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Publication Metadata only 3D coffee stains(Royal Soc Chemistry, 2017) N/A; N/A; Department of Electrical and Electronics Engineering; N/A; N/A; N/A; Department of Molecular Biology and Genetics; Department of Chemistry; Department of Chemistry; Department of Electrical and Electronics Engineering; Doğru-Yüksel, Itır Bakış; Söz, Çağla Koşak; Press, Daniel Aaron; Melikov, Rustamzhon; Begar, Efe; Çonkar, Deniz; Karalar, Elif Nur Fırat; Yılgör, Emel; Yılgör, İskender; Nizamoğlu, Sedat; PhD Student; PhD Student; Researcher; PhD Student; PhD Student; PhD Student; PhD Student; Faculty Member; Researcher; Faculty Member; Faculty Member; Department of Molecular Biology and Genetics; Department of Chemistry; Department of Electrical and Electronics Engineering; N/A; Koç University Surface Science and Technology Center (KUYTAM) / Koç Üniversitesi Yüzey Teknolojileri Araştırmaları Merkezi (KUYTAM); N/A; N/A; N/A; N/A; N/A; N/A; Koç University Surface Science and Technology Center (KUYTAM) / Koç Üniversitesi Yüzey Teknolojileri Araştırmaları Merkezi (KUYTAM); Koç University Surface Science and Technology Center (KUYTAM) / Koç Üniversitesi Yüzey Teknolojileri Araştırmaları Merkezi (KUYTAM); Koç University Surface Science and Technology Center (KUYTAM) / Koç Üniversitesi Yüzey Teknolojileri Araştırmaları Merkezi (KUYTAM); Graduate School of Sciences and Engineering; Graduate School of Sciences and Engineering; College of Engineering; Graduate School of Sciences and Engineering; Graduate School of Sciences and Engineering; Graduate School of Sciences and Engineering; College of Sciences; College of Sciences; College of Sciences; College of Engineering; N/A; N/A; N/A; N/A; N/A; N/A; 206349; N/A; 24181; 130295When a liquid droplet (e.g., coffee, wine, etc.) is splattered on a surface, the droplet dries in a ring-shaped stain. This widely observed pattern in everyday life occurs due to the phenomenon known as a coffee stain (or coffee ring) effect. While the droplet dries, the capillary flow moves and deposits the particles toward the pinned edges, which shows a 2D ring-like structure. Here we demonstrate the transition from a 2D to a 3D coffee stain that has a well-defined and hollow sphere-like structure, when the substrate surface is switched from hydrophilic to superhydrophobic. The 3D stain formation starts with the evaporation of the pinned aqueous colloidal droplet placed on a superhydrophobic surface that facilitates the particle flow towards the liquid-air interface. This leads to spherical skin formation and a cavity in the droplet. Afterwards the water loss in the cavity due to pervaporation leads to bubble nucleation and growth, until complete evaporation of the solvent. In addition to the superhydrophobicity of the surface, the concentration of the solution also has a significant effect on 3D coffee stain formation. Advantageously, 3D coffee stain formation in a pendant droplet configuration enables the construction of all-protein lasers by integrating silk fibroin with fluorescent proteins. No tools, components and/or human intervention are needed after the construction process is initiated; therefore, 3D coffee-stains hold promise for building self-assembled and functional 3D constructs and devices from colloidal solutions.Publication Open Access A cartridge based sensor array platform for multiple coagulation measurements from plasma(Royal Society of Chemistry (RSC), 2015) Bulut, Serpil; Yaralioglu, G. G.; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Çakmak, Onur; Ermek, Erhan; Kılınç, Necmettin; Barış, İbrahim; Kavaklı, İbrahim Halil; Ürey, Hakan; PhD Student; Other; Researcher; Teaching Faculty; Faculty Member; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; College of Engineering; Graduate School of Sciences and Engineering; College of Sciences; N/A; 109991; N/A; 111629; 40319; 8579This paper proposes a MEMS-based sensor array enabling multiple clot-time tests for plasma in one disposable microfluidic cartridge. The versatile LoC (Lab-on-Chip) platform technology is demonstrated here for real-time coagulation tests (activated Partial Thromboplastin Time (aPTT) and Prothrombin Time (PT)). The system has a reader unit and a disposable cartridge. The reader has no electrical connections to the cartridge. This enables simple and low-cost cartridge designs and avoids reliability problems associated with electrical connections. The cartridge consists of microfluidic channels and MEMS microcantilevers placed in each channel. The microcantilevers are made of electroplated nickel. They are actuated remotely using an external electro-coil and the read-out is also conducted remotely using a laser. The phase difference between the cantilever oscillation and the coil drive is monitored in real time. During coagulation, the viscosity of the blood plasma increases resulting in a change in the phase read-out. The proposed assay was tested on human and control plasma samples for PT and aPTT measurements. PT and aPTT measurements from control plasma samples are comparable with the manufacturer's datasheet and the commercial reference device. The measurement system has an overall 7.28% and 6.33% CV for PT and aPTT, respectively. For further implementation, the microfluidic channels of the cartridge were functionalized for PT and aPTT tests by drying specific reagents in each channel. Since simultaneous PT and aPTT measurements are needed in order to properly evaluate the coagulation system, one of the most prominent features of the proposed assay is enabling parallel measurement of different coagulation parameters. Additionally, the design of the cartridge and the read-out system as well as the obtained reproducible results with 10 mu l of the plasma samples suggest an opportunity for a possible point-of-care application.Publication Metadata only A chemically inducible organelle rerouting assay to probe primary cilium assembly, maintenance, and disassembly in cultured cells(Humana Press Inc., 2024) Department of Molecular Biology and Genetics; İşsezer, Fatma Başak Turan; Ercan, Muhammed Erdem; Karalar, Elif Nur Fırat; Department of Molecular Biology and Genetics; Graduate School of Sciences and Engineering; College of SciencesThe primary cilium is a conserved, microtubule-based organelle that protrudes from the surface of most vertebrate cells as well as sensory cells of many organisms. It transduces extracellular chemical and mechanical cues to regulate diverse cellular processes during development and physiology. Loss-of-function studies via RNA interference and CRISPR/Cas9-mediated gene knockouts have been the main tool for elucidating the functions of proteins, protein complexes, and organelles implicated in cilium biology. However, these methods are limited in studying acute spatiotemporal functions of proteins as well as the connection between their cellular positioning and functions. A powerful approach based on inducible recruitment of plus or minus end-directed molecular motors to the protein of interest enables fast and precise control of protein activity in time and in space. In this chapter, we present a chemically inducible heterodimerization method for functional perturbation of centriolar satellites, an emerging membrane-less organelle involved in cilium biogenesis and function. The method we present is based on rerouting of centriolar satellites to the cell center or the periphery in mammalian epithelial cells. We also describe how this method can be applied to study the temporal functions of centriolar satellites during primary cilium assembly, maintenance, and disassembly.Publication Open Access A new series of indeno[1,2-c]pyrazoles as EGFR TK inhibitors for NSCLC therapy(Multidisciplinary Digital Publishing Institute (MDPI), 2022) Özdemir, A.; Sever, B.; Tateishi, H.; Otsuka, M.; Fujita, M.; Altıntop, M.D.; Department of Molecular Biology and Genetics; Çiftçi, Halil İbrahim; Department of Molecular Biology and Genetics; College of SciencesNon-small cell lung cancer (NSCLC) is the leading cause of cancer-related death throughout the world. Due to the shortcomings of traditional chemotherapy, targeted therapies have come into prominence for the management of NSCLC. In particular, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy has emerged as a first-line therapy for NSCLC patients with EGFR-activating mutations. In this context, new indenopyrazoles, which were prepared by an efficient microwave-assisted method, were subjected to in silico and in vitro assays to evaluate their potency as EGFR TK-targeted anti-NSCLC agents. Compound 4 was the most promising antitumor agent towards A549 human lung adenocarcinoma cells, with an IC50 value of 6.13 µM compared to erlotinib (IC50 = 19.67 µM). Based on its low cytotoxicity to peripheral blood mononuclear cells (PBMCs), it can be concluded that compound 4 exerts selective antitumor action. This compound also inhibited EGFR TK with an IC50 value of 17.58 µM compared to erlotinib (IC50 = 0.04 µM) and induced apoptosis (56.30%). Taking into account in silico and in vitro data, compound 4 stands out as a potential EGFR TKI for the treatment of NSCLC.Publication Open Access A proximity mapping journey into the biology of the mammalian centrosome/cilium complex(Multidisciplinary Digital Publishing Institute (MDPI), 2020) Department of Molecular Biology and Genetics; Arslanhan, Melis Dilara; Gülensoy, Dila; Karalar, Elif Nur Fırat; Faculty Member; Department of Molecular Biology and Genetics; Graduate School of Sciences and Engineering; College of Sciences; N/A; N/A; 206349The mammalian centrosome/cilium complex is composed of the centrosome, the primary cilium and the centriolar satellites, which together regulate cell polarity, signaling, proliferation and motility in cells and thereby development and homeostasis in organisms. Accordingly, deregulation of its structure and functions is implicated in various human diseases including cancer, developmental disorders and neurodegenerative diseases. To better understand these disease connections, the molecular underpinnings of the assembly, maintenance and dynamic adaptations of the centrosome/cilium complex need to be uncovered with exquisite detail. Application of proximity-based labeling methods to the centrosome/cilium complex generated spatial and temporal interaction maps for its components and provided key insights into these questions. In this review, we first describe the structure and cell cycle-linked regulation of the centrosome/cilium complex. Next, we explain the inherent biochemical and temporal limitations in probing the structure and function of the centrosome/cilium complex and describe how proximity-based labeling approaches have addressed them. Finally, we explore current insights into the knowledge we gained from the proximity mapping studies as it pertains to centrosome and cilium biogenesis and systematic characterization of the centrosome, cilium and centriolar satellite interactomes.Publication Open Access AF10 (MLLT10) prevents somatic cell reprogramming through regulation of DOT1L-mediated H3K79 methylation(BioMed Central, 2021) Philpott, Martin; Oppermann, Udo; Department of Molecular Biology and Genetics; Önder, Tamer Tevfik; Uğurlu Çimen, Deniz; Sevinç, Kenan; Küçük, Nazlı Ezgi Özkan; Özçimen, Burcu; Demirtaş, Deniz; Enüstün, Eray; Faculty Member; Faculty Member; PhD Student; Department of Molecular Biology and Genetics; Koç University Research Center for Translational Medicine (KUTTAM) / Koç Üniversitesi Translasyonel Tıp Araştırma Merkezi (KUTTAM); School of Medicine; College of Sciences; Graduate School of Sciences and Engineering; Graduate School of Health Sciences; 42946; 105301; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/ABackground: the histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown. Results: we employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knock-out cells, re-expression wild-type AF10, but not a DOT1L binding-impaired mutant, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes. Conclusions: our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.Publication Open Access An advanced workflow for single-particle imaging with the limited data at an X-ray free-electron laser(International Union of Crystallography, 2020) Assalauova, Dameli; Kim, Young Yong; Bobkov, Sergey; Khubbutdinov, Ruslan; Rose, Max; Alvarez, Roberto; Andreasson, Jakob; Balaur, Eugeniu; Contreras, Alice; Gelisio, Luca; Hajdu, Janos; Hunter, Mark S.; Kurta, Ruslan P.; Li, Haoyuan; McFadden, Matthew; Nazari, Reza; Schwander, Peter; Teslyuk, Anton; Walter, Peter; Xavier, P. Lourdu; Yoon, Chun Hong; Zaare, Sahba; Ilyin, Viacheslav A.; Kirian, Richard A.; Hogue, Brenda G.; Aquila, Andrew; Vartanyants, Ivan A.; Department of Molecular Biology and Genetics; Demirci, Hasan; Faculty Member; Department of Molecular Biology and Genetics; College of Sciences; 307350An improved analysis for single-particle imaging (SPI) experiments, using the limited data, is presented here. Results are based on a study of bacteriophage PR772 performed at the Atomic, Molecular and Optical Science instrument at the Linac Coherent Light Source as part of the SPI initiative. Existing methods were modified to cope with the shortcomings of the experimental data: inaccessibility of information from half of the detector and a small fraction of single hits. The general SPI analysis workflow was upgraded with the expectation-maximization based classification of diffraction patterns and mode decomposition on the final virus-structure determination step. The presented processing pipeline allowed us to determine the 3D structure of bacteriophage PR772 without symmetry constraints with a spatial resolution of 6.9 nm. The obtained resolution was limited by the scattering intensity during the experiment and the relatively small number of single hits.Publication Open Access An LED-Based structured illumination microscope using a digital micromirror device and GPU accelerated image reconstruction(Public Library of Science, 2022) Aydın, Musa; Doğan, Buket; Department of Physics; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; Kiraz, Alper; Karalar, Elif Nur Fırat; Morova, Berna; Uysallı, Yiğit; Özgönül, Ekin; Faculty Member; Researcher; PhD Student; PhD Student; Department of Physics; Department of Electrical and Electronics Engineering; Department of Molecular Biology and Genetics; Koç University Research Center for Translational Medicine (KUTTAM) / Koç Üniversitesi Translasyonel Tıp Araştırma Merkezi (KUTTAM); Graduate School of Sciences and Engineering; College of Engineering; School of Medicine; 22542; 206349; N/A; N/A; N/AWhen combined with computational approaches, fluorescence imaging becomes one of the most powerful tools in biomedical research. It is possible to achieve resolution figures beyond the diffraction limit, and improve the performance and flexibility of high-resolution imaging systems with techniques such as structured illumination microscopy (SIM) reconstruction. In this study, the hardware and software implementation of an LED-based superresolution imaging system using SIM employing GPU accelerated parallel image reconstruction is presented. The sample is illuminated with two-dimensional sinusoidal patterns with various orientations and lateral phase shifts generated using a digital micromirror device (DMD). SIM reconstruction is carried out in frequency space using parallel CUDA kernel functions. Furthermore, a general purpose toolbox for the parallel image reconstruction algorithm and an infrastructure that allows all users to perform parallel operations on images without developing any CUDA kernel code is presented. The developed image reconstruction algorithm was run separately on a CPU and a GPU. Two different SIM reconstruction algorithms have been developed for the CPU as mono-thread CPU algorithm and multi-thread OpenMP CPU algorithm. SIM reconstruction of 1024 × 1024 px images was achieved in 1.49 s using GPU computation, indicating an enhancement by*28 and*20 in computation time when compared with mono-thread CPU computation and multi-thread OpenMP CPU computation, respectively.Publication Open Access BRD9-containing non-canonical BAF complex maintains somatic cell transcriptome and acts as a barrier to human reprogramming(Elsevier, 2022) Philpott, M.; Cribbs, A.P.; Dunford, J.E.; Sigua, L.H.; Qi, J.; Oppermann, U.; Department of Molecular Biology and Genetics; N/A; Sevinç, Kenan; Cavga, Ayşe Derya; Kelekçi, Simge; Can, Hazal; Yıldız, Abdullah Burak; Yılmaz, Alperen; Ayar, Enes Sefa; Ata, Deniz; Önder, Tamer Tevfik; Faculty Member; Department of Molecular Biology and Genetics; Koç University Research Center for Translational Medicine (KUTTAM) / Koç Üniversitesi Translasyonel Tıp Araştırma Merkezi (KUTTAM); School of Medicine; Graduate School of Sciences and Engineering; N/A; N/A; N/A; N/A; N/A; N/A; N/A; N/A; 42946Epigenetic reprogramming to pluripotency requires extensive remodeling of chromatin landscapes to silence existing cell-type-specific genes and activate pluripotency genes. ATP-dependent chromatin remodeling complexes are important regulators of chromatin structure and gene expression; however, the role of recently identified Bromodomain-containing protein 9 (BRD9) and the associated non-canonical BRG1-associated factors (ncBAF) complex in reprogramming remains unknown. Here, we show that genetic or chemical inhibition of BRD9, as well as ncBAF complex subunit GLTSCR1, but not the closely related BRD7, increase human somatic cell reprogramming efficiency and can replace KLF4 and c-MYC. We find that BRD9 is dispensable for human induced pluripotent stem cells under primed but not under naive conditions. Mechanistically, BRD9 inhibition downregulates fibroblast-related genes and decreases chromatin accessibility at somatic enhancers. BRD9 maintains the expression of transcriptional regulators MN1 and ZBTB38, both of which impede reprogramming. Collectively, these results establish BRD9 as an important safeguarding factor for somatic cell identity whose inhibition lowers chromatin-based barriers to reprogramming.Publication Metadata only Bud14 function is crucial for spindle pole body size maintenance(TUBITAK, 2024) Department of Molecular Biology and Genetics; Girgin, Sevilay Münire; Çaydaşı, Ayşe Koca; Department of Molecular Biology and Genetics; College of Sciences; Graduate School of Sciences and EngineeringBackground/aim: Spindle pole bodies (SPB), the functional equivalent of centrosomes in yeast, duplicate through generation of a new SPB next to the old one. However, SPBs are dynamic structures that can grow and exchange, and mechanisms that regulate SPB size remain largely unknown. This study aims to elucidate the role of Bud14 in SPB size maintenance in Saccharomyces cerevisiae. Materials and methods: We employed quantitative fluorescence microscopy to assess the relative and absolute amounts of SPB structural proteins at SPBs of wildtype cells and in cells lacking BUD14 (bud14∆). Quantifications were performed using asynchronous cell cultures, as well as cultures synchronously progressing through the cell cycle and upon different cell cycle arrests. We also utilized mutants that allow the separation of Bud14 functions. Results: Our results indicate that higher levels of SPB inner, outer, and central plaque proteins are present at the SPBs of bud14∆ cells compared to wildtype cells during anaphase, as well as during nocodazole-induced M-phase arrest. However, during α-factor mediated G1 arrest, inner and outer plaque proteins responded differently to the absence of BUD14. A Bud14 mutant that cannot interact with the Protein Phosphatase 1 (Glc7) phenocopied bud14∆ in terms of SPB-bound levels of the inner plaque protein Spc110, whereas disruption of Bud14-Kel1-Kel2 complex did not alter Spc110 levels at SPBs. In cells synchronously released from α-factor arrest, lack of Bud14-Glc7 caused increase of Spc110 at the SPBs at early stages of the cell cycle. Conclusion: We identified Bud14 as a critical protein for SPB size maintenance. The interaction of Bud14 with Glc7, but not with the Kelch proteins, is indispensable for restricting levels of Spc110 incorporated into the SPBs. © TÜBİTAK.