partly via induction of EMT. Our results imply CYR61 as a prominent therapeutic candidate for a subpopulation of breast tumors with high Notch activity.Article Citation - WoS: 53Citation - Scopus: 581.55 Å-Resolution Structure of Ent-Copalyl Diphosphate Synthase and Exploration of General Acid Function by Site-Directed Mutagenesis(Elsevier, 2014) Köksal, Mustafa; Christianson, David W.; Peters, Reuben John; Potter, KevinBackground The diterpene cyclase ent-copalyl diphosphate synthase (CPS) catalyzes the first committed step in the biosynthesis of gibberellins. The previously reported 2.25 Å resolution crystal structure of CPS complexed with (S)-15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (1) established the αβγ domain architecture, but ambiguities regarding substrate analog binding remained. Method Use of crystallization additives yielded CPS crystals diffracting to 1.55 Å resolution. Additionally, active site residues that hydrogen bond with D379, either directly or through hydrogen bonded water molecules, were probed by mutagenesis. Results This work clarifies structure-function relationships that were ambiguous in the lower resolution structure. Well-defined positions for the diphosphate group and tertiary ammonium cation of 1, as well as extensive solvent structure, are observed. Conclusions Two channels involving hydrogen bonded solvent and protein residues lead to the active site, forming hydrogen bonded "proton wires" that link general acid D379 with bulk solvent. These proton wires may facilitate proton transfer with the general acid during catalysis. Activity measurements made with mutant enzymes indicate that N425, which donates a hydrogen bond directly to D379, and T421, which hydrogen bonds with D379 through an intervening solvent molecule, help orient D379 for catalysis. Residues involved in hydrogen bonds with the proton wire, R340 and D503, are also important. Finally, conserved residue E211, which is located near the diphosphate group of 1, is proposed to be a ligand to Mg2 + required for optimal catalytic activity. General significance This work establishes structure-function relationships for class II terpenoid cyclases.