Identification of the Candidate mGlu2 Allosteric Modulator THRX-195518 through In Silico Method and Evaluation of Its Neuroprotective Potential against Glutamate-Induced Neurotoxicity in SH-SY5Y Cell Line.

Glutamate (Glu) toxicity has been an important research topic in toxicology and neuroscience studies. In vitro and in vivo studies have shown that Group II metabotropic Glu2 (mGlu2) activators have cell viability effects. This study aims to determine a candidate ligand with high mGlu2 allosteric region activity among cytotoxicity-safe molecules using the in silico positioning method and to evaluate its cell viability effect in vitro. We investigated the candidate molecule's cell viability effect on the SH-SY5Y human neuroblastoma cell line by MTT analysis. In the study, LY 379268 (agonist) and JNJ-46281222 (positive allosteric modulator; PAM) were used as control reference molecules. Drug bank screening yielded THRX-195518 (docking score being -12.4 kcal/mol) as a potential novel drug candidate that has a high docking score and has not been mentioned in the literature so far. The orthosteric agonist LY 379268 exhibited a robust protective effect in our study. Additionally, our findings demonstrate that JNJ-46281222 and THRX-195518, identified as activating the mGlu2 allosteric region through in silico methods, preserve cell viability against Glu toxicity. Therefore, our study not only emphasizes the positive effects of this compound on cell viability against Glu toxicity but also sheds light on the potential of THRX-195518, acting as a mGlu2 PAM, based on in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) data, as a candidate drug molecule. These findings underscore the potential utility of THRX-195518 against both neurotoxicity and Central Nervous System (CNS) disorders, providing valuable insights.

Enhancing CAR-T cells: unleashing lasting impact potential with phytohemagglutinin activation in in vivo leukemia model.

Chimeric antigen receptor T (CAR-T) cell therapy holds great promise as an innovative immunotherapeutic approach for cancer treatment. To optimize the production and application of CAR-T cells, we evaluated the in vivo stability and efficacy capacities of CAR-T cells developed under different conditions. In this study, CAR-T cells were activated using Phytohemagglutinin (PHA) or anti-CD3&anti-CD28 and were compared in an in vivo CD19+B-cell cancer model in mouse groups. Our results demonstrated that CAR-T cells activated with PHA exhibited higher stability and anti-cancer efficacy compared to those activated with anti-CD3&anti-CD28. Specifically, CAR19BB-T cells activated with PHA exhibited continuous proliferation and long-term persistence without compromising their anti-cancer efficacy. Kaplan-Meier survival analysis revealed prolonged overall survival in the CAR-T cell-treated groups compared to the only tumor group. Furthermore, specific LTR-targeted RT-PCR analysis confirmed the presence of CAR-T cells in the treated groups, with significantly higher levels observed in the CAR19BB-T (PHA) group compared to other groups. Histopathological analysis of spleen, kidney, and liver tissue sections indicated reduced inflammation and improved tissue integrity in the CAR-T cell-treated groups. Our findings highlight the potential benefits of using PHA as a co-stimulatory method for CAR-T cell production, offering a promising strategy to enhance their stability and persistence. These results provide valuable insights for the development of more effective and enduring immunotherapeutic approaches for cancer treatment. CAR-T cells activated with PHA may offer a compelling therapeutic option for advancing cancer immunotherapy in clinical applications.

Microglia Mediated Neuroinflammation in Parkinson's Disease.

Parkinson's Disease (PD) is the second most common neurodegenerative disorder seen, especially in the elderly. Tremor, shaking, movement problems, and difficulty with balance and coordination are among the hallmarks, and dopaminergic neuronal loss in substantia nigra pars compacta of the brain and aggregation of intracellular protein α-synuclein are the pathological characterizations. Neuroinflammation has emerged as an involving mechanism at the initiation and development of PD. It is a complex network of interactions comprising immune and non-immune cells in addition to mediators of the immune response. Microglia, the resident macrophages in the CNS, take on the leading role in regulating neuroinflammation and maintaining homeostasis. Under normal physiological conditions, they exist as "homeostatic" but upon pathological stimuli, they switch to the "reactive state". Pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes are used to classify microglial activity with each phenotype having its own markers and released mediators. When M1 microglia are persistent, they will contribute to various inflammatory diseases, including neurodegenerative diseases, such as PD. In this review, we focus on the role of microglia mediated neuroinflammation in PD and also signaling pathways, receptors, and mediators involved in the process, presenting the studies that associate microglia-mediated inflammation with PD. A better understanding of this complex network and interactions is important in seeking new therapies for PD and possibly other neurodegenerative diseases.

Proposing novel natural compounds against Alzheimer's disease targeting acetylcholinesterase.

Alzheimer's disease (AD) is a neurodegenerative disorder considered as a global public health threat influencing many people. Despite the concerning rise in the affected population, there is still a shortage of potent and safe therapeutic agents. The aim of this research is to discover novel natural source molecules with high therapeutic effects, stability and less toxicity for the treatment of AD, specifically targeting acetylcholinesterase (AChE). This research can be divided into two steps: in silico search for molecules by systematic simulations and in vitro experimental validations. We identified five leading compounds, namely Queuine, Etoperidone, Thiamine, Ademetionine and Tetrahydrofolic acid by screening natural molecule database, conducting molecular docking and druggability evaluations. Stability of the complexes were investigated by Molecular Dynamics simulations and free energy calculations were conducted by Molecular Mechanics Generalized Born Surface Area method. All five complexes were stable within the binding catalytic site (CAS) of AChE, with the exception of Queuine which remains stable on the peripheral site (PAS). On the other hand Etoperidone both interacts with CAS and PAS sites showing dual binding properties. Binding free energy values of Queuine and Etoperidone were -71.9 and -91.0 kcal/mol respectively, being comparable to control molecules Galantamine (-71.3 kcal/mol) and Donepezil (-80.9 kcal/mol). Computational results were validated through in vitro experiments using the SH-SY5Y(neuroblastoma) cell line with Real Time Cell Analysis (RTCA) and cell viability assays. The results showed that the selected doses were effective with half inhibitory concentrations estimated to be: Queuine (IC50 = 70,90 µM), Etoperidone (IC50 = 712,80 µM), Thiamine (IC50 = 18780,34 µM), Galantamine (IC50 = 556,01 µM) and Donepezil (IC50 = 222,23 µM), respectively. The promising results for these molecules suggest the development of the next step in vivo animal testing and provide hope for natural therapeutic aids in the treatment of AD.

Polyamidoamine Dendron-Bearing Lipids as Drug-Delivery Excipients.

An amine-terminated polyamidoamine (PAMAM) dendron and two long alkyl groups were designed as a novel drug carrier that possesses an interior for the encapsulation of drugs and a biocompatible surface. We synthesized three dendron-bearing lipids, DL-G1, DL-G2, and DL-G3, which included first, second, and third generation polyamidoamine dendrons, respectively. The synthesized dendrimer encapsulating anticancer drug, 5-fluorouracil (5-FU), was prepared by extraction with chloroform from mixtures of the dendrimers and varying amounts of the drug. In vitro cytotoxicity of PAMAM conjugated di-n-dodecylamine micelles (G1, G2, G3) were analyzed on human gastric adenocarcinoma cells (AGS) by water-soluble tetrazolium-1 (WST-1) cell proliferation assay. Upon exposure to 5-FU loaded micelles, the viability of the cells decreased gradually in all generations. Cytotoxicity increased with increasing generation and reached its highest rate of 69.8 ± 3.2% upon 15 µM 5FU-loaded 25 µM PAMAM DL-3 micelle treatment. These results demonstrate that 5FU-loaded PAMAM conjugated di-n-dodecylamine treatment inhibits the proliferation of AGS cells in a generation-dependent manner.

Knockdown of c-MYC Controls the Proliferation of Oral Squamous Cell Carcinoma Cells in vitro via Dynamic Regulation of Key Apoptotic Marker Genes.

Oral squamous cell carcinoma (OSCC) is the most common malignant epithelial cancer occurring in the oral cavity, where it accounts for nearly 90% of all oral cavity neoplasms. The c-MYC transcription factor plays an important role in the control of programmed cell death, normal-to-malignant cellular transformation, and progression of the cell cycle. However, the role of c-MYC in controlling the proliferation of OSCC cells is not well known. In this study, c-MYC gene was silenced in OSCC cells (ORL-136T), and molecular and cellular responses were screened. To identify the pathway through which cell death occurred, cytotoxicity, colony formation, western blotting, caspase-3, and RT-qPCR analyzes were performed. Results indicated that knockdown of c-MYC has resulted in a significant decrease in the cell viability and c-MYC protein synthesis. Furthermore, caspase-3 was shown to be upregulated leading to apoptosis via the intrinsic pathway. In response to c-MYC knockdown, eight cell proliferation-associated genes showed variable expression profiles: c-MYC (-21.2), p21 (-2.5), CCNA1(1.8), BCL2 (-1.4), p53(-3.7), BAX(1.1), and CYCS (19.3). p27 expression was dramatically decreased in c-MYC-silenced cells in comparison with control, and this might indicate that the relative absence of c-MYC triggered intrinsic apoptosis in OSCC cells via p27 and CYCS.

Down regulation of DNA topoisomerase IIß exerts neurodegeneration like effect through Rho GTPases in cellular model of Parkinson's disease by Down regulating tyrosine hydroxylase.

Initiating the transcriptional activation of neuronal genes, DNA topoisomerase IIß (topo IIß) has a crucial role in neural differentiation and brain development. Inhibition of topo IIß activity causes shorter axons and deteriorated neuronal connections common in neurodegenerative diseases. We previously reported that topo IIß silencing could give rise to neurodegeneration through dysregulation of Rho GTPases and may contribute to pathogenesis of neurodegenerative diseases. Although there are several studies available proposing a link between Parkinson's Disease (PD) and Rho GTPases, there have been no reports analyzing the topo IIß-dependent association of PD and Rho GTPases. Here, for the first time, we identified that topo IIß has a regulatory role on Rho GTPases contributing to PD-like pathology. We analyzed the association between topo IIß and PD by comparing topo IIß expression levels of Retinoic Acid (RA) and Brain-derived neutrophic factor (BDNF) induced and MPP+-intoxicated SH-SY5Y cells used as an in vitro PD model. While both mRNA and protein levels of topo IIß increase in neural differentiated cells, a significant decrease is detected in the PD model. Additionally, silencing of topo IIß by specific siRNAs caused phenotypic alterations like deteriorated neural connections and transcriptional regulations such as upregulation of RhoA and downregulation of Cdc42, Rac1, and tyrosine hydroxylase gene expressions. Our results suggest that topo IIß downregulation may cause neurodegeneration through dysregulation of Rho-GTPases leading to PD-like pathology.

DNA topoisomerase IIß stimulates neurite outgrowth in neural differentiated human mesenchymal stem cells through regulation of Rho-GTPases (RhoA/Rock2 pathway) and Nurr1 expression.

BACKGROUND: DNA topoisomerase IIß (topo IIß) is known to regulate neural differentiation by inducing the neuronal genes responsible for critical neural differentiation events such as neurite outgrowth and axon guidance. However, the pathways of axon growth controlled by topo IIß have not been clarified yet. Microarray results of our previous study have shown that topo IIß silencing in neural differentiated primary human mesenchymal stem cells (hMSCs) significantly alters the expression pattern of genes involved in neural polarity, axonal growth, and guidance, including Rho-GTPases. This study aims to further analyze the regulatory role of topo IIß on the process of axon growth via regulation of Rho-GTPases. METHODS AND RESULTS: For this purpose, topo IIß was silenced in neurally differentiated hMSCs. Cells lost their morphology because of topo IIß deficiency, becoming enlarged and flattened. Additionally, a reduction in both neural differentiation efficiency and neurite length, upregulation in RhoA and Rock2, downregulation in Cdc42 gene expression were detected. On the other hand, cells were transfected with topo IIß gene to elucidate the possible neuroprotective effect of topo IIß overexpression on neural-induced hMSCs. Topo IIß overexpression prompted all the cells to exhibit neural cell morphology as characterized by longer neurites. RhoA and Rock2 expressions were downregulated, whereas Cdc42 expression was upregulated. Nurr1 expression level correlated with topo IIß in both topo IIß-overexpressed and -silenced cells. Furthermore, differential translocation of Rho-GTPases was detected by immunostaining in response to topo IIß. CONCLUSION: Our results suggest that topo IIß deficiency could give rise to neurodegeneration through dysregulation of Rho-GTPases. However, further in-vivo research is needed to demonstrate if re-regulation of Rho GTPases by topo IIß overexpression could be a neuroprotective treatment in the case of neurodegenerative diseases.

Preparation and characterization of amine functional nano-hydroxyapatite/chitosan bionanocomposite for bone tissue engineering applications.

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.

Boronic Acid vs. Folic Acid: A Comparison of the bio-recognition performances by Impedimetric Cytosensors based on Ferrocene cored dendrimer.

A comparative study is reported where folic acid (FA) and boronic acid (BA) based cytosensors and their analytical performances in cancer cell detection were analyzed by using electrochemical impedance spectroscopy (EIS) method. Cytosensors were fabricated using self-assembled monolayer principle by modifying Au electrode with cysteamine (Cys) and immobilization of ferrocene cored polyamidiamine dendrimers second generation (Fc-PAMAM (G2)), after which electrodes were modified with FA and BA. Au/Fc-PAMAM(G2)/FA and Au/Fc-PAMAM(G2)/BA based cytosensors showed extremely good analytical performances in cancer cell detection with linear range of 1×102 to 1×106cellsml-1, detection limit of 20cellsml-1 with incubation time of 20min for FA based electrode, and for BA based electrode detection limit was 28cellsml-1 with incubation time of 10min. Next to excellent analytical performances, cytosensors showed high selectivity towards cancer cells which was demonstrated in selectivity study using human embryonic kidney 293 cells (HEK 293) as normal cells and Au/Fc-PAMAM(G2)/FA electrode showed two times better selectivity than BA modified electrode. These cytosensors are promising for future applications in cancer cell diagnosis.

Cellular Model of Alzheimer's Disease: Aß1-42 Peptide Induces Amyloid Deposition and a Decrease in Topo Isomerase IIß and Nurr1 Expression.

BACKGROUND: DNA topoisomerase IIß (topo IIß) plays a crucial role in neural differentiation and axonogenesis. Inhibition of topo IIß 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 IIß in the pathogenesis of AD remains to be elucidated. OBJECTIVES: We aimed to investigate the role of topo IIß 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-ß 1-42 (Aß1-42) for 48 hours with cultured cerebellar granule neurons (CGNs) isolated from post-natal eight-day rats. The regulatory role of topo IIß on the transcription of Nurr1 was analyzed in topo IIß silenced CGNs, and also topo IIß silenced and overexpressed in a neurally-differentiated human mesenchymal (hMSC) cell line. RESULTS: Aß1-42 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 IIß 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 IIß deficiency, and was increased in topo IIß overexpressing hMSCs. CONCLUSION: Our findings suggest that topo IIß 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 IIß with AD.

Highly sensitive detection of cancer cells with an electrochemical cytosensor based on boronic acid functional polythiophene.

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.

Preparation and in vitro evaluation of 5-flourouracil loaded magnetite-zeolite nanocomposite (5-FU-MZNC) for cancer drug delivery applications.

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.

DNA topoisomerase IIß as a molecular switch in neural differentiation of mesenchymal stem cells.

Two isoforms of DNA topoisomerase II (topo II) have been identified in mammalian cells, named topo IIα and topo IIß. Topo IIα plays an essential role in segregation of daughter chromosomes and thus for cell proliferation in mammalian cells. Unlike its isozyme topo IIα, topo IIß is greatly expressed upon terminal differentiation of neuronal cells. Although there have been accumulating evidence about the crucial role of topo IIß 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 IIß 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 IIß 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 IIß 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 IIß. For this purpose, topo IIß was silenced by specific small interfering RNAs in hMSCs and cells were induced to differentiate into neuron-like cells. Differentiation and silencing of topo IIß were monitored by real-time cell analysis and also expressions of topo II isoforms were analyzed. Change in transcription patterns of genes upon topo IIß 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 IIß silencing suggesting that topoIIß 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 IIß in neural differentiation.

Delivery of lipophilic porphyrin by liposome vehicles: preparation and photodynamic therapy activity against cancer cell lines.

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.

Donor age and long-term culture affect differentiation and proliferation of human bone marrow mesenchymal stem cells.

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 (ß 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 IIß 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.

Comparison of long-term retinoic acid-based neural induction methods of bone marrow human mesenchymal stem cells.

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.

Selective silencing of DNA topoisomerase IIß in human mesenchymal stem cells by siRNAs (small interfering RNAs).

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 IIß 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 IIß selectively. A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIß-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.

Steady and disturbed flow effects on human umbilical vein endothelial cells (HUVECs) in vascular system: an experimental study.

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.

The SUMO pathway is required for selective degradation of DNA topoisomerase IIbeta induced by a catalytic inhibitor ICRF-193(1).

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.