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In this study, chitosan-based novel scaffolds containing zeolite A were fabricated by freeze-drying technique. The nanocomposite scaffolds were prepared from chitosan and zeolite A nanocrystals with different amounts (0.5, 1.0, and 2.0%) in aqueous media. The zeolite A nanocrystals and nanocomposite scaffolds were characterized by using FTIR, X-ray powder diffraction, scanning electron microscope, and thermogravimetric analysis. The scaffolds were seeded with bone marrow-derived human mesenchymal stem cell line (UE7T-13), and cell attachment, viability, and cytotoxicity assays were performed. In vitro cytotoxicity of scaffolds toward human mesenchymal stem cell line was evaluated through the evaluation of cell viability and cell attachment assays.

In this work, super paramagnetic magnetite nanoparticles were synthesized onto/into zeolite, then loaded with anti-cancer drug 5-fluorouracil (5-FU). The physical properties of the prepared nanocomposite and drug loaded nanocomposite were characterized using different techniques. The drug loading and releasing behavior of the magnetic nanocarrier was investigated and the drug-loaded nanoparticles exhibited a sustained release of drug without any burst release phenomenon. Furthermore, 5-FU loaded MZNC were evaluated for its biological characteristics. The functional 5-FU-MZNC has been triggered intra-cellular release of the cancer therapeutic agent 5-fluorouracil (5-FU). Cytotoxic effects of 5-FU loaded MZNC on human gastric carcinoma (AGS) cells were determined by real time cell analysis and colorimetric WST-1 cell viability assay. Apoptosis of cells was further investigated by Annexin-V staining which indicates the loss of cell membrane integrity. According to our results, 5-FU-MZNC showed a concentration-dependent cell proliferation inhibitory function against AGS cells. Morphologic and apoptotic images were consistent with the cytotoxicity results. In conclusion, 5-FU loaded MZNC efficiently inhibit the proliferation of AGS cells in vitro through apoptotic mechanisms, and may be a beneficial agent against cancer, however further animal study is still required. (C) 2015 Elsevier Masson SAS. All rights reserved. Bezmiâlem Vakıf Üniversitesi

In this study, three different types of scaffolds including a uniquely modified composite scaffold - namely chitosan (CTS), nano-hydroxyapatite/chitosan composite (CTS + nHAP), and amine group (NH2) modified nano-hydroxyapatite/chitosan composite (CTS + nHAP-NH2) scaffolds- were synthesized for bone tissue engineering (BTE) purposes. As results of the study, it was found that all scaffold types were biodegradable with CTS and CTS + nHAP scaffolds losing up to 15% of their initial weight, while the CTS + nHAP-NH2 scaffold showing 10% of weight loss after six weeks of lysozyme treatment. In addition, all three types of scaffolds were shown to be biocompatible, and amongst them CTS + nHAP-NH2 scaffolds supported the most cell proliferation in WST-1 assay and expressed the least and acceptable level of cytotoxicity in lactate dehydrogenase (LDH) test for human bone mesenchymal stem cells (hBM-MSCs). Finally, during osteoinductivity assessment, CTS + nHAP-NH2 nearly tripled initial alkaline phosphatase (ALP) activity when whereas both CTS and CTS + nHAP scaffolds only doubled. These results indicate that all synthesized scaffold types under investigation have certain potential to be used in bone tissue engineering approaches with CTS + nHAP-NH2 scaffold being the most promising and applicable one. In the future, we plan to intensify our studies on osteogenic differentiation on our scaffolds on a detailed molecular level and to include in vivo studies for pre-clinical purposes. (C) 2017 Elsevier Ltd. All rights reserved. Türkiye Bilimsel Ve Teknolojik Araştırma Kurumu ( Tübitak )

Two isoforms of DNA topoisomerase II (topo II) have been identified in mammalian cells, named topo II alpha and topo II beta. Topo II alpha plays an essential role in segregation of daughter chromosomes and thus for cell proliferation in mammalian cells. Unlike its isozyme topo II alpha, topo II beta is greatly expressed upon terminal differentiation of neuronal cells. Although there have been accumulating evidence about the crucial role of topo II beta in neural development through activation or repression of developmentally regulated genes at late stages of neuronal differentiation, there have been no reports that analyzed the roles of topo II beta in the neural trans differentiation process of multipotent stem cells. Terminal differentiation of neurons and transdifferentiation of Mesenchymal Stem Cells (MSCs) are two distinct processes. Therefore, the functional significance of topo II beta may also be different in these differentiation systems. MSC transdifferentiation into neuron-like cells represents an useful model to further validate the role of topo II beta in neuronal differentiation. The aim of this study is to evaluate the subset of genes that are regulated in neural transdifferentiation of bone marrow-derived human MSCs (BM-hMSCs) in vitro and find genes related with topo II beta. For this purpose, topo II beta was silenced by specific small interfering RNAs in hMSCs and cells were induced to differentiate into neuron-like cells. Differentiation and silencing of topo II beta were monitored by real-time cell analysis and also expressions of topo II isoforms were analyzed. Change in transcription patterns of genes upon topo II beta silencing was identified by DNA microarray analysis, and apparently genes involved in regulation of several ion channels and transporters, vesicle function, and cell calcium metabolism were particularly affected by topo II beta silencing suggesting that topoII beta silencing can significantly alter the gene expression pattern of genes involved in variety of biological processes and signal transduction pathways including transcription, translation, cell trafficking, vesicle function, transport, cell morphology, neuron guidance, growth, polarity, and axonal growth. It appears that the deregulation of these pathways may contribute to clarify the further role of topo II beta in neural differentiation. Türkiye Bilimsel Ve Teknolojik Araştırma Kurumu ( Tübitak )

Novel series of asymmetric Poly(amidoamine) (PAMAM) dendrons containing a single ferrocene unit located as the focal point has been prepared. First, second, and third generation ferrocene-PAMAM dendrons with branched amine periphery and focal ferrocene functionality were synthesized via divergent pathways. The synthesized dendrons were characterized by FT-IR and NMR spectroscopy. Real-time cell analysis, cell viability, and anti-cancer activity of ferrocene-PAMAM dendrons were evaluated in AGS cells. While cell viability decreased, anti-cancer activity increased gradually in a dose-dependent manner in one, two, and three generations.

The detection of cancer cells through important molecular recognition target such as sialic acid is significant for the clinical diagnosis and treatment. There are many electrochemical cytosensors developed for cancer cells detection but most of them have complicated fabrication processes which results in poor reproducibility and reliability. In this study, a simple, low-cost, and highly sensitive electrochemical cytosensor was designed based on boronic acid-functionalized polythiophene. In cytosensors fabrication simple single-step procedure was used which includes coating pencil graphite electrode (PGE) by means of electro-polymerization of 3-Thienyl boronic acid and Thiophen. Electrochemical impedance spectroscopy and cyclic voltammetry were used as an analytical methods to optimize and measure analytical performances of PGE/P(TBA0.5Th0.5) based electrode. Cytosensor showed extremely good analytical performances in detection of cancer cells with linear rage of 1×101 to 1×106 cellsmL-1 exhibiting low detection limit of 10 cellsmL-1 and incubation time of 10min. Next to excellent analytical performances, it showed high selectivity towards AGS cancer cells when compared to HEK 293 normal cells and bone marrow mesenchymal stem cells (BM-hMSCs). This method is promising for future applications in early stage cancer diagnosis.

Background: DNA topoisomerase IIβ (topo IIβ) plays a crucial role in neural differentiation and axonogenesis. Inhibition of topo IIβ activity in vitro and in vivo results in shorter axons and increased DNA damage. These molecular events also involve in Alzheimer's disease (AD); however, the role of topo IIβ in the pathogenesis of AD remains to be elucidated. Objectives: We aimed to investigate the role of topo IIβ association with Nuclear receptor related 1 protein (Nurr1) in the onset of AD. Methods: In vitro AD model was established by the incubation of fibrillar amyloid-β 1-42 (Aβ1-42) for 48 hours with cultured cerebellar granule neurons (CGNs) isolated from post-natal eight-day rats. The regulatory role of topo IIβ on the transcription of Nurr1 was analyzed in topo IIβ silenced CGNs, and also topo IIβ silenced and overexpressed in a neurally-differentiated human mesenchymal (hMSC) cell line. Results: Aβ1-42 fibrils led to the upregulation of Presenilin1 and Cofilin1 genes as measured at mRNA levels and hyperphosphorylation of tau protein, all are distinctive characteristics of AD pathology. A significant decrease in topo IIβ expression at mRNA and protein levels and Nurr1 at mRNA level was also observed. In both cell types, Nurr1 expression was dramatically down-regulated due to topo IIβ deficiency, and was increased in topo IIβ overexpressing hMSCs. Conclusion: Our findings suggest that topo IIβ could be a down-stream target of signaling pathways contributing to AD-like pathology. However, further studies must be carried out in vivo to elucidate the precise association topo IIβ with AD.

As a major component of the Mediterranean diet, olive and olive products have protective effects against several human diseases, such as cardiovascular diseases, neurodegenerative diseases and cancer, via their antioxidant activity. Olive’s beneficial effect is attributed to its phenolic content and fatty acid profile. In this study, effects of different types of olive extracts on cancer cell lines were evaluated. Proliferative, cytotoxic and apoptotic effects of olive extracts were investigated and gene expression profiles of cancer cells treated with olive extracts were analysed in AGS, Caco-2 cell lines and bone marrow human mesenchymal stem cells (hMSCs). Since topo IIα is essential for chromosome segregation in mitotic cells, the expression level of topo IIα can be used as a marker of cell proliferation. Expression level of topo IIα was checked by using RT-PCR. In vitro results revealed that AGS cell lines were more sensitive to treatment of olive extracts than were colon carcinoma models, Caco-2, and healthy human cells.

The photoinactivation of three different human cancer cell lines, HeLa, neuroblastoma and U87 and antimicrobial activities against Gram-positive (Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) by protoporphyrin IX and protoporphyrin–dopamine conjugate were examined. The antimicrobial activities were screened by optical density measurement at 600 nm. Also, the cytotoxicity and proliferation assays were performed with LDH measurements at 490 nm and WST-1 assays at 450 nm, respectively, with and without irradiation.

The differentiation of human mesenchymal stem cells (hMSCs) into neural cells in vitro provides a potential tool to be utilized for cell therapy of neurodegenerative disorders. Although previous studies repeated different protocols for the induction of neural cells from hMSCs in vitro, the results were not in complete agreement. In this study, we have attempted to compare three of these neural induction methods; retinoic acid (RA) treatment, RA treatment in serum reduced conditions, and treatment using other chemical compounds (dimethyl sulfoxide and potassium chloride) along with RA by real-time cell analysis and immunofluorescent staining of neural markers. RA treatment led to a slow progression of cells into neural-like morphology with the expression of neural protein neurofilament whereas reducing serum during RA treatment caused a much more extended differentiation process. Additionally, neural-like morphology was persistent in the later periods of differentiation in RA treatment. On the other hand, chemical induction caused cell shrinkages mimicking neural-like morphology in a short time and loss of this morphology along with increased cell death in later periods. Among the three methods compared, RA treatment was the most reliable one in terms of stability of differentiation and neural protein expressions.

hMSCs (human mesenchymal stem cells) express two isoforms of DNA topo II (topoisomerase II). Although both isoforms have the same catalytic activity, they are specialized for different functions in the cell: while topo IIα is essential for chromosome segregation in mitotic cells, topo IIβ is involved in more specific cellular functions. A number of inhibitors are available that inhibit the catalytic activity of both topo II isoforms. However, in order to investigate the isoform-specific inhibition of these two enzymes, it is necessary to use other techniques such as siRNA (small interfering RNA) interference to selectively silence either one of the isoforms individually. Depending on the lipid charge densities and protein varieties of the cell membrane, previous studies have demonstrated that transfection efficiencies of siRNAs to hMSCs are very low. In the study reported here, we demonstrate the use of Lipofectamine RNAiMAX as an efficient transfection reagent to introduce siRNAs into human mesenchymal stem cells with significantly great efficiency to silence topo IIβ selectively. A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos. Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs. The reagent induced minimal cytotoxicity (3.5-4.5%), and cell viability of the transfected hMSCs decreased 20-30% compared with untreated cells, depending on the concentration of the reagent.

In this study, effect of a novel LED-based light source developed for 96-well-plates cell culture applications, was tried on AGS stomach cancer cell line, in combination with Poly(amido amine) (PAMAM) modified – porhyrin molecule. For each 4 generation of modified PpIX molecule 5 different concentrations tried. According to results PAMAM molecule doesnt have any photosensitizer property also didn’t show any toxic effect even if higher concentrations. Morphology and real time monitoring analysis results hold up each other and confirmed that, PpIX molecules with and without modificated high concentrations (>100μM) caused cell death via toxicicity this reason optimal concentration for PAMAM modified PpIX should be between 25 - 50 μm concentration .

Gold nanoparticles (GNPs) are favorable nanostructures for several biological applications due to their easy synthesis and biocompatible properties. Commonly studied GNP shapes are nanosphere (AuNS), nanorod (AuNR), and nanocage (AuNC). In addition to distinct geometries and structural symmetries, these shapes have different photophysical properties detected by surface plasmon resonances. Therefore, choosing the best shaped GNP for a specific purpose is crucial to the success of the application. In this study, all three shapes of GNP were investigated for their potency to interact with cell surface receptors. Anti-HER2 antibody was conjugated to the surface of nanoparticles. MCF-7 breast adenocarcinoma and hMSC human mesenchymal cell lines were treated with GNPs and analyzed for cellular uptake and bioimaging efficiencies using the UV–vis spectroscopy and dark-field microscopy.

DNA topoisomerase I and II have been shown to be modified with a ubiquitin-like protein SUMO in response to their specific inhibitors called 'poisons'. These drugs also damage DNA by stabilizing the enzyme-DNA cleavable complex and induce a degradation of the enzymes through the 26S proteasome system. A plausible link between sumoylation and degradation has not yet been elucidated. We demonstrate here that topoisomerase IIbeta, but not its isoform IIalpha, is selectively degraded through proteasome by exposure to the catalytic inhibitor ICRF-193 which does not damage DNA. The beta isoform immunoprecipitated from ICRF-treated cells was modified by multiple modifiers, SUMO-2/3, SUMO-1, and polyubiquitin. When the SUMO conjugating enzyme Ubc9 was conditionally knocked out, the ICRF-induced degradation of topoisomerase IIbeta did not occur, suggesting that the SUMO modification pathway is essential for the degradation.

Porphyrin photosensitizers are mostly used components in photodynamic therapy (PDT). The poor solubility of porphyrins in aqueous medium is the problem to be solved for the in vivo applications. The delivery of photosensitizers to the tumor cells using liposome vehicles can help to overcome this problem. In this work, we have first functionalized the protoporphyrin IX with lipophilic oleylamine arms and encapsulated it into 1,2 dioleyl-sn-glycero-phosphatidylcholine (DOPC) liposomes. The appropriate sizes of liposomes are about 140 nm and have the characteristic Soret and Q band absorptions at 405 nm (Soret), 507 nm, 541 nm, 577 nm and 631 nm (Q bands), respectively. In the photodynamic activity studies, the liposomal porphyrins were irradiated with light (375 nm, 10 mW) in the presence of cancer cell lines, HeLa and AGS. We have found that both liposomal porphyrins and oleylamine conjugated porphyrins are much more effective than PpIX. This result can be attributed to the drug delivery characteristic of the liposomes which plays effective role in endocytosis. We also found that, in AGS cells, liposomal PpIX-Ole induced apoptosis more than HeLa cells under light conditions.

The matrix attachment region (MAR) is a distinctive genomic DNA involved in a variety of nuclear processes through association with the nuclear matrix. Recent studies suggest that nuclear matrix is altered in the process of apoptosis and presented to the immune system, leading to the production of autoantibodies against its protein components. To see whether MARs are also recognized by autoantibodies, a collection of human sera containing antinuclear antibodies was screened for the presence of binding activities against cloned MARs. We found that MAR-binding activities are quite common in these sera. There was a positive correlation among the MAR-binding titers for three different MAR probes. As expected, the MAR-binding activity was copurified with serum IgG, and subclass analysis with affinity-purified IgG on MAR-Sepharose showed a predominance of IgG2 isotype. Several lines of evidence implied that the anti-MAR antibodies detected here is distinct from the ordinary anti-DNA antibodies that are reactive to bulk DNA.

Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) represent a promising cell-based therapy for a number of degenerative conditions. Many applications require cell expansion and involve the treatment of diseases and conditions found in an aging population. Therefore, the effects of donor age and long-term passage must be clarified. In this study, the effects of donor age and long-term passage on the morphology, proliferation potential, characteristics, mesodermal differentiation ability, and transdifferentiation potential of hMSCs towards neurogenic lineage were evaluated. Cells from child donors (0–12 years, n = 6) maintained their fibroblast-like morphology up to higher passages and proliferated in a greater number than those from adult (25–50 years, n = 6) and old (over 60 years, n = 6) donors. Adipogenic, osteogenic, and neurogenic differentiation potential decreased with age, while chondrogenic potential did not change. Long-term passage affected the morphology and proliferation of hMSCs from all ages. With increasing passage number, proliferation rate decreased and cells lost their typical morphology. Expression levels of neural markers (β III tubulin and NSE) and topo II isoforms in populations of nondifferentiated hMSCs were investigated by reverse transcription polymerase chain reaction analysis. While neural marker and topo IIβ expression levels increased due to increasing passage number in adult hMSCs compared to child hMSCs, topo IIα decreased in both. These results indicated that, even under highly standardized culture conditions, donor age and long-term passage have effects on hMSC characteristics, which should be taken into account prior to stem cell-based therapies.

Coronary artery disease is still one of the most important reasons of the death in the world. The endothelium is the membrane of special cells which lines the interior surface of blood vessels forming an interface between circulating blood in the lumen and the rest of the vessel wall. Endothelial cells (ECs) line the entire circulatory system, from the heart to the smallest capillary. ECs dysfunction has been linked with atherosclerosis through their response to fluid forces. ECs change their morphology when exposed to mechanical stresses. The morphological responses include reorientation, elongation, and rearrangement of adhering molecules. Atherosclerotic lesions are formed in specific arterial regions, where low and oscillatory endothelial shear stress (ESS) occur. In this study, the effects of steady and disturbed flow over human umbilical vein endothelial cells (HUVECs) at different flow rates and periods were determined. Steady flow experiments were performed at flow rate of 1000 cm3/min for twenty four hours. Disturbed flow experiments simulating the flow in branching regions of arterial systems were carried out at flow rates of 250 cm3/min for five hours. The results obtained testified to the morphological changes easily observed. The directional alignment of the cells was determined in the steady flow experiments. Under disturbed flow conditions we observed not only the cell movement at the stagnation point but also the polygonal cell shape downstream the flow field.

Objective: We aimed to improve an ICG (Indocyanine Green) based nanoparticle, a light source and an application method that is centred to photodynamic therapy of MCF-7 cells. Methods: MCF-7 cells were obtained from ATCC, expanded in DMEM, 10% FBS and subjected to tissue culture E-Plate that was used to generate dynamic real-time data by measuring electrical impedance across interdigitated micro-electrodes on the bottom of the plate. ICG (has a high absorption at 770-810 nm) embedded and functional folic acid groups covered magnetic mesoporous nanoparticles that can be directed by magnetic field was prepared at various (50, 25, 10, 5, 2, 5 µg/ml) concantrations and applied to the E-plate. Light source (LEDs) was designed as a system that fit to 96 well-plate was irradiated 785 nm for 20 minute. To confirm the xCelligence system's data, proliferation assay was performed according to the manufacture's instructions (WST-1 reagent, ROCHE). Afterwards absorbance was measured at 450 nm using an ELISA reader. Phosphatidylserine accumulation was determined with the Apoptosis Detection Kit, Annexin-V-Alexa 568 (ROCHE). Results: According to xCelligence system analysis, the effect of optimal dose of irradiated MNP-MS-ICG-PAMAM-Folic Acid molecule was more potent at reducing proliferation of MCF-7 cells than MNP-MS-ICG-PAMAM. Also these results were confirmed by WST-1 assay was used in dark and light conditions for MCF-7 cells. The irradiation light itself did not cause any cytotoxic effect in the absence of photosensitizers. Our results showed that in-vitro application of MNP-MS-ICG-PAMAM-Folic Acid-PDT cause apoptosis in the MCF-7 cell line. Apoptotic cells were observed after 24 hours treatment under the irradiation. Conclusion: This study revealed that MNP-MS-ICG-PAMAM- Folic Acid –PDT is more cytotoxic and thereby lethal than the nanoparticle without Folic Acid integration. This indicates high efficiency for future in vivo studies aiming anti-cancer treatment strategies instead of using the latter.