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  1. Article ; Online: SELEX against whole-cell bacteria resulted in lipopolysaccharide binding aptamers.

    Yılmaz, Deniz / Muslu, Tuğdem / Parlar, Ayhan / Kurt, Hasan / Yüce, Meral

    Journal of biotechnology

    2022  Volume 354, Page(s) 10–20

    Abstract: Nucleic acid aptamers are target-specific oligonucleotides selected from combinatorial libraries through an iterative in vitro screening process known as Systemic Evolution of Ligands by Exponential Enrichment (SELEX). In this report, the selection of ... ...

    Abstract Nucleic acid aptamers are target-specific oligonucleotides selected from combinatorial libraries through an iterative in vitro screening process known as Systemic Evolution of Ligands by Exponential Enrichment (SELEX). In this report, the selection of bacteria differentiating ssDNA aptamer candidates from a combinatorial library through the whole-cell SELEX method was performed. The enriched SELEX pool was sequenced using Illumina Next-Generation Sequencing (NGS) technology and analyzed for the most abundant sequences using CLC Genomics Workbench. The sequencing data resulted in several oligonucleotide families from which three individual sequences were chosen per SELEX based on the copy numbers. The binding performance of the selected aptamers was assessed by flow cytometry and fluorescence spectroscopy, and the binding constants were estimated using binding saturation curves. Varying results were obtained from two independent SELEX procedures where the SELEX against the model gram-negative bacterium Escherichia coli provided more selective sequences while the SELEX library used against gram-positive bacterium Listeria monocytogenes did not evolve as expected. The sequences that emerged from E. coli SELEX were shown to bind Lipopolysaccharide residues (LPS) and inhibit LPS-induced macrophage polarization. Thus, it can be said that, performed whole-cell SELEX could be resulted as the selection of aptamers which can bind LPS and inhibit LPS induced inflammation response and thus can be candidates for the inhibition of bacterial infections. In future studies, the selected aptamer sequences could be structurally and chemically modified and exploited as potential diagnostic tools and therapeutic agents as LPS antagonists.
    MeSH term(s) Aptamers, Nucleotide/chemistry ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Gene Library ; Humans ; Ligands ; Lipopolysaccharides ; SELEX Aptamer Technique/methods
    Chemical Substances Aptamers, Nucleotide ; Ligands ; Lipopolysaccharides
    Language English
    Publishing date 2022-06-11
    Publishing country Netherlands
    Document type Journal Article
    ZDB-ID 843647-2
    ISSN 1873-4863 ; 0168-1656 ; 1389-0352
    ISSN (online) 1873-4863
    ISSN 0168-1656 ; 1389-0352
    DOI 10.1016/j.jbiotec.2022.06.001
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  2. Article: SELEX against whole-cell bacteria resulted in lipopolysaccharide binding aptamers

    Yılmaz, Deniz / Muslu, Tuğdem / Parlar, Ayhan / Kurt, Hasan / Yüce, Meral

    Journal of biotechnology. 2022 Aug. 10, v. 354

    2022  

    Abstract: Nucleic acid aptamers are target-specific oligonucleotides selected from combinatorial libraries through an iterative in vitro screening process known as Systemic Evolution of Ligands by Exponential Enrichment (SELEX). In this report, the selection of ... ...

    Abstract Nucleic acid aptamers are target-specific oligonucleotides selected from combinatorial libraries through an iterative in vitro screening process known as Systemic Evolution of Ligands by Exponential Enrichment (SELEX). In this report, the selection of bacteria differentiating ssDNA aptamer candidates from a combinatorial library through the whole-cell SELEX method was performed. The enriched SELEX pool was sequenced using Illumina Next-Generation Sequencing (NGS) technology and analyzed for the most abundant sequences using CLC Genomics Workbench. The sequencing data resulted in several oligonucleotide families from which three individual sequences were chosen per SELEX based on the copy numbers. The binding performance of the selected aptamers was assessed by flow cytometry and fluorescence spectroscopy, and the binding constants were estimated using binding saturation curves. Varying results were obtained from two independent SELEX procedures where the SELEX against the model gram-negative bacterium Escherichia coli provided more selective sequences while the SELEX library used against gram-positive bacterium Listeria monocytogenes did not evolve as expected. The sequences that emerged from E. coli SELEX were shown to bind Lipopolysaccharide residues (LPS) and inhibit LPS-induced macrophage polarization. Thus, it can be said that, performed whole-cell SELEX could be resulted as the selection of aptamers which can bind LPS and inhibit LPS induced inflammation response and thus can be candidates for the inhibition of bacterial infections. In future studies, the selected aptamer sequences could be structurally and chemically modified and exploited as potential diagnostic tools and therapeutic agents as LPS antagonists.
    Keywords Escherichia coli ; Gram-negative bacteria ; Gram-positive bacteria ; Listeria monocytogenes ; biotechnology ; flow cytometry ; fluorescence emission spectroscopy ; inflammation ; lipopolysaccharides ; macrophages ; oligonucleotides ; systematic evolution of ligands by exponential enrichment ; therapeutics
    Language English
    Dates of publication 2022-0810
    Size p. 10-20.
    Publishing place Elsevier B.V.
    Document type Article
    ZDB-ID 843647-2
    ISSN 1873-4863 ; 0168-1656 ; 1389-0352
    ISSN (online) 1873-4863
    ISSN 0168-1656 ; 1389-0352
    DOI 10.1016/j.jbiotec.2022.06.001
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  3. Article: Silencing of survivin and cyclin B1 through siRNA-loaded arginine modified calcium phosphate nanoparticles for non-small-cell lung cancer therapy

    Kara, Goknur / Parlar, Ayhan / Cakmak, Melike Cokol / Cokol, Murat / Denkbas, Emir Baki / Bakan, Feray

    Colloids and surfaces. 2020 Aug. 16,

    2020  

    Abstract: With the development of nanotechnology, various drug delivery systems including inorganic nanoparticles, liposomes, polymers, etc. have been developed over the past decade. Some of these nanoparticles are also forthcoming candidates for the successful ... ...

    Abstract With the development of nanotechnology, various drug delivery systems including inorganic nanoparticles, liposomes, polymers, etc. have been developed over the past decade. Some of these nanoparticles are also forthcoming candidates for the successful delivery of small interfering RNA (siRNA) for targeted gene silencing. Upon its discovery, siRNA was perceived as a highly promising agent in the treatment of various diseases. However, it could not exhibit the expected clinical outcomes owing to the unfavorable challenges during delivery. One such challenge was identified as the lack of an effective carrier. Among the carriers, calcium phosphate (CaP) nanoparticles have attracted remarkable attention due to the superior biochemical properties and hold great promise for siRNA. It is well known that synthesis conditions influence the types of crystalline phases of CaPs as well as morphology. In this study, to address the influence of these parameters on the success of siRNA delivery, three different arginine (Arg) modified CaP nanoparticles having different chemical and morphological characteristics were synthesized as being the carriers of two specific siRNAs against survivin and cyclin B1. The functioning of CaP surfaces with Arg results in positive zeta potential on the surfaces. Functionalized nanoparticles have a higher loading capacity compared to unmodified particles, as they have a cationic surface that can be easily attached to negatively charged siRNAs. The gene silencing ability and the consequent in vitro antitumor activity of these CaP-Arg-siRNA complexes were investigated using A549 non-small-cell lung cancer cells. We found that high survivin and cyclin B1 expression is associated with worse survival in patients with lung cancer based on the Kaplan-Meier database. Considering the promoting role of survivin and cyclin B1 in cancer development and progression, CaP-Arg-siRNA mediated suppression of these genes resulted in a significant decrease in cell growth and induction of apoptosis. Our data suggest that all three CaP-Arg nanoparticles synthesized in this work can be used as safe and efficient nanocarriers for siRNA delivery, offering the opportunity to develop new therapeutic strategies for the treatment of lung cancer.
    Keywords antineoplastic activity ; apoptosis ; arginine ; calcium phosphates ; cancer therapy ; carcinogenesis ; cell growth ; cyclins ; databases ; gene silencing ; genes ; lung neoplasms ; nanocarriers ; nanoparticles ; neoplasm cells ; patients ; polymers ; pro-apoptotic proteins ; small interfering RNA ; zeta potential
    Language English
    Dates of publication 2020-0816
    Publishing place Elsevier B.V.
    Document type Article
    Note Pre-press version
    ZDB-ID 1500523-9
    ISSN 1873-4367 ; 0927-7765
    ISSN (online) 1873-4367
    ISSN 0927-7765
    DOI 10.1016/j.colsurfb.2020.111340
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  4. Article: Structural and Functional Analysis of CEX Fractions Collected from a Novel Avastin

    Gurel, Busra / Berksoz, Melike / Capkin, Eda / Parlar, Ayhan / Pala, Meltem Corbacioglu / Ozkan, Aylin / Capan, Yılmaz / Daglikoca, Duygu Emine / Yuce, Meral

    Pharmaceutics

    2022  Volume 14, Issue 8

    Abstract: ... ...

    Abstract Avastin
    Language English
    Publishing date 2022-07-28
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2527217-2
    ISSN 1999-4923
    ISSN 1999-4923
    DOI 10.3390/pharmaceutics14081571
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  5. Article ; Online: Silencing of survivin and cyclin B1 through siRNA-loaded arginine modified calcium phosphate nanoparticles for non-small-cell lung cancer therapy.

    Kara, Goknur / Parlar, Ayhan / Cakmak, Melike Cokol / Cokol, Murat / Denkbas, Emir Baki / Bakan, Feray

    Colloids and surfaces. B, Biointerfaces

    2020  Volume 196, Page(s) 111340

    Abstract: With the development of nanotechnology, various drug delivery systems including inorganic nanoparticles, liposomes, polymers, etc. have been developed over the past decade. Some of these nanoparticles are also forthcoming candidates for the successful ... ...

    Abstract With the development of nanotechnology, various drug delivery systems including inorganic nanoparticles, liposomes, polymers, etc. have been developed over the past decade. Some of these nanoparticles are also forthcoming candidates for the successful delivery of small interfering RNA (siRNA) for targeted gene silencing. Upon its discovery, siRNA was perceived as a highly promising agent in the treatment of various diseases. However, it could not exhibit the expected clinical outcomes owing to the unfavorable challenges during delivery. One such challenge was identified as the lack of an effective carrier. Among the carriers, calcium phosphate (CaP) nanoparticles have attracted remarkable attention due to the superior biochemical properties and hold great promise for siRNA. It is well known that synthesis conditions influence the types of crystalline phases of CaPs as well as morphology. In this study, to address the influence of these parameters on the success of siRNA delivery, three different arginine (Arg) modified CaP nanoparticles having different chemical and morphological characteristics were synthesized as being the carriers of two specific siRNAs against survivin and cyclin B1. The functioning of CaP surfaces with Arg results in positive zeta potential on the surfaces. Functionalized nanoparticles have a higher loading capacity compared to unmodified particles, as they have a cationic surface that can be easily attached to negatively charged siRNAs. The gene silencing ability and the consequent in vitro antitumor activity of these CaP-Arg-siRNA complexes were investigated using A549 non-small-cell lung cancer cells. We found that high survivin and cyclin B1 expression is associated with worse survival in patients with lung cancer based on the Kaplan-Meier database. Considering the promoting role of survivin and cyclin B1 in cancer development and progression, CaP-Arg-siRNA mediated suppression of these genes resulted in a significant decrease in cell growth and induction of apoptosis. Our data suggest that all three CaP-Arg nanoparticles synthesized in this work can be used as safe and efficient nanocarriers for siRNA delivery, offering the opportunity to develop new therapeutic strategies for the treatment of lung cancer.
    MeSH term(s) Arginine ; Calcium Phosphates ; Carcinoma, Non-Small-Cell Lung/drug therapy ; Carcinoma, Non-Small-Cell Lung/genetics ; Cyclin B1/genetics ; Humans ; Lung Neoplasms/drug therapy ; Lung Neoplasms/genetics ; Nanoparticles ; RNA, Small Interfering/genetics ; Survivin/genetics
    Chemical Substances Calcium Phosphates ; Cyclin B1 ; RNA, Small Interfering ; Survivin ; Arginine (94ZLA3W45F)
    Language English
    Publishing date 2020-08-27
    Publishing country Netherlands
    Document type Journal Article
    ZDB-ID 1500523-9
    ISSN 1873-4367 ; 0927-7765
    ISSN (online) 1873-4367
    ISSN 0927-7765
    DOI 10.1016/j.colsurfb.2020.111340
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  6. Article ; Online: Fractionated charge variants of biosimilars: A review of separation methods, structural and functional analysis.

    Yüce, Meral / Sert, Fatma / Torabfam, Milad / Parlar, Ayhan / Gürel, Büşra / Çakır, Nilüfer / Dağlıkoca, Duygu E / Khan, Mansoor A / Çapan, Yılmaz

    Analytica chimica acta

    2021  Volume 1152, Page(s) 238189

    Abstract: The similarity between originator and biosimilar monoclonal antibody candidates are rigorously assessed based on primary, secondary, tertiary, quaternary structures, and biological functions. Minor differences in such parameters may alter target-binding, ...

    Abstract The similarity between originator and biosimilar monoclonal antibody candidates are rigorously assessed based on primary, secondary, tertiary, quaternary structures, and biological functions. Minor differences in such parameters may alter target-binding, potency, efficacy, or half-life of the molecule. The charge heterogeneity analysis is a prerequisite for all biotherapeutics. Monoclonal antibodies are prone to enzymatic or non-enzymatic structural modifications during or after the production processes, leading to the formation of fragments or aggregates, various glycoforms, oxidized, deamidated, and other degraded residues, reduced Fab region binding activity or altered FcR binding activity. Therefore, the charge variant profiles of the monoclonal antibodies must be regularly and thoroughly evaluated. Comparative structural and functional analysis of physically separated or fractioned charged variants of monoclonal antibodies has gained significant attention in the last few years. The fraction-based charge variant analysis has proved very useful for the biosimilar candidates comprising of unexpected charge isoforms. In this report, the key methods for the physical separation of monoclonal antibody charge variants, structural and functional analyses by liquid chromatography-mass spectrometry, and surface plasmon resonance techniques were reviewed.
    MeSH term(s) Antibodies, Monoclonal ; Biosimilar Pharmaceuticals ; Chromatography, Liquid ; Mass Spectrometry ; Surface Plasmon Resonance
    Chemical Substances Antibodies, Monoclonal ; Biosimilar Pharmaceuticals
    Language English
    Publishing date 2021-01-12
    Publishing country Netherlands
    Document type Journal Article ; Review
    ZDB-ID 1483436-4
    ISSN 1873-4324 ; 0003-2670
    ISSN (online) 1873-4324
    ISSN 0003-2670
    DOI 10.1016/j.aca.2020.12.064
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  7. Article ; Online: Engineering antigen-specific NK cell lines against the melanoma-associated antigen tyrosinase via TCR gene transfer.

    Parlar, Ayhan / Sayitoglu, Ece Canan / Ozkazanc, Didem / Georgoudaki, Anna-Maria / Pamukcu, Cevriye / Aras, Mertkaya / Josey, Benjamin J / Chrobok, Michael / Branecki, Suzanne / Zahedimaram, Pegah / Ikromzoda, Lolai / Alici, Evren / Erman, Batu / Duru, Adil D / Sutlu, Tolga

    European journal of immunology

    2019  Volume 49, Issue 8, Page(s) 1278–1290

    Abstract: Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes ...

    Abstract Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL-TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off-target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen-specific TCR complex on NK cell lines NK-92 and YTS, demonstrated by using a TCR against the HLA-A2-restricted tyrosinase-derived melanoma epitope, Tyr
    MeSH term(s) Antigens, Neoplasm/immunology ; Cell Line ; Cytotoxicity, Immunologic ; HLA-A2 Antigen/metabolism ; Humans ; Immunotherapy, Adoptive/methods ; Killer Cells, Natural/physiology ; Killer Cells, Natural/transplantation ; Melanoma/immunology ; Melanoma/therapy ; Monophenol Monooxygenase/immunology ; Peptides/immunology ; Protein Engineering ; Receptors, Antigen, T-Cell, alpha-beta/genetics ; Receptors, Chimeric Antigen/genetics
    Chemical Substances Antigens, Neoplasm ; HLA-A*02 antigen ; HLA-A2 Antigen ; Peptides ; Receptors, Antigen, T-Cell, alpha-beta ; Receptors, Chimeric Antigen ; Monophenol Monooxygenase (EC 1.14.18.1)
    Language English
    Publishing date 2019-05-17
    Publishing country Germany
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 120108-6
    ISSN 1521-4141 ; 0014-2980
    ISSN (online) 1521-4141
    ISSN 0014-2980
    DOI 10.1002/eji.201948140
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